HLA-B57 screening test not done at inclusion

Set up of study

randomised dubble blind placebo controlled

randomised blinded for backbone

randomised open label

randomised dubble blind

randomised open label

dubble blind

randomised dubble blind placebo controlled

AIDS and death (clinical failure).

FTC 301 (16) randomised

Acronime of study

HEAT Sudy #(10)

ACTG 5202# (11,12)

Gilead 934 Study (13)

CNA30024# (14)

GESIDA (15)

Gilead 903 Study(17)

#### **3.1 Grade 3 and 4, serious adverse events**

The prevalence and incidence of grade 3 (severe adverse event) and grade 4 (potentially life threatening adverse event) adverse events (table according to NIAID, Division of AIDS) (19) provide information on the safety of anti-retrovirals. It is not always possible to determine which adverse events are caused by an individual drug or by the combination of drugs in a regimen. Grade 3-4 adverse events have major consequences for the patient and may lead to significant morbidity, hospitalisation and mortality. The medication must almost always be stopped, leading to an increased risk of resistant mutations, decrease in CD4 cell count and resulting impairment of physical condition and a lower quality of life **(22,23,24,31).**

Adverse events in the studies with mainly naive patients show highly variable incidences of grade 3 and 4 adverse events, ranging from 0-57%. On the basis of these studies, it is not possible to determine a certain pattern of side-effects. It is not clear which side-effects can be linked to the individual NRTI, to the backbone or other drugs. In some studies all grade 2-4 side-effects are summarised without full details **(10,14,16).** In other studies report only a selection of grade 3 and 4 clinical and or only laboratory adverse events are reported **(11,12).**  The percentage of patients stopping study drugs because of adverse events ranged up to 34% (see table1). Several adverse events with changing severity have been associated with NRTIs (**34).**

Anemia, neutropenia and thrombocytopenia

Anemia and neutropenia (granulocytopenia) occur are 1.1-9.7% of the patients treated with ZVD. Higher percentages is seen in patients with AIDS, 15-61% **(35).** Serious grade 3 of 4 anemia and neutropenia are relatively rare with earlier initiation of treatment. Hematologic toxicity is more often seen with ZVD and rarely to never in treatment with other NRTIs. Combinations of NRTIs with other hemato-toxic medication may increase the incidence of serious anemia and neutropenia **(35).** Serious grade 3 of 4 thrombocytopenia due to cART, necessitating thrombocytes transfusions have been reported **(35).**

InforMatrix Nucleoside/Nucleotide ReverseTranscriptase Inhibitors "Backbones" 369

monotherapy and in 2.2 cases per 100 person-years for ZVD/ddl **(43).** Sensoric neuropathy

Drug hypersensitivity reactions are an important cause of morbidity in HIV-infected patients. The hypersensitivity reactions can be caused by each of the antiretroviral drugs in

ABC is known for its hypersensitivity reaction usually appearing in the first six week after starting ABC therapy. Symptoms of this ABC related hypersensitivity reaction are nonspecific and can be difficult to distinguish from reactions to other drugs or conditions and can lead to serious complications and death if not recognized. The presence of HLA-B5701 antigen is highly predictive for the chance of developing this hypersensitivity reaction. ABC hypersensitivity reaction affects 5-8% of patients during the first six weeks of

Hypersensitivity reactions to 3TC, FTC, ddI, TDF have been reported but occur very rarely **(45).** Other forms of hypersensitivity reactions and rashes are related to the use of NNRTIs

Other serious adverse events like rhabdomyolysis, myopathy and cardiomyopathy are very

Chronic kidney disease in HIV-positive persons can be caused by both HIV and traditional or non-HIV-related factors and antiretroviral drugs. Tenofovir has been associated with

Tenofovir is mainly cleared by glomerular filtration and active tubular secretion through tubular transport proteins. Interactions and competition of different anti-retrovirals with these transport proteins can lead to renal toxicity and increased blood levels. The combination of TDF/ddI leads to ~ 30% increased ddI levels and ddI related toxicity. Up to February 2006, 27 individual cases of renal failure with or without proteinuria or Fanconi syndrome (renal tubular acidosis) have been described with the use of TDF **(47).** During a follow-up of 144 weeks of 600 patients in the 903 Study **(17,50)** comparing d4T/3TC/EFV with TDF/3TC/EFV no significant increases in mean creatinine level were seen in the 299 patients treated with tenofovir. In the Heat study **(10)** comparing ABC/3TC and TDF/FTC and in Study 934 **(13)** comparing ZVD/3TC and TDF/FTC during an obserervation period of respectively 96 weeks and 48 weeks no significant difference in

In the Swiss HIV Cohort Study **(48)** 363 treatment-naive patients or patients with treatment interruptions of more than 12 months starting either a TDF-based cART and 715 patients on a TDF-sparing regime were compared for the time to reach a 10 ml/min reduction in calculated GFR (cGFR). Apart from diabetes mellitus, higher baseline cGFR (by 10 ml/min), TDF use and boosted PI use were significantly associated with an increased risk for reaching

During a median follow-up of 3.7 years in the EuroSida Study Group **(49)** 225 (3.3%) persons progressed to chronic kidney disease during 21.482 person-years follow-up, an incidence of 1.05 per 100 person-years follow-up. After adjustment for traditional factors associated with chronic kidney disease, increasing cumulative exposure to TDF and the PIs, indinavir, atazanavir and lopinavir/ritonavir were associated with a significantly increased rate of decline in renal function. No other antiretroviral drugs were associated with increased

a 10 ml/min reduction in cGFR during an observation time of two years.

has not been associated with the use of ABC,3TC, FTC and TDF.

the cART regimen or by other concomitend prescribed drugs.

Rash (hypersensitivity reaction) and muscle disorders

and PIs and are usually mild and self-limiting **(46).**

rare complications, which are not clearly related to NRTIs. **(46).**

treatment **(18).**

Renal failure

decline in renal funtion **(47,48,49).**

renal function could be shown.

incidence of chronic kidney disease.

Pancreatitis:

In the older studies pancreatitis was seen in 4-7% of patients treated with ddI and d4T. In 6% of these patients pancreatis was fatal **(35).** Combinations of ddI/d4T double the risk and ddI/TDF also increase the risk of development of pancreatitis **(36)**.

In the ACTG studies from October 1989 through July 1999 the overall pancreatitis rates were 0.61 per 100 person-years clinical and 2.23 per 100 person-years clinical/laboratory **(36).** The incidence of pancreatitis in the EuroSida cohort decreased over the years with earlier start of therapy and with higher CD4 cell counts. The incidence was 0.127 per 100 person-years over the years 2001-2006 **(38).**

Lactate acidosis (mitochondrial toxicity):

Lactate acidosis is a serious complication of the treatment with NRTIs, which occurs in around 0.9 times per 1000 years of treatment and often leads to serious morbidity and mortality **(35).**

In vitro studies have shown that inhibition of DNA polymerase gamma and other mitochondrial enzymes by NRTIs may lead to mitochondrial disfunction and cellular toxicity.

The clinical symptomatology of NRTI-induced mitochondrial toxicity consists of steatosis, hepatitis, lactate acidosis, myopathy, nephrotoxicity, peripheral neuropathy and pancreatitis. Studies with NRTIs in enzym assays and in cell cultures have shown that the following NRTIs are responsible for this mitochondrial toxicity, to a decreasing extent: ddI > d4T > ZVD **(39)**. DNA polymerase gamma inhibition is not found in normal concentrations of ABC, 3TC and TDF **(39).**

Lipodystrophy:

Three forms of lipodystrophy are distinguished: lipoatrophy caused by loss of subcutaneous fat, fat accumulation or lipohypertrophy and mixed forms. It was described with cART in 1997 and may occur in all combinations of cART medication with a prevalence between 2-84%. Lipo-accumumulation like buffelo-hump and "crix belli" may be caused by PIs, lipoatrophy by NRTIs. Lipoatrophy may occur shortly after initiation of cART. In a large observational cohort, 62% of the patients who developed lipoatrophy has symptoms within one year. In this cohort a highly significant correlation was found between lipoatrophy and the use of d4T **(40)**. It has been shown in observational cohort studies, clinical trials and in pathologic studies that lipoatrophy is specifically related to the use of NRTI especially d4T and in to a lesser extent to ZVD. Host factors have a modulating effect on the risk and the severity of lipoatrophy **(2).** 

The development of lipoatrophy is a serious complication which often leads to a marked decrease of the quality of life. In clinical studies is was shown that lipoatrophy improved by the replacement of ZVD or d4T by ABC **(41)** or TDF **(42).** 

Peripheral neuropathy

The occurrence of peripheral neuropathy is a well known complication of HIV-infection itself or due to a toxic effect on mitochondria induced by some NRTIs. Its incidence increases with the extent of immunodeficiency and older age. Patients who develop peripheral neuropathy tend to do so shortly after exposure to antiretroviral therapy and certain subgroup of patients are found to be more susceptible than others **(43).**

With ddI use the incidence of medication related sensoric neuropathy was 6.8 cases per 100 person years. In one study the relatieve risk is 1.4 fold higher in d4T use and 3.5 times higher in the combination of ddI/d4T compared to other NRTIs **(44).** In an other study peripheral neuropathy was reported in 3.0 cases per 100 person-years for ZVD monotherapy and in 2.2 cases per 100 person-years for ZVD/ddl **(43).** Sensoric neuropathy has not been associated with the use of ABC,3TC, FTC and TDF.

Rash (hypersensitivity reaction) and muscle disorders

Drug hypersensitivity reactions are an important cause of morbidity in HIV-infected patients. The hypersensitivity reactions can be caused by each of the antiretroviral drugs in the cART regimen or by other concomitend prescribed drugs.

ABC is known for its hypersensitivity reaction usually appearing in the first six week after starting ABC therapy. Symptoms of this ABC related hypersensitivity reaction are nonspecific and can be difficult to distinguish from reactions to other drugs or conditions and can lead to serious complications and death if not recognized. The presence of HLA-B5701 antigen is highly predictive for the chance of developing this hypersensitivity reaction. ABC hypersensitivity reaction affects 5-8% of patients during the first six weeks of treatment **(18).**

Hypersensitivity reactions to 3TC, FTC, ddI, TDF have been reported but occur very rarely **(45).** Other forms of hypersensitivity reactions and rashes are related to the use of NNRTIs and PIs and are usually mild and self-limiting **(46).**

Other serious adverse events like rhabdomyolysis, myopathy and cardiomyopathy are very rare complications, which are not clearly related to NRTIs. **(46).**

Renal failure

368 Recent Translational Research in HIV/AIDS

In the older studies pancreatitis was seen in 4-7% of patients treated with ddI and d4T. In 6% of these patients pancreatis was fatal **(35).** Combinations of ddI/d4T double the risk and

In the ACTG studies from October 1989 through July 1999 the overall pancreatitis rates were 0.61 per 100 person-years clinical and 2.23 per 100 person-years clinical/laboratory **(36).** The incidence of pancreatitis in the EuroSida cohort decreased over the years with earlier start of therapy and with higher CD4 cell counts. The incidence was 0.127 per 100 person-years

Lactate acidosis is a serious complication of the treatment with NRTIs, which occurs in around 0.9 times per 1000 years of treatment and often leads to serious morbidity and

In vitro studies have shown that inhibition of DNA polymerase gamma and other mitochondrial enzymes by NRTIs may lead to mitochondrial disfunction and cellular

The clinical symptomatology of NRTI-induced mitochondrial toxicity consists of steatosis, hepatitis, lactate acidosis, myopathy, nephrotoxicity, peripheral neuropathy and pancreatitis. Studies with NRTIs in enzym assays and in cell cultures have shown that the following NRTIs are responsible for this mitochondrial toxicity, to a decreasing extent: ddI > d4T > ZVD **(39)**. DNA polymerase gamma inhibition is not found in normal

Three forms of lipodystrophy are distinguished: lipoatrophy caused by loss of subcutaneous fat, fat accumulation or lipohypertrophy and mixed forms. It was described with cART in 1997 and may occur in all combinations of cART medication with a prevalence between 2-84%. Lipo-accumumulation like buffelo-hump and "crix belli" may be caused by PIs, lipoatrophy by NRTIs. Lipoatrophy may occur shortly after initiation of cART. In a large observational cohort, 62% of the patients who developed lipoatrophy has symptoms within one year. In this cohort a highly significant correlation was found between lipoatrophy and the use of d4T **(40)**. It has been shown in observational cohort studies, clinical trials and in pathologic studies that lipoatrophy is specifically related to the use of NRTI especially d4T and in to a lesser extent to ZVD. Host factors have a modulating

The development of lipoatrophy is a serious complication which often leads to a marked decrease of the quality of life. In clinical studies is was shown that lipoatrophy improved

The occurrence of peripheral neuropathy is a well known complication of HIV-infection itself or due to a toxic effect on mitochondria induced by some NRTIs. Its incidence increases with the extent of immunodeficiency and older age. Patients who develop peripheral neuropathy tend to do so shortly after exposure to antiretroviral therapy and

With ddI use the incidence of medication related sensoric neuropathy was 6.8 cases per 100 person years. In one study the relatieve risk is 1.4 fold higher in d4T use and 3.5 times higher in the combination of ddI/d4T compared to other NRTIs **(44).** In an other study peripheral neuropathy was reported in 3.0 cases per 100 person-years for ZVD

certain subgroup of patients are found to be more susceptible than others **(43).**

ddI/TDF also increase the risk of development of pancreatitis **(36)**.

Pancreatitis:

mortality **(35).**

Lipodystrophy:

Peripheral neuropathy

toxicity.

over the years 2001-2006 **(38).**

Lactate acidosis (mitochondrial toxicity):

concentrations of ABC, 3TC and TDF **(39).**

effect on the risk and the severity of lipoatrophy **(2).** 

by the replacement of ZVD or d4T by ABC **(41)** or TDF **(42).** 

Chronic kidney disease in HIV-positive persons can be caused by both HIV and traditional or non-HIV-related factors and antiretroviral drugs. Tenofovir has been associated with decline in renal funtion **(47,48,49).**

Tenofovir is mainly cleared by glomerular filtration and active tubular secretion through tubular transport proteins. Interactions and competition of different anti-retrovirals with these transport proteins can lead to renal toxicity and increased blood levels. The combination of TDF/ddI leads to ~ 30% increased ddI levels and ddI related toxicity.

Up to February 2006, 27 individual cases of renal failure with or without proteinuria or Fanconi syndrome (renal tubular acidosis) have been described with the use of TDF **(47).**

During a follow-up of 144 weeks of 600 patients in the 903 Study **(17,50)** comparing d4T/3TC/EFV with TDF/3TC/EFV no significant increases in mean creatinine level were seen in the 299 patients treated with tenofovir. In the Heat study **(10)** comparing ABC/3TC and TDF/FTC and in Study 934 **(13)** comparing ZVD/3TC and TDF/FTC during an obserervation period of respectively 96 weeks and 48 weeks no significant difference in renal function could be shown.

In the Swiss HIV Cohort Study **(48)** 363 treatment-naive patients or patients with treatment interruptions of more than 12 months starting either a TDF-based cART and 715 patients on a TDF-sparing regime were compared for the time to reach a 10 ml/min reduction in calculated GFR (cGFR). Apart from diabetes mellitus, higher baseline cGFR (by 10 ml/min), TDF use and boosted PI use were significantly associated with an increased risk for reaching a 10 ml/min reduction in cGFR during an observation time of two years.

During a median follow-up of 3.7 years in the EuroSida Study Group **(49)** 225 (3.3%) persons progressed to chronic kidney disease during 21.482 person-years follow-up, an incidence of 1.05 per 100 person-years follow-up. After adjustment for traditional factors associated with chronic kidney disease, increasing cumulative exposure to TDF and the PIs, indinavir, atazanavir and lopinavir/ritonavir were associated with a significantly increased rate of decline in renal function. No other antiretroviral drugs were associated with increased incidence of chronic kidney disease.

InforMatrix Nucleoside/Nucleotide ReverseTranscriptase Inhibitors "Backbones" 371

associated with a higher risk of acute MI within each CVD risk category defined by the Framingham Risk Score **(62).** Exposure to ABC within the most recent 6 months was associated with a 1.90 relative risk of acute MI. Subsequent analysis suggested cumulative use of ABC may also been associated with increased MI risk, although to a lesser extent than recent use **(63).** Since that first publication, several reports on MI risk associated with ABC have appeared, and some of these analyses have not implicated ABC as an MI risk factor. Several studies have focused on possible mechanisms that may explain the increased risk on MI in patients taking ABC. In the largest analysis, SMART study investigators found higher levels of hsCRP and IL-6 in patients taking ABC than in patients not taking ABC **(64)** However, a study of 13 biomarkers in virologically suppressed patients taking ABC/3TC vs TDF/FTC found no significant change in either group after 48 weeks **(65).** The results of the DAD study **(62)** could have been confounded by te so-called allocation biases such as high cardiovascular risk and renal function. In the Veterans Affairs Study a weak correlation between ABC use and MI was found, disappearing entirely after statistical adjustment for

In November 2008, DHHS guidelines reclassified abacavir from a preferred first-line agent

In the DAD study correcting the increased relative risk for antiretroviral-associated CVD for lipds attenuated this CVD risk by around 10% **(55).** In the ACTG 5202 study **(11,12)** fasting lipids at week 48 had increased more in the ABC/3TC arm than in the TDF/FTC arm (respectively; total cholesterol 0,87 mmol/L versus 0,67 mmol/L and triglycerides 0.28 mmol/L versus 0.03 mmol/L) with no significant difference between groups in the change in the ratio of total cholesterol to HDL cholesterol. In a systemic review of 7 clinical trials with a total of 3,807 paticipants, studying initial treatment in naïve subjects receiving 2NRTIs/efavirenz regimens the mean change in total cholesterol from baseline to 48 weeks

Many studies have documented an increased prevalence of osteopenia in HIV-infected individuals with dual x-ray absorptiometry bone densitometry (DEXA) scans. This finding is important since bone mineral density (BMD) predicts fracture risk **(68).** A higher fracture rate has been demonstrated among HIV-infected subjects compared with controls in a large healthcare system**.(69).** Many factors may play a role in the increased prevalence of osteopenia like vitamine D deficiency, low body mass, aging, corticosteroid use, alcoholism

Decreased BMD has been found in both treatment-naive and treated HIV-infected patients. Ongoing BMD loss over time has been observed in some treatment studies, although it is uncertain whether it is due to drug toxicity since it is difficult to differentiate between effects associated with antiretrovirals and other factors. In addition the presence and strength of antiviral-related factors is difficult to ascertain as combinations of classes of antiretroviral

In the GS 903 study comparing d4T/3TC and TDF/3TC, each combined with efavirenz in treatment-naive patients, after an an initial decrease in BMD was found in both study arms but stabilized after 24 weeks. By week 144, the mean decrease in BMD of the spine was significantly different 0.9% in the d4T/3TC arm and 2.2% in the TDF/3TC arm. At baseline there was a relatively high incidence of both osteopenia and osteoporosis in both study arms, but there was no significant difference in rates of new-onset osteopenia or progression

to an alternate agent, partly because of these data on cardiovascular risk.

was significantly greater in patients taking a non-TDF containing regimen **(67).**

Bone mineral density loss associated with HIV infection and cART

renal disease **(66).** 

and HIV-infection.

drugs are used.

to osteoporosis through week 144 **(70).**

In a prospective observational cohort study at Johns Hopkins **(51)** patients taking both TDF and NRTIs experienced an initial decline in cGFR during the first 180 days of therapy, but cGFR stabilized between 180 and 720 days. In this study there was no difference between TDF and NRTI use in more than 25% or 50% decline in cGFR at 1 or 2 years or in change in cGFR at 6, 12, or 24 months. Those taking TDF and a PI/ritonavir had a greater median decline in cGFR than those taking TDF and a NNRTI at 6 months. There was no difference in median cGFR decline between those on an NRTI/PI/ritonavir versus those on an NRTI/NNRTI regimen.

The reversibility of TDF-related nephrotoxicity in 24 male patients who ceased TDF for renal impairment by retrospective assessment were determined **(52).** Median eGFR pre-TDF was 74 mL/min/1.73 m2 (using the Modified Diet in Renal Disease equation) and fell to 51 mL/min/1.73 m2 at TDF cessation and increased to 58 mL/min/1.73 m2 in a median of 13 months after TDF cessation. This decline in cGFR, most recent versus pre-TDF is significant. Results were similar using the Cockcroft-Gault equation for cGFR. Only 10 patients reached their pre-TDF cGFR.

Many patients on antiretroviral therapy have multiple medical problems and may take other potentially nephrotoxic drugs. It has been clearly shown that taking TDF in combination with PI may increase a decline in renal function.

In a systematic review **(53)** of a total of 17 studies (including 9 randomized, controlled trials) a significantly greater loss of kidney function was seen among patients using TDF, compared with control subjects (mean difference in eGFR was 3.92 mL/min, as well as a greater risk of acute renal failure. There was no evidence that TDF use led to increased risk of severe proteinuria, hypophosphatemia, or fractures.

Thus in some well designed randomized prospective trials **(10,17,50)** no decline in renal function during treatment with TDF has been noted. Some observational stusies have found evidence of mild decrease in kidney function in TDF treated patients and when TDF related renal toxicity was present it was not always fully reversible**.** 

Cardiovascular risk and lipids

The risk of cardio vascular disease (CVD) and other non-AIDS conditions increases with age, but prevalence of these diseases by age is greater in HIV-positive populations. In a casecontrol study of HIV-infected patients and healthy HIV-negative individuals from an observational database comparing rates of 6 comorbidities,CVD, hypertension, renal failure, osteoporosis, diabetes, and hypothyroidism to be higher in HIV-infected patients **(54).**

Numerous large observational cohort studies in Europe and the USA have found higher rates of acute myocardial infarction (MI) or coronary heart disease (CHD) in patients with HIV. (5-10) In a cross-sectional study of HIV-infected participants and controls without preexisting CVD preclinical atherosclerosis assessed by carotid intima-medial thickness measurements in the internal/bulb and common carotid regions in HIV-infected participants and controls after adjusting for traditional CVD risk factors showed that HIV infection was accompanied by more extensive atherosclerosis **(61).**

The higher risk among patients with HIV-infected patients held true for every age group analyzed and in multivariate analysis adjusting for demographics and common cardiovascular risk factors confirm that HIV infection is an independent predictor of acute MI, conferring nearly a two-fold risk. The risk of myocardial infarcion is found to be associated with the cumulative use of PIs in these studies **(55,56,59,60).** 

In the D.A.D cohort, a lage observational prospective cohort study with more than 30,000 HIV-infected patients in 212 clinics since 1999, it was found that ABC and ddI were

In a prospective observational cohort study at Johns Hopkins **(51)** patients taking both TDF and NRTIs experienced an initial decline in cGFR during the first 180 days of therapy, but cGFR stabilized between 180 and 720 days. In this study there was no difference between TDF and NRTI use in more than 25% or 50% decline in cGFR at 1 or 2 years or in change in cGFR at 6, 12, or 24 months. Those taking TDF and a PI/ritonavir had a greater median decline in cGFR than those taking TDF and a NNRTI at 6 months. There was no difference in median cGFR decline between those on an NRTI/PI/ritonavir versus those on an

The reversibility of TDF-related nephrotoxicity in 24 male patients who ceased TDF for renal impairment by retrospective assessment were determined **(52).** Median eGFR pre-TDF was 74 mL/min/1.73 m2 (using the Modified Diet in Renal Disease equation) and fell to 51 mL/min/1.73 m2 at TDF cessation and increased to 58 mL/min/1.73 m2 in a median of 13 months after TDF cessation. This decline in cGFR, most recent versus pre-TDF is significant. Results were similar using the Cockcroft-Gault equation for cGFR. Only 10 patients reached

Many patients on antiretroviral therapy have multiple medical problems and may take other potentially nephrotoxic drugs. It has been clearly shown that taking TDF in combination

In a systematic review **(53)** of a total of 17 studies (including 9 randomized, controlled trials) a significantly greater loss of kidney function was seen among patients using TDF, compared with control subjects (mean difference in eGFR was 3.92 mL/min, as well as a greater risk of acute renal failure. There was no evidence that TDF use led to increased risk

Thus in some well designed randomized prospective trials **(10,17,50)** no decline in renal function during treatment with TDF has been noted. Some observational stusies have found evidence of mild decrease in kidney function in TDF treated patients and when TDF related

The risk of cardio vascular disease (CVD) and other non-AIDS conditions increases with age, but prevalence of these diseases by age is greater in HIV-positive populations. In a casecontrol study of HIV-infected patients and healthy HIV-negative individuals from an observational database comparing rates of 6 comorbidities,CVD, hypertension, renal failure, osteoporosis, diabetes, and hypothyroidism to be higher in HIV-infected patients **(54).** Numerous large observational cohort studies in Europe and the USA have found higher rates of acute myocardial infarction (MI) or coronary heart disease (CHD) in patients with HIV. (5-10) In a cross-sectional study of HIV-infected participants and controls without preexisting CVD preclinical atherosclerosis assessed by carotid intima-medial thickness measurements in the internal/bulb and common carotid regions in HIV-infected participants and controls after adjusting for traditional CVD risk factors showed that HIV

The higher risk among patients with HIV-infected patients held true for every age group analyzed and in multivariate analysis adjusting for demographics and common cardiovascular risk factors confirm that HIV infection is an independent predictor of acute MI, conferring nearly a two-fold risk. The risk of myocardial infarcion is found to be

In the D.A.D cohort, a lage observational prospective cohort study with more than 30,000 HIV-infected patients in 212 clinics since 1999, it was found that ABC and ddI were

NRTI/NNRTI regimen.

their pre-TDF cGFR.

Cardiovascular risk and lipids

with PI may increase a decline in renal function.

of severe proteinuria, hypophosphatemia, or fractures.

renal toxicity was present it was not always fully reversible**.** 

infection was accompanied by more extensive atherosclerosis **(61).**

associated with the cumulative use of PIs in these studies **(55,56,59,60).** 

associated with a higher risk of acute MI within each CVD risk category defined by the Framingham Risk Score **(62).** Exposure to ABC within the most recent 6 months was associated with a 1.90 relative risk of acute MI. Subsequent analysis suggested cumulative use of ABC may also been associated with increased MI risk, although to a lesser extent than recent use **(63).** Since that first publication, several reports on MI risk associated with ABC have appeared, and some of these analyses have not implicated ABC as an MI risk factor. Several studies have focused on possible mechanisms that may explain the increased risk on MI in patients taking ABC. In the largest analysis, SMART study investigators found higher levels of hsCRP and IL-6 in patients taking ABC than in patients not taking ABC **(64)** However, a study of 13 biomarkers in virologically suppressed patients taking ABC/3TC vs TDF/FTC found no significant change in either group after 48 weeks **(65).** The results of the DAD study **(62)** could have been confounded by te so-called allocation biases such as high cardiovascular risk and renal function. In the Veterans Affairs Study a weak correlation between ABC use and MI was found, disappearing entirely after statistical adjustment for renal disease **(66).** 

In November 2008, DHHS guidelines reclassified abacavir from a preferred first-line agent to an alternate agent, partly because of these data on cardiovascular risk.

In the DAD study correcting the increased relative risk for antiretroviral-associated CVD for lipds attenuated this CVD risk by around 10% **(55).** In the ACTG 5202 study **(11,12)** fasting lipids at week 48 had increased more in the ABC/3TC arm than in the TDF/FTC arm (respectively; total cholesterol 0,87 mmol/L versus 0,67 mmol/L and triglycerides 0.28 mmol/L versus 0.03 mmol/L) with no significant difference between groups in the change in the ratio of total cholesterol to HDL cholesterol. In a systemic review of 7 clinical trials with a total of 3,807 paticipants, studying initial treatment in naïve subjects receiving 2NRTIs/efavirenz regimens the mean change in total cholesterol from baseline to 48 weeks was significantly greater in patients taking a non-TDF containing regimen **(67).**

Bone mineral density loss associated with HIV infection and cART

Many studies have documented an increased prevalence of osteopenia in HIV-infected individuals with dual x-ray absorptiometry bone densitometry (DEXA) scans. This finding is important since bone mineral density (BMD) predicts fracture risk **(68).** A higher fracture rate has been demonstrated among HIV-infected subjects compared with controls in a large healthcare system**.(69).** Many factors may play a role in the increased prevalence of osteopenia like vitamine D deficiency, low body mass, aging, corticosteroid use, alcoholism and HIV-infection.

Decreased BMD has been found in both treatment-naive and treated HIV-infected patients. Ongoing BMD loss over time has been observed in some treatment studies, although it is uncertain whether it is due to drug toxicity since it is difficult to differentiate between effects associated with antiretrovirals and other factors. In addition the presence and strength of antiviral-related factors is difficult to ascertain as combinations of classes of antiretroviral drugs are used.

In the GS 903 study comparing d4T/3TC and TDF/3TC, each combined with efavirenz in treatment-naive patients, after an an initial decrease in BMD was found in both study arms but stabilized after 24 weeks. By week 144, the mean decrease in BMD of the spine was significantly different 0.9% in the d4T/3TC arm and 2.2% in the TDF/3TC arm. At baseline there was a relatively high incidence of both osteopenia and osteoporosis in both study arms, but there was no significant difference in rates of new-onset osteopenia or progression to osteoporosis through week 144 **(70).**

InforMatrix Nucleoside/Nucleotide ReverseTranscriptase Inhibitors "Backbones" 373

significant influence on compliance and quality of life **(22,26)** and on the durability of a certain combination. It is not always evident which drug in a cART regimen is responsible for which side-effect. The HIV-infection as such or complications of opportunistic infections

General symptoms as fatigue, pain, anorexia, sleep and concentration disturbances occur

It is difficult to give a reliable estimation of the relative incidence of different grade of adverse events, based on the EMEA and FDA data **(76)**, because of the relative lack of randomised

Cohort studies yield better insight as to why patients switch or stop certain antiretroviral drugs and how long they keep using the same regimen, in comparison with randomised

In the older cohort studies high rates of toxicity driven changes in antiretroviral drugs were common. For instance in the Swiss HIV Cohort Study, with 2,674 patients, 35% stopped treatment with at least one drug during the observation period of 3.2 years because of adverse events and/or intolerability and 41% stopped the combination of anti-retroviral

In the Italian ICONA-cohort **(78),** 36% of the 862 patients stopped because of side-effects

Earlier initiation of cART, lower pill burden and dosing schemes of once or twice daily,

In the Athena-cohort the incidence per 100 patient years of toxicity driven changes of cART during the first 3 years after the start of therapy decreased from 29% in 1996 to 15% in 2008. Significant decline in toxicity driven changes of cART started to be apparent after calendar year 2000. The incidence of toxicity driven changes of cART is highest in the first 3 month

The combinations of ABC/3TC, TDF/FTC and ddI/FTC or 3TC can be given once daily. ZVD/3TC (Combivir® )has to be given twice daily. The other combinations are given once or twice daily. The combinations of ABCbacavir/Lamivudine (Kivexa®, Epzicom®) and TDT/FTC (Truvada ®) can be given as one tablet per day. TDT/FTC in combination with

DdI is given 2 hours before or after food. The rest of the drugs can be taken irrespective of

Abacavir is not significantly metabolised by CYP450, which makes serious reactions regarding inhibition or induction of CYP450 enzymes unlikely **(80).** No interactions were

may lead to symptoms marked as adverse events of anti-retroviral medication.

comparative studies with extended follow-up and asufficient number of participants.

drugs at least once or completely changed to another combination **(77).**

together with declining toxicity, have improved tolerability.

**5.1 Ease of use (dosage frequency, number of tablets per day)** 

efavirenz can be given in one tablet (Atripla ®)

**6.1 Availability of different formulations** 

Liquid or dispersible formulations are available for ddI.

seen with adefovir, amprenavir, indinavir, ZVD and 3TC **(50).**

during study period of 45 weeks and only 5% because of virologic failure.

studies which usually have a limited follow-up time.

frequently **(46).**

after initiation.

**5. Easy of use** 

**6. Applicability** 

**6.2 Drug interactions** 

food.

Abacavir

In the STEAL study, 360 virologically suppressed patients were randomized to switch their current NRTIs to either ABC/3TC or TDF/FTC. No significant change in spine or hip T scores were observed in the ABC/3TC arm, but BMD at spine and hip decreased in the TDF/FTC arm, and the difference between the regimens was statistically significant at weeks 48 and 96 **(71).** 

In a study comparing the effect of TDF versus ABC based regimens on BMD, BMD decreased early during therapy in both arms before stabilizing. The mean loss of BMD was statistically greater with TDF and the loss correlated with biomarkers of bone turnover **(72).** Similar results were obtained in an other study comparing the safety aspects of ABC/3TC and TDF/FTC in 385 treatment-naive patients **(73).**

In the ACTG 5202 metabolic substudy, there was an initial reduction in BMD in all stady arms, which stabilized after 48 weeks. A significantly greater loss of BMD was seen at week 96 with TDF/FTC versus ABC/3TC. This included a significant 2% greater reduction in lumbar spine BMD and a significant 1.5% greater reduction in hip BMD. No difference was found in fracture rates between study arms at week 48 **(74).**

In the bone substudy of this trial, the initiation of antiretroviral therapy was associated with a decrease in bone mass of 2% to 4% that was independent of the regimen selected and stabilized by week 48; this decrease was greatest in patients who started a regimen that contained TDF **(75).**

Thus overall, BMD appears to decline to some degree during the first several months after initiation of cART, regardless of regimen, but the decline may be slightly greater with TDF containing regimens. However, there are no conclusive data showing that therapyassociated reductions in bone mineral density are also associated with an increased rate of fractures.

#### **3.2 Documentation**

The clinical documentation of the combinations is summarised in Table 2


Table 2. Documentation

\* according to the definition of National Institute of Health/PubMed (www.ncbi.nih.gov)

#### **4. Tolerability of anti-retroviral backbones**

#### **4.1 Grade 1 and 2, mild to moderate side-effects**

The tolerability of a cART regimens is an important predictor of durability and long-term succes. Grade 1(mild adverse event) and grade 2 (moderate adverse event) **(19)** may have a significant influence on compliance and quality of life **(22,26)** and on the durability of a certain combination. It is not always evident which drug in a cART regimen is responsible for which side-effect. The HIV-infection as such or complications of opportunistic infections may lead to symptoms marked as adverse events of anti-retroviral medication.

General symptoms as fatigue, pain, anorexia, sleep and concentration disturbances occur frequently **(46).**

It is difficult to give a reliable estimation of the relative incidence of different grade of adverse events, based on the EMEA and FDA data **(76)**, because of the relative lack of randomised comparative studies with extended follow-up and asufficient number of participants.

Cohort studies yield better insight as to why patients switch or stop certain antiretroviral drugs and how long they keep using the same regimen, in comparison with randomised studies which usually have a limited follow-up time.

In the older cohort studies high rates of toxicity driven changes in antiretroviral drugs were common. For instance in the Swiss HIV Cohort Study, with 2,674 patients, 35% stopped treatment with at least one drug during the observation period of 3.2 years because of adverse events and/or intolerability and 41% stopped the combination of anti-retroviral drugs at least once or completely changed to another combination **(77).**

In the Italian ICONA-cohort **(78),** 36% of the 862 patients stopped because of side-effects during study period of 45 weeks and only 5% because of virologic failure.

Earlier initiation of cART, lower pill burden and dosing schemes of once or twice daily, together with declining toxicity, have improved tolerability.

In the Athena-cohort the incidence per 100 patient years of toxicity driven changes of cART during the first 3 years after the start of therapy decreased from 29% in 1996 to 15% in 2008. Significant decline in toxicity driven changes of cART started to be apparent after calendar year 2000. The incidence of toxicity driven changes of cART is highest in the first 3 month after initiation.

#### **5. Easy of use**

372 Recent Translational Research in HIV/AIDS

In the STEAL study, 360 virologically suppressed patients were randomized to switch their current NRTIs to either ABC/3TC or TDF/FTC. No significant change in spine or hip T scores were observed in the ABC/3TC arm, but BMD at spine and hip decreased in the TDF/FTC arm, and the difference between the regimens was statistically significant at

In a study comparing the effect of TDF versus ABC based regimens on BMD, BMD decreased early during therapy in both arms before stabilizing. The mean loss of BMD was statistically greater with TDF and the loss correlated with biomarkers of bone turnover **(72).** Similar results were obtained in an other study comparing the safety aspects of ABC/3TC

In the ACTG 5202 metabolic substudy, there was an initial reduction in BMD in all stady arms, which stabilized after 48 weeks. A significantly greater loss of BMD was seen at week 96 with TDF/FTC versus ABC/3TC. This included a significant 2% greater reduction in lumbar spine BMD and a significant 1.5% greater reduction in hip BMD. No difference was

In the bone substudy of this trial, the initiation of antiretroviral therapy was associated with a decrease in bone mass of 2% to 4% that was independent of the regimen selected and stabilized by week 48; this decrease was greatest in patients who started a regimen that

Thus overall, BMD appears to decline to some degree during the first several months after initiation of cART, regardless of regimen, but the decline may be slightly greater with TDF containing regimens. However, there are no conclusive data showing that therapyassociated reductions in bone mineral density are also associated with an increased rate of

> Number of clinical trials\*

532/23 >10

165/10 >10

160/15 > 10

78/115 >10

\* according to the definition of National Institute of Health/PubMed (www.ncbi.nih.gov)

The tolerability of a cART regimens is an important predictor of durability and long-term succes. Grade 1(mild adverse event) and grade 2 (moderate adverse event) **(19)** may have a

Years since

registration

Emt: 8

Emt: 8

Emt: 8

>10

The clinical documentation of the combinations is summarised in Table 2

weeks 48 and 96 **(71).** 

contained TDF **(75).**

**3.2 Documentation** 

fractures.

and TDF/FTC in 385 treatment-naive patients **(73).**

found in fracture rates between study arms at week 48 **(74).**

Zidovudine/Lamivudine or

Didanosine/Lamivudine or

Abacavir/Lamivudine or

Tenofovir/emtricitabine or

**4. Tolerability of anti-retroviral backbones 4.1 Grade 1 and 2, mild to moderate side-effects** 

emtricitabine

emtricitabine

emtricitabine

Lamivudine

Table 2. Documentation

#### **5.1 Ease of use (dosage frequency, number of tablets per day)**

The combinations of ABC/3TC, TDF/FTC and ddI/FTC or 3TC can be given once daily. ZVD/3TC (Combivir® )has to be given twice daily. The other combinations are given once or twice daily. The combinations of ABCbacavir/Lamivudine (Kivexa®, Epzicom®) and TDT/FTC (Truvada ®) can be given as one tablet per day. TDT/FTC in combination with efavirenz can be given in one tablet (Atripla ®)

DdI is given 2 hours before or after food. The rest of the drugs can be taken irrespective of food.

#### **6. Applicability**

#### **6.1 Availability of different formulations**

Liquid or dispersible formulations are available for ddI.

#### **6.2 Drug interactions**

#### Abacavir

Abacavir is not significantly metabolised by CYP450, which makes serious reactions regarding inhibition or induction of CYP450 enzymes unlikely **(80).** No interactions were seen with adefovir, amprenavir, indinavir, ZVD and 3TC **(50).**

InforMatrix Nucleoside/Nucleotide ReverseTranscriptase Inhibitors "Backbones" 375

Zidovudine is mainly glucuronidated. The drug may theoretically show interactions with a large number of drugs which are also excreted through glucuronidation, like aspirin, NSAIDs, penicillins and oxazepam. Very limited data on the relevance of these possible

The bioavailability of zidovudine may be decreased to a limited extent (22%) by

The renal clearance of zidovudine decreases by 50% during simultaneous use of cotrimoxazole (**96).** This interaction is only relevant in disturbed glucuronidation of zidovudine. Rifampicin lowers the AUC of zidovudine by 50%, an interaction with rifabutin is not very

Zidovudine may cause an unpredictable interaction with phenytoin (increase of decrease of

Atovaquone increases the AUC of zidovudine by 35%. Valproic acid and methadone may

Nephrotoxic or myelosuppressive drugs may increase potential side-effects of ZVD (SPC on

There are no major differences in the approved indications. The applicability in children is

Lamivudine, emtricitabine and tenofovir also have anti hepatitis B virus activity. An advantage of these drugs is that "two in one" treatment is possible It is recommended that lamivudine or emtricitabine should be combined with tenofovir (97) in case of hepatitis B

Zidovudine/lamivudine (Combivir) and abacavir/lamivudine (Kivexa) can be used in

A dose reduction is necessary in case of renal function impaiment. Abacavir/Lamivudine should not be used when the creatinine clearance is lower than 50 ml/min. Tenofovir/Emtricitabine should bot be used when the creatinine clearance is lower than

children from 12 years. The individual components can be used from 3 months.

relevant, a 14% decrease of the AUC of zidovudine was seen (**96)**. The AUC of ZVD increases by 75% in combination with fluconazole (**96).**

co-infection. Only lamivudine is approved for this indication.

All drugs are contra-indicated in case of hypersensitivity.

Hypersensitivity to abacavir may be very serious.

No dose adjustments are necessary in the elderly.

Tenofovir and emcitabine are only applicable in adults.

the phenytoin levels). Phenytoin levels have to be checked on a regular basis.

also lead to an increase in the AUC of zidovudine, but little data are available. Zidovudine is antagonistic in combination with ribavirine or stavudine.

Zidovudine

zidovudine).

described in 5.5. Treatment co-infections

**6.3 Approved indications** 

**6.4 Contra-indications** 

30 ml/min.

**6.5 Use in children and elderly** 

Lamivudine can be used from 3 months Didanosine tablets can be used from 6 years.

**6.6 Use in renal and hepatic disease** 

interactions is available (**95).**

simultaneouse intake with food (**96).**

Enzymeinducers like rifampicin, phenobarbital and phenytoin may decrease the plasma concentrations of abacavir to a minor extent through an effect on UDPglucuronyltransferases [72].

Alcohol may decrease the AUC of abacavir by 40% **(81,82).**

Didanosine

The AUC of ddI doubles during simultaneous use of ganciclovir. Didanosine has no significant effect on the pharmacokinetics of zidovudine **(83).**

No clinically relevant interaction occurs between ddI with ritonavir, nevirapine, emtricitabine and nelfinavir **(84).** 

Ribavirine may increase intracellular levels of ddI. The relevance of this is unknown.

Didanosine decreases the bioavailability of ciprofloxacin during simultaneous intake. It is recommended to take ciprofloxacin an hour before or at least 4 hours after ddI **(84).** Didanosine showed no interaction with indinavir and fluconazole. Ketoconazole and itraconazole increase the AUC of ddI, maar these interactions do not appear te be very relevant.

The AUC of ddI increases by 50% in combination with tenofovir often leading to ddI toxicity **(3).** 

Xanthine oxidase plays a role in the metabolism of didanosine, interactions with inhibitors of xanthine oxidase, like allopurinol, may theoretically decrease the clearance of didanosine. Emtricitabine

Tenofovir and FTC do not affect each other's pharmacokinetics **(85, 86).** Emtricitabine is metabolised to a limited extent and is excreted unchanged in the urine through glomerular filtration and active tubular secretion**(85).** Interactions regarding to inhibition of active tubular secretion cannot be excluded, maar have not been studied **(85).**

Emcitabrine shows no pharmacokinetic interactions with protease inhibitors or with ddI **(85).** Lamivudine

Lamivudine shows few metabolic interactions. The drug is excreted in an unchanged form through glomerular filtration and active tubular secretion **(87, 88).**

No interaction is seen with ZVD and ddI **(87, 88)**.

Trimethoprim may decrease active tubular secretion, increasing the AUC of lamivudine by 40% **(87, 88)**. Applications of high dose co-trimoxazole in pneumocystis carinii infections should not be combined with lamivudine **(87)**. There is inadequate documentation on a possible interaction with intravenous ganciclovir or foscarnet. This combination should be avoided.

Tenofovir

Tenofovir is mainly excreted uchanged in the urine through glomerular filtration and active tubular secretion **(90,91)**. Interactions regarding to inhibition of active tubular secretion cannot be excluded, but have not been studied **(90).**

Tenofovir and FTC have no effect on each other's pharmacokinetics **(85, 86,91).**

The AUC of TDF increases by 30% in combination with lopinavir and ritonavir or atazanavir **(92,93).** Tenofovir may decrease the AUC of atazanavir by 25%. The AUC of lopinavir increases by 15% by tenofovir. Tenofovir shows no interaction with saquinavir (**91)**.

The AUC of ddI increases by 50% in combination with tenofovir **(91)**. This may increase the risk of pancreatitis and other ddI related toxicity. The AUC of atazanavir decreases by 25% in combination with TDF **(86).**

Tenofovir showed no interactions with indinavir, methadon, ribavirine or rifampicin**(91,93, 94).**

Zidovudine

374 Recent Translational Research in HIV/AIDS

Enzymeinducers like rifampicin, phenobarbital and phenytoin may decrease the plasma concentrations of abacavir to a minor extent through an effect on UDP-

The AUC of ddI doubles during simultaneous use of ganciclovir. Didanosine has no

No clinically relevant interaction occurs between ddI with ritonavir, nevirapine,

The AUC of ddI increases by 50% in combination with tenofovir often leading to ddI

Xanthine oxidase plays a role in the metabolism of didanosine, interactions with inhibitors of xanthine oxidase, like allopurinol, may theoretically decrease the clearance of didanosine.

Tenofovir and FTC do not affect each other's pharmacokinetics **(85, 86).** Emtricitabine is metabolised to a limited extent and is excreted unchanged in the urine through glomerular filtration and active tubular secretion**(85).** Interactions regarding to inhibition of active

Emcitabrine shows no pharmacokinetic interactions with protease inhibitors or with ddI **(85).**

Lamivudine shows few metabolic interactions. The drug is excreted in an unchanged form

Trimethoprim may decrease active tubular secretion, increasing the AUC of lamivudine by 40% **(87, 88)**. Applications of high dose co-trimoxazole in pneumocystis carinii infections should not be combined with lamivudine **(87)**. There is inadequate documentation on a possible interaction with intravenous ganciclovir or foscarnet. This

Tenofovir is mainly excreted uchanged in the urine through glomerular filtration and active tubular secretion **(90,91)**. Interactions regarding to inhibition of active tubular secretion

The AUC of TDF increases by 30% in combination with lopinavir and ritonavir or atazanavir **(92,93).** Tenofovir may decrease the AUC of atazanavir by 25%. The AUC of lopinavir

The AUC of ddI increases by 50% in combination with tenofovir **(91)**. This may increase the risk of pancreatitis and other ddI related toxicity. The AUC of atazanavir decreases by 25%

Tenofovir showed no interactions with indinavir, methadon, ribavirine or rifampicin**(91,93, 94).**

Tenofovir and FTC have no effect on each other's pharmacokinetics **(85, 86,91).**

increases by 15% by tenofovir. Tenofovir shows no interaction with saquinavir (**91)**.

tubular secretion cannot be excluded, maar have not been studied **(85).**

through glomerular filtration and active tubular secretion **(87, 88).**

No interaction is seen with ZVD and ddI **(87, 88)**.

cannot be excluded, but have not been studied **(90).**

combination should be avoided.

in combination with TDF **(86).**

Ribavirine may increase intracellular levels of ddI. The relevance of this is unknown. Didanosine decreases the bioavailability of ciprofloxacin during simultaneous intake. It is recommended to take ciprofloxacin an hour before or at least 4 hours after ddI **(84).** Didanosine showed no interaction with indinavir and fluconazole. Ketoconazole and itraconazole increase the AUC of ddI, maar these interactions do not appear te be very

glucuronyltransferases [72].

emtricitabine and nelfinavir **(84).** 

Didanosine

relevant.

toxicity **(3).** 

Emtricitabine

Lamivudine

Tenofovir

Alcohol may decrease the AUC of abacavir by 40% **(81,82).**

significant effect on the pharmacokinetics of zidovudine **(83).**

Zidovudine is mainly glucuronidated. The drug may theoretically show interactions with a large number of drugs which are also excreted through glucuronidation, like aspirin, NSAIDs, penicillins and oxazepam. Very limited data on the relevance of these possible interactions is available (**95).**

The bioavailability of zidovudine may be decreased to a limited extent (22%) by simultaneouse intake with food (**96).**

The renal clearance of zidovudine decreases by 50% during simultaneous use of cotrimoxazole (**96).** This interaction is only relevant in disturbed glucuronidation of zidovudine.

Rifampicin lowers the AUC of zidovudine by 50%, an interaction with rifabutin is not very relevant, a 14% decrease of the AUC of zidovudine was seen (**96)**.

The AUC of ZVD increases by 75% in combination with fluconazole (**96).**

Zidovudine may cause an unpredictable interaction with phenytoin (increase of decrease of the phenytoin levels). Phenytoin levels have to be checked on a regular basis.

Atovaquone increases the AUC of zidovudine by 35%. Valproic acid and methadone may also lead to an increase in the AUC of zidovudine, but little data are available.

Zidovudine is antagonistic in combination with ribavirine or stavudine.

Nephrotoxic or myelosuppressive drugs may increase potential side-effects of ZVD (SPC on zidovudine).

#### **6.3 Approved indications**

There are no major differences in the approved indications. The applicability in children is described in 5.5.

Treatment co-infections

Lamivudine, emtricitabine and tenofovir also have anti hepatitis B virus activity. An advantage of these drugs is that "two in one" treatment is possible It is recommended that lamivudine or emtricitabine should be combined with tenofovir (97) in case of hepatitis B co-infection. Only lamivudine is approved for this indication.

#### **6.4 Contra-indications**

All drugs are contra-indicated in case of hypersensitivity. Hypersensitivity to abacavir may be very serious.

#### **6.5 Use in children and elderly**

No dose adjustments are necessary in the elderly.

Zidovudine/lamivudine (Combivir) and abacavir/lamivudine (Kivexa) can be used in children from 12 years. The individual components can be used from 3 months.

Lamivudine can be used from 3 months

Didanosine tablets can be used from 6 years.

Tenofovir and emcitabine are only applicable in adults.

#### **6.6 Use in renal and hepatic disease**

A dose reduction is necessary in case of renal function impaiment. Abacavir/Lamivudine should not be used when the creatinine clearance is lower than 50 ml/min. Tenofovir/Emtricitabine should bot be used when the creatinine clearance is lower than 30 ml/min.

InforMatrix Nucleoside/Nucleotide ReverseTranscriptase Inhibitors "Backbones" 377

nephrotoxic drugs is not recommended (3TC)

recommended beause of possible virologic failure.

nephrotoxisc drugs is not recommended (TDF) No not use in case of the HIV-1 K65R mutation (TDF) Tenofovir may lower the bone mineral density (TDF)

factors for liverdiseases (3TC)

Do not combine with lamivudine

The tablet contains lactose.

hepatic side-effects (TDF)

children (ddI)

patients who also have hepatitis B. Didanosine/Emtricitabine Great caution with pancreatitis in the anamnesis (ddI)

Lipodystrophy may occur (ddI)

hepatic side-effects (ddI) Lipodystrophy may occur (ddI)

Acquisition cost excluded for VAT in Euro ("vergoedingsprijs", Z-Index July 2011)

300 mg in 1-2 doses

1 dd 245 mg 300 mg in 1-2 doses

200 mg 1 dose

Peripheral neuropathy may occur (ddI)

Use with great caution in case of hepatomegaly, hepatitis or risk

Renal function should be checked. Combination with

Combination with a third nucleoside analogue is not

Renal function should be checked. Combination with

Patients with hepatitis B or C have an increased risk on serious

Cessation of TDF/FTC may lead to increased symptoms in

Changes in the retina and N.opticus are to be checked in

Lactic acidosis has been described. Therapy should be stopped

Patients with hepatitis B of C have an increased risk on serious

Cost per month in

Euro

306/336

510

313/343

in case of hyperlactatemia or metabolic acidosis (ddI)

2 dd 300/150 mg 379

1 dd 600/300 mg 422

1 dd 200/245 mg 510

1 dd 400 or 250 mg (weight based)

1 dd 400 or 250 mg (weight based)

also have hepatitis B.

Tenofovir/Emtricitabine

**7. Acquisition cost** 

Zidovudine/Lamivudine

Didanosine ER (Videx) Lamivudine (Epivir)

Abacavir/Lamivudine (Kivexa, Epzicom)

Tenofovir/Emtricitabine

Didanosine ER (Videx) Emtricitabine (Emtriva)

Tenofovir (Viread) Lamivudine (Epivir)

(Combivir)

(Truvada)

(Truvada)

No dose adjustments are usually necessary in patients with liver disease.

#### **6.7 Use in pregancy and lactation**

A variable extent of mitochondrial damage may occur during in utero exposition to nucleoside-analogues. This may lead to hematologic toxicity or metabolic disturbances.

All drugs should be avoided during lactation. None of the combinations is recommended in case of pregnancy, but they are usually not absolutely contra-indicated.

#### **6.8 Special precautions**



### **7. Acquisition cost**

376 Recent Translational Research in HIV/AIDS

A variable extent of mitochondrial damage may occur during in utero exposition to nucleoside-analogues. This may lead to hematologic toxicity or metabolic disturbances. All drugs should be avoided during lactation. None of the combinations is recommended in

Monitoring of hematologic parameters (ZVD)

in case of hyperlactatemia or metabolic acidosis

factors for liverdiseases (3TC)

Didanosine/Lamivudine Great caution with pancreatitis in the anamnesis (ddI and 3TC) Peripheral neuropathy may occur (ddI)

riskfactors for liverdiseases (3TC)

hepatic side-effects (ddI) Lipodystrophy may occur (ddI)

factors for liverdiseases (3TC) Lipodystrophy may occur (3TC)

also have hepatitis B.

hepatic side-effects

Tenofovir/Lamivudine Tenofovir may lower the BMD (TDF)

who also have hepatitis B.

also have hepatitis B.

children (ddI)

Lowering of the dosage of ZVD in abnormal hematologic

Use with great caution in case of hepatomegaly, hepatitis or risk

Cessation of L may lead to increased symptoms in patients who

Changes in the retina and N.opticus are to be checked in

Use with great caution in case of hepatomegaly, hepatitis or

Lactic acidosis has been described. Therapy should be stopped in case of hyperlactatemia or metabolic acidosis (ddI and 3TC) Patients with hepatitis B or C have an increased risk on serious

Cessation of L may lead to increased symptoms in patients who

Cessation of therapy during signs of pancreatitis (ABC and 3TC) Lactic acidosis has been described. Therapy should be stopped

Use with great caution in case of hepatomegaly, hepatitis or risk

Patients with hepatitis B or C have an increased risk on serious

Cessation of 3TC may lead to increased symptoms in patients

Lactic acidosis has been described. Therapy should be stopped

Cessation of L may lead to increased symptoms in patients who

No not use in case of the HIV-1 K65R mutation (TDF)

in case of hyperlactatemia or metabolic acidosis (TDF)

in case of hyperlactatemia or metabolic acidosis

Therapy cessation during signs of pancreatitis (ZVD and 3TC) Lactic acidosis has been described. Therapy should be stopped

No dose adjustments are usually necessary in patients with liver disease.

case of pregnancy, but they are usually not absolutely contra-indicated.

parameters

**6.7 Use in pregancy and lactation** 

**6.8 Special precautions** 

Zidovudine/Lamivudine

Abacavir/Lamivudine

(Kivexa)

(Combivir)

Acquisition cost excluded for VAT in Euro ("vergoedingsprijs", Z-Index July 2011)


InforMatrix Nucleoside/Nucleotide ReverseTranscriptase Inhibitors "Backbones" 379

[7] Thompson MA, Aberg JA, Cahn P, et al. Antiretroviral treatment of adult HIV infection:

[8] Gulick RM, Ribaudo HJ, Shikuma CM, et al. Triple-nucleoside regimens versus

[9] Gras L, Kesselring AM, Griffin JT, et al. CD4 cell counts of 800 cells/mm3 or greater after

[11] Sax P, Tierney C, Collier A, and ACTG A5202 Study Team Abacavir-lamivudine versus tenofovir-emtricitabine for initial HIV-1 therapy New Engl J Med 2009;361:2230-40 [12] Daar E, Tierney C, Fischl M, and ACTG A5202 Study Team, ACTG 5202: Final Results

[13] Gallant J, DeJesus E, Arribas JR, et al. Tenofovir DF, emtricitabine and efavirenz vs zidovudine, lamivudine and EFV for HIV. N Engl J Med 2006;354:251-60 [14] DeJesus E, Herrera G, Teofilo E, et al Abacavir versus Zidovudine combined with

[15] Berenguer J, Gonzales J, Ribera E, et al. Didanosine, lamivudine, and efavirenz vs

[16] Saag M, Cahn P, Raffi F, et al. Efficacy and safety of emtricitabine vs stavudine in

[17] Cassetti I, Madruga JV, Suleiman JM, et al The safety and efficacy of tenofovir DF in

[18] Mallal S, Phillips E, Caras G, et al HLA-B5701 screening for hypersensitivity to abacavir.

[21] Hill AM, Sawyer WS. Effects of NRTI backbone on efficacy of first-line boosted PI-based

[19] National Institute of Allrgy and Infectious Diseases, Division of AIDS (DAIDS)

lopinavir/ritonavir for initial HIV treatment. AIDS 2009;23:1547-56

Opportunistic Infections San Francisco, CA, USA, 2010

HIV-1-infected patients. HIV Clin Trials 2007;8:164-72

adults Clin Infect Dis 2004;48:1038-46

Infect Dis 2008:47:1083-32

New Engl J Med 2008;358:568-79

International AIDS Conference 2008

2008. Washington DC, USA.

2004;292:180-90

2010;304:321-33.

Med. 2004;350:1850-61.

2010 recommendations of the International AIDS Society-USA panel. JAMA.

efavirenz-containing regimens for the initial treatment of HIV-1 infection. N Engl J

7 years of highly active antiretroviral therapy are feasible in most patients starting with 350 cells/mm3 or greater. J Acquir Immune Defic Syndr. 2007;45:183-92. [10] Smith K, Patel P, Fine DM, et al Randomized, double-blind, placebo-matched,

multicenter trial of abacavir/lamivudine or tenofovir/emtricitabine with

of ABC/3TC or TDF/FTC with either EFV or ATV/r in Treatment-naive HIVinfected Patients, Abstract 59LB 17th Conference on Retroviruses and

lamivudine and efavirenz, for the treatment of antiretroviral-naive HIV-infected

zidovudine, lamivudine, and efavirenz, for initial treatment of HIV infection: Final analysis of a prospective randomized noninferiority clinical trial, GESIDA 3903 Clin

combination therapy in antiretroviral-naive patients: a randomized trial JAMA

combination with lamivudine and efavirenz through 6 years in antiretroviral-naïve

table for grading severity of adult adverse experiences http://www.niaid.nih.gov [20] Pappa K, Hernandez J, Ha1 B, et al Abacavir/lamivudine shows robust virologic

responses in ART-naïve patients for baseline viral loads (VL) of >100,000c/mL and <100,000c/mL by endpoint used in ACTG5202, Abstract THABO304, XVII

HAART--meta-analysis of 12 clinical trials in 4896 patients. Abstract H-1254. 48th Annual International Conference on Antimicrobial Agents and Chemotherapy

#### **8. Conclusion**

Optimal care requires individualized management and ongoing attention to relevant scientific and clinical information. The availability of new antiretroviral drugs since the introduction of the fist cART has expanded treatment choices. Guidelines are presented as recommendations if the supporting evidence warrants routine use in a particular situation and as considerations if data are preliminary or incomplete but suggestive. But the importance of adherence, emerging long-term complications of therapy, recognition and management of antiretroviral failure is often underestimated and there is but to often little data to guide our choices.

The judgement of the relative efficacy and safety of the various NRTI backbones in the treament of HIV infection is hindered by the fact that there are only few direct comparative studies. This makes it difficult to make firm statements concerning the pros and cons of the individual drugs concerning efficacy and safety.

In this InforMatrix manuscript, no firm conclusions are drawn by the authors. The purpose of this manuscript is to facilitate discussion on the properties of each treatment option for HIV by using only clinically relevant selection criteria by providing an up-to-date overview. The InforMatrix program will be made available in an interactive format on www.informatrix.nl. By means of the program, the user can assign a relative weight to each main selection criterion (with a total of 30 points to be distributed) and can judge the properties of each therapeutic option per criterion on the basis of his own personal expertise and/or the present document. Zero to ten points can be assigned to each treatment option on each criterion. The program is freely accessible.

The present InforMatrix manuscript is specific for the Netherlands, because the Dutch available formulations and Dutch prices were used. The most important part of the paper (efficacy, safety and tolerability) is internationally valid. Local adjustments are necessary for an optimal use of the method in other countries. This could also include price-adjustments for the individual hospitals in other countries.

#### **9. References**


Optimal care requires individualized management and ongoing attention to relevant scientific and clinical information. The availability of new antiretroviral drugs since the introduction of the fist cART has expanded treatment choices. Guidelines are presented as recommendations if the supporting evidence warrants routine use in a particular situation and as considerations if data are preliminary or incomplete but suggestive. But the importance of adherence, emerging long-term complications of therapy, recognition and management of antiretroviral failure is often underestimated and there is but to often little

The judgement of the relative efficacy and safety of the various NRTI backbones in the treament of HIV infection is hindered by the fact that there are only few direct comparative studies. This makes it difficult to make firm statements concerning the pros and cons of the

In this InforMatrix manuscript, no firm conclusions are drawn by the authors. The purpose of this manuscript is to facilitate discussion on the properties of each treatment option for HIV by using only clinically relevant selection criteria by providing an up-to-date overview. The InforMatrix program will be made available in an interactive format on www.informatrix.nl. By means of the program, the user can assign a relative weight to each main selection criterion (with a total of 30 points to be distributed) and can judge the properties of each therapeutic option per criterion on the basis of his own personal expertise and/or the present document. Zero to ten points can be assigned to each treatment option

The present InforMatrix manuscript is specific for the Netherlands, because the Dutch available formulations and Dutch prices were used. The most important part of the paper (efficacy, safety and tolerability) is internationally valid. Local adjustments are necessary for an optimal use of the method in other countries. This could also include price-adjustments

[1] Bartlett J FM, DeMasi R, Quinn J, et al. An Updated Meta-analysis of Triple Combination

[2] Hammond E, McKinnon E, Nolan D. Human immunodeficiency virus treatment-

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Therapy in Antiretroviral-naive HIV-infected Adults. Abstract 586 12th Conference

induced adipose tissue pathology and lipoatrophy: prevalence and metabolic

didanosine + efavirenz combination in HIV-positive patients upon starting

underlying mechanisms and therapeutic options. J Antimicrob Chemother.

**8. Conclusion** 

data to guide our choices.

individual drugs concerning efficacy and safety.

on each criterion. The program is freely accessible.

for the individual hospitals in other countries.

**9. References** 

2008;62:648-60.


InforMatrix Nucleoside/Nucleotide ReverseTranscriptase Inhibitors "Backbones" 381

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**Part 7** 

**New Therapy Strategies** 


## **Part 7**

**New Therapy Strategies** 

384 Recent Translational Research in HIV/AIDS

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1608.

properties and therapeutic efficacy in the management of HIV infection. Drugs

**19** 

*USA* 

**Crippling of HIV at Multiple Stages with** 

*1Laboratory of Surgical Oncology & Developmental Therapeutics, Department of Surgery,* 

Acquired immunodeficiency syndrome (AIDS) is a disease caused by the infection of human immunodeficiency virus-1 (HIV-1) that primarily impairs immune function by reducing the CD4 T-lymphocyte count. More than two decades after the first clinical evidence of AIDS was reported, AIDS continues to be a major public health problem worldwide with millions of people infected and new infections rising in an alarming rate in third world countries especially in Asia and sub-Saharan Africa.(1, 2) AIDS has become one of the most devastating diseases that the scientific community has ever faced, struggling till today to come up with a therapeutic strategy that successfully controls the disease. AIDS is now the leading cause of death in sub-Saharan Africa, and is presently the fourth biggest killer worldwide. AIDS-related deaths totaled over 5 million by 2009 reaching a cumulative death toll of over 30 million since the beginning of the epidemic. More than 75 million people have been infected with HIV-1, and roughly 2.7 million new HIV-1 infections were diagnosed in

To date, there is no effective treatment and the number of individuals infected with HIV-1 is growing dramatically in the eastern part of the world. Considering its infection rate, it is imperative to devise newer strategies to control progression of the disease. Although newer approaches such as highly active antiretroviral therapy (HAART) have proven to be effective in prolonging life, other constraints associated with their use underscores the need for development of other effective therapies. Protease inhibitors appear to be successful at controlling the viral replication immediately following budding of immature virus particle, but the development of drug resistant viral mutants and toxicity after prolonged therapy contributes to their failure.(4) HAART has considerable toxicity and its inability to

Oksana V. Gruzdyn1, Aamer M. Qazi1,2, Assaad Y. Semaan1, Shelly M. Seward1, Christopher P. Steffes1,

*1Laboratory of Surgical Oncology & Developmental Therapeutics, Department of Surgery, Wayne State* 

2009(3) even though this rate has decreased from one decade ago.

David L. Bouwman1, Donald W. Weaver1 and Scott A. Gruber2

*2John D. Dingell VA Medical Center, Detroit, MI, USA* 

**1. Introduction** 

 \*

*University, Detroit, MI, USA* 

**Recombinant Adeno-Associated Viral** 

**Mediated RNA Interference** 

*2John D. Dingell VA Medical Center, Detroit, MI* 

Ramesh B. Batchu1,2 et al.\*

*Wayne State University, Detroit, MI* 

### **Crippling of HIV at Multiple Stages with Recombinant Adeno-Associated Viral Mediated RNA Interference**

Ramesh B. Batchu1,2 et al.\*

*1Laboratory of Surgical Oncology & Developmental Therapeutics, Department of Surgery, Wayne State University, Detroit, MI 2John D. Dingell VA Medical Center, Detroit, MI USA* 

#### **1. Introduction**

Acquired immunodeficiency syndrome (AIDS) is a disease caused by the infection of human immunodeficiency virus-1 (HIV-1) that primarily impairs immune function by reducing the CD4 T-lymphocyte count. More than two decades after the first clinical evidence of AIDS was reported, AIDS continues to be a major public health problem worldwide with millions of people infected and new infections rising in an alarming rate in third world countries especially in Asia and sub-Saharan Africa.(1, 2) AIDS has become one of the most devastating diseases that the scientific community has ever faced, struggling till today to come up with a therapeutic strategy that successfully controls the disease. AIDS is now the leading cause of death in sub-Saharan Africa, and is presently the fourth biggest killer worldwide. AIDS-related deaths totaled over 5 million by 2009 reaching a cumulative death toll of over 30 million since the beginning of the epidemic. More than 75 million people have been infected with HIV-1, and roughly 2.7 million new HIV-1 infections were diagnosed in 2009(3) even though this rate has decreased from one decade ago.

To date, there is no effective treatment and the number of individuals infected with HIV-1 is growing dramatically in the eastern part of the world. Considering its infection rate, it is imperative to devise newer strategies to control progression of the disease. Although newer approaches such as highly active antiretroviral therapy (HAART) have proven to be effective in prolonging life, other constraints associated with their use underscores the need for development of other effective therapies. Protease inhibitors appear to be successful at controlling the viral replication immediately following budding of immature virus particle, but the development of drug resistant viral mutants and toxicity after prolonged therapy contributes to their failure.(4) HAART has considerable toxicity and its inability to

<sup>\*</sup> Oksana V. Gruzdyn1, Aamer M. Qazi1,2, Assaad Y. Semaan1, Shelly M. Seward1, Christopher P. Steffes1, David L. Bouwman1, Donald W. Weaver1 and Scott A. Gruber2

*<sup>1</sup>Laboratory of Surgical Oncology & Developmental Therapeutics, Department of Surgery, Wayne State University, Detroit, MI, USA* 

*<sup>2</sup>John D. Dingell VA Medical Center, Detroit, MI, USA* 

Crippling of HIV at Multiple Stages with

against virus in secondary lymphoid tissue.

**3. Newer approaches to therapy** 

failure to enter the clinic.(27)

**4. RNA interference (RNA***i***): A natural way of gene silencing** 

Diseases, for which a foreign gene can be identified as the cause, such as in the case of viral infections, are potentially treatable by blocking its expression that will cripple the causative agent. Over the last decade, small non-coding RNA molecules such as short interfering RNA (siRNA), micro RNA (miRNA) and piwi RNA (*pi*RNA), collectively known as RNA interference (RNA*i*), emerged as critical regulators in mammalian gene expression and hold the promise of selectively inhibiting expression of disease-causing genes.(28, 29) RNA*i* is an evolutionarily conserved mechanism of gene inhibition or silencing first described in Caenorhabditis elegans and was shown to produce sequence-specific gene silencing.(30) In 1999, it was recognized as the natural cellular process to destroy unwanted foreign genes such as those causing viral infections(31). In 2001, for the first time, *the use of synthetic siRNA to silence genes in mammalian cells was demonstrated, and was referred to* as 'Biotech's billion dollar break through.(32) In short, RNA*i* has the potential to revolutionize the treatment approach to various diseases. Over the years, it has become clear that RNA*i* is a highly conserved molecular mechanism used by eukaryotic organisms to control gene expression during development and to defend their genomes against invaders, such as transposons and RNA viruses. siRNAs are primarily exogenous in origin, derived directly from the virus or transposon. siRNAs are 21 to 23 double-stranded RNA molecules that recognize the cognate

Recombinant Adeno-Associated Viral Mediated RNA Interference 389

analogs for HIV-1, though effective in selected patients in prolonging life, unfortunately generate drug resistant viral mutants, unacceptable levels of drug toxicity, and are ineffective

Introduction of molecules that are able to dominantly interfere with intracellular replication of HIV-1 is known as ''intracellular immunization''. Intracellular immunization by gene therapy strategies offers a promising alternative approach for controlling and managing HIV-1 disease. These include protein-based approaches such as trans-dominant strategies to inhibit HIV-1: toxins, zinc finger nucleases and single-chain antibodies.(18) Protein-based strategies have been the single largest area of anti-HIV-1 gene transfer trials in humans in the recent past.(19) Other RNA-based intracellular immunization approaches include the use of ribozymes and decoys. These second generation ribozymes are RNA molecules that cleave viral transcripts such as *tat*, *rev*, and *gag* at specific sequences targeting HIV-1 at critical stages, and have been shown to reduce HIV-1 levels *in vitro*.(20-22) RNA decoys are RNA homologues, such as TAR and RRE that bind viral proteins and compete with native ligands necessary for replication. They were also shown to inhibit HIV-1 *in vitro*.(20, 23, 24) In comparison with protein-based strategies, RNA-based approaches may have the advantage of not being immunogenic. Both viral or host cellular factors can be targeted, the latter potentially mitigating the possibility of escape mutants, but nevertheless, these transdominant approaches had shown initial promise but fell short of practical utility in providing adequate protection. DNA-based vaccines have shown partial success.(23, 25, 26) Anti-sense molecules were shown by several groups to inhibit HIV-1 *in vitro* when targeted towards critical HIV-1 genes such as *tat*, *rev*, and integrase, but the need for large amounts for *in vivo* studies apart from the problems associated with stability contributed to their

effectively act on virus in secondary lymphoid tissue is a significant drawback. Vast majority of people with AIDS live in poorer countries. HAART is expensive and unreachable to low and middle-income countries.(5) Many of these places do not have access to HAART, or if they do, supply can be intermittent. The finding that infections with drug-resistant HIV-1 are increasing further underscores the need to develop inhibitors of HIV-1 that are effective, affordable and universally accessible.

With the discovery of RNA interference (RNA*i*) phenomenon, that operates in mammalian cells and is highly effective in selective gene silencing, new, potent, small interfering RNA (siRNA) molecules have become available to add to control HIV-1. By analyzing the challenges of HIV-1 drug development, we review novel and multi-faceted therapies by simultaneously targeting multiple regions of HIV-1 so as to effectively cripple of the virus. The targets include essential cellular genes to avoid viral escape through mutations; multiple regions at various phases of the viral life cycle for a synergistic effect; and antisense approach as well to avoid viral escape strategies of HIV-1 against RNA*i*. Current challenges facing the advancement of RNA*i* therapy are its safety and inefficient delivery *in vivo*. Self-complimentary recombinant adeno-associated viral (rAAV-sc) vectors can overcome shortcomings associated with RNA*i*-mediated gene silence therapy.(6) AAV vectors are safe and clinically proven. New generation vectors with mutant capsids circumvent pitfalls of ubiquitin-proteasome mediated degradation leading to high-efficiency transduction at low doses ideally suited to be part of a new arsenal for in vivo RNA*i* delivery to fight HIV-1.(7) Unlike present drugs in the clinical trial or R&D stage, the multitargeted AAV mediated RNA*i* approach not only kills the virus but also prevents the development of escape strategies and emergence of resistant viruses by simultaneous attack at multiple targets employing multiple technologies.

#### **2. Traditional AIDS therapies**

Anti-retroviral drugs such as nucleoside reverse transcriptase inhibitors and non-nucleoside inhibitors are first generation drugs successful in reducing the viral burden.(8-13) Although they prolong life in selected patients, these agents have significant side effects and generate drug resistant viral mutants. Protease inhibitors appear to be most effective at blocking HIV-1 replication, substantially reducing AIDS-related hospital admissions and death rates.(14) Present-day therapy uses a combination of nucleoside analogues and protease inhibitors known as HAART.(15) HAART has been shown to be effective in controlling the spread of the virus by reducing the plasma viral load to undetectable levels and to some extent depleting the pool of virus in lymphoid tissues. In the past decade, HAART has become more effective with the introduction of several protease inhibitors, but the treatment is expensive and unavailable in poor countries.(5, 15) Approximately 30 million HIV-1 positive people of whom the vast majority live in low- and middle-income countries do not have access to proper treatment. This underscores the need for the development of inexpensive, yet effective drugs that can reach the majority of patients. Despite the apparent success of HAART therapy, the capacity of HIV-1 to establish latent infection of CD4+ T cells allows viral particles to persist in tissues. Some studies indicated that the therapy does not completely eliminate viral replication in secondary lymphoid tissues. HIV-1 was routinely isolated from lymphoid organs of patients even after years of therapy due to continued replication.(16) Moreover, the initiation of HAART even as early as days after the onset of AIDS symptoms, could not prevent the establishment of a pool of latently-infected T lymphocytes.(17)These observations clearly indicate that traditional combinatorial therapies with protease inhibitors and nucleoside analogs for HIV-1, though effective in selected patients in prolonging life, unfortunately generate drug resistant viral mutants, unacceptable levels of drug toxicity, and are ineffective against virus in secondary lymphoid tissue.

#### **3. Newer approaches to therapy**

388 Recent Translational Research in HIV/AIDS

effectively act on virus in secondary lymphoid tissue is a significant drawback. Vast majority of people with AIDS live in poorer countries. HAART is expensive and unreachable to low and middle-income countries.(5) Many of these places do not have access to HAART, or if they do, supply can be intermittent. The finding that infections with drug-resistant HIV-1 are increasing further underscores the need to develop inhibitors of

With the discovery of RNA interference (RNA*i*) phenomenon, that operates in mammalian cells and is highly effective in selective gene silencing, new, potent, small interfering RNA (siRNA) molecules have become available to add to control HIV-1. By analyzing the challenges of HIV-1 drug development, we review novel and multi-faceted therapies by simultaneously targeting multiple regions of HIV-1 so as to effectively cripple of the virus. The targets include essential cellular genes to avoid viral escape through mutations; multiple regions at various phases of the viral life cycle for a synergistic effect; and antisense approach as well to avoid viral escape strategies of HIV-1 against RNA*i*. Current challenges facing the advancement of RNA*i* therapy are its safety and inefficient delivery *in vivo*. Self-complimentary recombinant adeno-associated viral (rAAV-sc) vectors can overcome shortcomings associated with RNA*i*-mediated gene silence therapy.(6) AAV vectors are safe and clinically proven. New generation vectors with mutant capsids circumvent pitfalls of ubiquitin-proteasome mediated degradation leading to high-efficiency transduction at low doses ideally suited to be part of a new arsenal for in vivo RNA*i* delivery to fight HIV-1.(7) Unlike present drugs in the clinical trial or R&D stage, the multitargeted AAV mediated RNA*i* approach not only kills the virus but also prevents the development of escape strategies and emergence of resistant viruses by simultaneous attack

Anti-retroviral drugs such as nucleoside reverse transcriptase inhibitors and non-nucleoside inhibitors are first generation drugs successful in reducing the viral burden.(8-13) Although they prolong life in selected patients, these agents have significant side effects and generate drug resistant viral mutants. Protease inhibitors appear to be most effective at blocking HIV-1 replication, substantially reducing AIDS-related hospital admissions and death rates.(14) Present-day therapy uses a combination of nucleoside analogues and protease inhibitors known as HAART.(15) HAART has been shown to be effective in controlling the spread of the virus by reducing the plasma viral load to undetectable levels and to some extent depleting the pool of virus in lymphoid tissues. In the past decade, HAART has become more effective with the introduction of several protease inhibitors, but the treatment is expensive and unavailable in poor countries.(5, 15) Approximately 30 million HIV-1 positive people of whom the vast majority live in low- and middle-income countries do not have access to proper treatment. This underscores the need for the development of inexpensive, yet effective drugs that can reach the majority of patients. Despite the apparent success of HAART therapy, the capacity of HIV-1 to establish latent infection of CD4+ T cells allows viral particles to persist in tissues. Some studies indicated that the therapy does not completely eliminate viral replication in secondary lymphoid tissues. HIV-1 was routinely isolated from lymphoid organs of patients even after years of therapy due to continued replication.(16) Moreover, the initiation of HAART even as early as days after the onset of AIDS symptoms, could not prevent the establishment of a pool of latently-infected T lymphocytes.(17)These observations clearly indicate that traditional combinatorial therapies with protease inhibitors and nucleoside

HIV-1 that are effective, affordable and universally accessible.

at multiple targets employing multiple technologies.

**2. Traditional AIDS therapies** 

Introduction of molecules that are able to dominantly interfere with intracellular replication of HIV-1 is known as ''intracellular immunization''. Intracellular immunization by gene therapy strategies offers a promising alternative approach for controlling and managing HIV-1 disease. These include protein-based approaches such as trans-dominant strategies to inhibit HIV-1: toxins, zinc finger nucleases and single-chain antibodies.(18) Protein-based strategies have been the single largest area of anti-HIV-1 gene transfer trials in humans in the recent past.(19) Other RNA-based intracellular immunization approaches include the use of ribozymes and decoys. These second generation ribozymes are RNA molecules that cleave viral transcripts such as *tat*, *rev*, and *gag* at specific sequences targeting HIV-1 at critical stages, and have been shown to reduce HIV-1 levels *in vitro*.(20-22) RNA decoys are RNA homologues, such as TAR and RRE that bind viral proteins and compete with native ligands necessary for replication. They were also shown to inhibit HIV-1 *in vitro*.(20, 23, 24) In comparison with protein-based strategies, RNA-based approaches may have the advantage of not being immunogenic. Both viral or host cellular factors can be targeted, the latter potentially mitigating the possibility of escape mutants, but nevertheless, these transdominant approaches had shown initial promise but fell short of practical utility in providing adequate protection. DNA-based vaccines have shown partial success.(23, 25, 26) Anti-sense molecules were shown by several groups to inhibit HIV-1 *in vitro* when targeted towards critical HIV-1 genes such as *tat*, *rev*, and integrase, but the need for large amounts for *in vivo* studies apart from the problems associated with stability contributed to their failure to enter the clinic.(27)

#### **4. RNA interference (RNA***i***): A natural way of gene silencing**

Diseases, for which a foreign gene can be identified as the cause, such as in the case of viral infections, are potentially treatable by blocking its expression that will cripple the causative agent. Over the last decade, small non-coding RNA molecules such as short interfering RNA (siRNA), micro RNA (miRNA) and piwi RNA (*pi*RNA), collectively known as RNA interference (RNA*i*), emerged as critical regulators in mammalian gene expression and hold the promise of selectively inhibiting expression of disease-causing genes.(28, 29) RNA*i* is an evolutionarily conserved mechanism of gene inhibition or silencing first described in Caenorhabditis elegans and was shown to produce sequence-specific gene silencing.(30) In 1999, it was recognized as the natural cellular process to destroy unwanted foreign genes such as those causing viral infections(31). In 2001, for the first time, *the use of synthetic siRNA to silence genes in mammalian cells was demonstrated, and was referred to* as 'Biotech's billion dollar break through.(32) In short, RNA*i* has the potential to revolutionize the treatment approach to various diseases. Over the years, it has become clear that RNA*i* is a highly conserved molecular mechanism used by eukaryotic organisms to control gene expression during development and to defend their genomes against invaders, such as transposons and RNA viruses. siRNAs are primarily exogenous in origin, derived directly from the virus or transposon. siRNAs are 21 to 23 double-stranded RNA molecules that recognize the cognate

Crippling of HIV at Multiple Stages with

minimize the escape of the resistant virus.

**6. Cellular targets of HIV for RNA***i* 

studying strain variants.

siRNAs provided marked protection from HIV-1 infection.(63)

**7. Escape strategies of HIV-1 from siRNA** 

the cytoplasm due to the availability of target transcripts.

Recombinant Adeno-Associated Viral Mediated RNA Interference 391

were present before the viral infection. This is because the vulnerability of genomic HIV RNA for RNA*i*-mediated knockdown is much greater immediately after viral entry into

A crucial finding was that a high degree of specificity of the RNA*i* for the sequence of its target was required. Even one base pair change dramatically lowers the potency of RNA*i*mediated inhibition.(58)This becomes important, given the high error rate of HIV-1 reverse transcriptase that leads to the emergence of RNA*i* escape mutants. The HIV-1 virus often becomes resistant to RNAi therapy as a result of the appearance of mutant variants. Because of these mutations, although siRNA directed against various HIV-1 genes shows initial success, the virus may soon escape inhibition within weeks. (44) Silencing evasion can also result from loss of target sequences within viral genomes, owing to the high viral mutation rates. In lymphocytes, for example, the effects of anti-HIV-1 siRNAs were progressively dampened by the emergence of viral quasi species that harbor mutations within the siRNA target sequence.(59) RNA*i*-mediated inhibition with single target has not yet been shown to protect cells against HIV-1 in long-term. RNA*i* could become a realistic therapeutic option, however, if used in a combined fashion while targeting multiple genes to prevent the emergence of mutant viruses. Simultaneous attacks by siRNA on various targets will

An essential cellular HIV receptor or co-receptor target may have more appeal than viral targets, which are prone to mutations. Cellular mRNAs that encode critical proteins involved in HIV-1 replication may circumvent pitfalls associated with viral escape mechanisms. Targeting cellular genes that are an essential part of the HIV-1 life cycle could therefore be advantageous. CD4 is the primary cellular receptor for HIV-1 entry on T lymphocytes. In addition to the CD4 primary receptor, the cellular chemokine receptors CCR5 and CXCR4, which function as co-receptors for HIV-1, have provided new therapeutic targets and a better understanding of the progression of viral infection. Several investigators targeted cellular proteins necessary for the HIV-1 life cycle by siRNAs and produced decreased levels of virus production.(60, 61) Preliminary observations from various laboratories have demonstrated that siRNAs specific for CD4 receptor do indeed inhibit HIV-1 replication.(62, 63) After transfection of cultured T cells with siRNA against the mRNA for CD4, HIV production, after exposure of the cells to the virus, decreased substantially. (64) CCR5, an HIV-1 co-receptor for the M-tropic HIV-1 variant, providing an attractive cellular target for siRNAs since homozygous deletions of CCR5 effectively confer protection from HIV-1 without any serious deleterious effects in immune function.(65) At least one group has taken advantage of this target for RNA*i*-mediated gene silencing, demonstrating that in vitro knockdown of CCR5 by

Although suppression of the primary receptor CD4 may be restricted by its normal role in the immune system, CCR5 seems dispensable for normal life.(66) Unfortunately, not all HIV-1 strains require CCR5, and the inhibition of CCR5 may result in the selection of HIV-1 variants that use CXCR4 as a co-receptor. It is critical to identify this particular aspect by

One of the hallmarks of RNA*i* is its sequence-specific knockdown of the target transcript, but unfortunately, it also presents a way out for HIV-1, since single nucleotide substitutions

mRNA with complementary sequence and cleave by naturally occurring cellular mechanisms.(31) *In vivo* silencing occurs after the formation of long double-stranded RNAs that are processed into short interfering RNAs (siRNAs) by an enzyme called Dicer, forming a ribonucleo-protein complex called RNA induced silencing complex (RISC). In the RISC, the anti-sense strand of the siRNA serves as a guide for the degradation of the homologous RNA target. In recent years, siRNA has emerged as a method of choice for specific and efficient gene silencing.

Since discovery of their mechanism, chemically synthesized siRNA molecules are being used to target abnormally elevated genes in many diseases. Since siRNA is a natural biological mechanism against viruses, it can elicit specific intracellular antiviral resistance that may provide a therapeutic strategy against human viruses. siRNAs have been shown to inhibit viral replication or block gene expression in cell culture systems for several viruses. In one study, pre-treatment of cells siRNAs specific to the poliovirus genome promoted the clearance of the virus from most of the infected cells.(33) Shlomai et al observed significant reduction in hepatitis B virus (HBV) by siRNA-producing vectors.(34) A number of groups have demonstrated that siRNAs interfere with hepatitis C virus (HCV) gene expression and replication.(35-39) Over 90% of human cervical cancers are positive for human papilloma virus (HPV) and siRNA-mediated silencing of E6 and E7, the viral genes necessary for the HPV life cycle, completely inhibited them in mammalian cells.(40)

#### **5. Targeting HIV-1 with RNA***i*

Since siRNA can elicit specific knockdown of transcripts and they have been successfully used against human viruses, this ancient defense mechanism can be recruited as a weapon in the fight against HIV-1. Several laboratories have shown that the introduction of siRNAs specific for HIV-1 transcripts has shown viral RNA degradation and inhibition of replication. (41-43) Stable and modified promoters for the expression of siRNA molecules have further shown to increase the potency of HIV *in vitro*.(44) The successful silencing of HIV-1 replication by several investigators through siRNA-mediated targeted knockdown of viral proteins made RNA interference a weapon of choice against HIV-1. (41, 45, 46) HIV-1 has a total of 15 proteins encoded by 9 genes.(47) Essentially, these can be grouped into four potential target sets for siRNA knockdown. The first potential target for gene silencing is the viral genomic RNA upon viral entry. Jacque et al demonstrated siRNAmediated destruction of incoming HIV-1(48), although other studies of RNA*i* inhibition of retroviral infection suggested that incoming genomic RNA may not be the best target for siRNAs.(49) Once viral DNA is integrated, the viral mRNA transcripts as well as the unspliced genomic RNA can be potential target. Early transcripts of HIV-1 such as *rev*, *nef*  and *tat* are an important second group of targets for gene silencing with RNA*i* because they not only regulate the subsequent expression of the structural genes, *gag*, *pol*, and *env* but also the synthesis of full length viral genomic RNA. siRNA targeting the *nef* gene has been demonstrated to provide efficient silencing in a transient-transfection system.(50) There have been efficient demonstrations of silencing of the expression of various regulatory genes in a transient-transfection system.(44, 51, 52) siRNAs targeted to the TAR regulatory region and nef of the HIV-1 genome have also been shown to be effective at silencing the level of virus replication and inhibiting reverse transcription intermediates.(53, 54) After regulatory genes, structural genes also represent a potential target group.(55-57) It was found that inhibition was more significant when the siRNA

mRNA with complementary sequence and cleave by naturally occurring cellular mechanisms.(31) *In vivo* silencing occurs after the formation of long double-stranded RNAs that are processed into short interfering RNAs (siRNAs) by an enzyme called Dicer, forming a ribonucleo-protein complex called RNA induced silencing complex (RISC). In the RISC, the anti-sense strand of the siRNA serves as a guide for the degradation of the homologous RNA target. In recent years, siRNA has emerged as a method of choice for specific and

Since discovery of their mechanism, chemically synthesized siRNA molecules are being used to target abnormally elevated genes in many diseases. Since siRNA is a natural biological mechanism against viruses, it can elicit specific intracellular antiviral resistance that may provide a therapeutic strategy against human viruses. siRNAs have been shown to inhibit viral replication or block gene expression in cell culture systems for several viruses. In one study, pre-treatment of cells siRNAs specific to the poliovirus genome promoted the clearance of the virus from most of the infected cells.(33) Shlomai et al observed significant reduction in hepatitis B virus (HBV) by siRNA-producing vectors.(34) A number of groups have demonstrated that siRNAs interfere with hepatitis C virus (HCV) gene expression and replication.(35-39) Over 90% of human cervical cancers are positive for human papilloma virus (HPV) and siRNA-mediated silencing of E6 and E7, the viral genes necessary for the

Since siRNA can elicit specific knockdown of transcripts and they have been successfully used against human viruses, this ancient defense mechanism can be recruited as a weapon in the fight against HIV-1. Several laboratories have shown that the introduction of siRNAs specific for HIV-1 transcripts has shown viral RNA degradation and inhibition of replication. (41-43) Stable and modified promoters for the expression of siRNA molecules have further shown to increase the potency of HIV *in vitro*.(44) The successful silencing of HIV-1 replication by several investigators through siRNA-mediated targeted knockdown of

HIV-1 has a total of 15 proteins encoded by 9 genes.(47) Essentially, these can be grouped into four potential target sets for siRNA knockdown. The first potential target for gene silencing is the viral genomic RNA upon viral entry. Jacque et al demonstrated siRNAmediated destruction of incoming HIV-1(48), although other studies of RNA*i* inhibition of retroviral infection suggested that incoming genomic RNA may not be the best target for siRNAs.(49) Once viral DNA is integrated, the viral mRNA transcripts as well as the unspliced genomic RNA can be potential target. Early transcripts of HIV-1 such as *rev*, *nef*  and *tat* are an important second group of targets for gene silencing with RNA*i* because they not only regulate the subsequent expression of the structural genes, *gag*, *pol*, and *env* but also the synthesis of full length viral genomic RNA. siRNA targeting the *nef* gene has been demonstrated to provide efficient silencing in a transient-transfection system.(50) There have been efficient demonstrations of silencing of the expression of various regulatory genes in a transient-transfection system.(44, 51, 52) siRNAs targeted to the TAR regulatory region and nef of the HIV-1 genome have also been shown to be effective at silencing the level of virus replication and inhibiting reverse transcription intermediates.(53, 54) After regulatory genes, structural genes also represent a potential target group.(55-57) It was found that inhibition was more significant when the siRNA

viral proteins made RNA interference a weapon of choice against HIV-1. (41, 45, 46)

HPV life cycle, completely inhibited them in mammalian cells.(40)

efficient gene silencing.

**5. Targeting HIV-1 with RNA***i* 

were present before the viral infection. This is because the vulnerability of genomic HIV RNA for RNA*i*-mediated knockdown is much greater immediately after viral entry into the cytoplasm due to the availability of target transcripts.

A crucial finding was that a high degree of specificity of the RNA*i* for the sequence of its target was required. Even one base pair change dramatically lowers the potency of RNA*i*mediated inhibition.(58)This becomes important, given the high error rate of HIV-1 reverse transcriptase that leads to the emergence of RNA*i* escape mutants. The HIV-1 virus often becomes resistant to RNAi therapy as a result of the appearance of mutant variants. Because of these mutations, although siRNA directed against various HIV-1 genes shows initial success, the virus may soon escape inhibition within weeks. (44) Silencing evasion can also result from loss of target sequences within viral genomes, owing to the high viral mutation rates. In lymphocytes, for example, the effects of anti-HIV-1 siRNAs were progressively dampened by the emergence of viral quasi species that harbor mutations within the siRNA target sequence.(59) RNA*i*-mediated inhibition with single target has not yet been shown to protect cells against HIV-1 in long-term. RNA*i* could become a realistic therapeutic option, however, if used in a combined fashion while targeting multiple genes to prevent the emergence of mutant viruses. Simultaneous attacks by siRNA on various targets will minimize the escape of the resistant virus.

#### **6. Cellular targets of HIV for RNA***i*

An essential cellular HIV receptor or co-receptor target may have more appeal than viral targets, which are prone to mutations. Cellular mRNAs that encode critical proteins involved in HIV-1 replication may circumvent pitfalls associated with viral escape mechanisms. Targeting cellular genes that are an essential part of the HIV-1 life cycle could therefore be advantageous. CD4 is the primary cellular receptor for HIV-1 entry on T lymphocytes. In addition to the CD4 primary receptor, the cellular chemokine receptors CCR5 and CXCR4, which function as co-receptors for HIV-1, have provided new therapeutic targets and a better understanding of the progression of viral infection. Several investigators targeted cellular proteins necessary for the HIV-1 life cycle by siRNAs and produced decreased levels of virus production.(60, 61) Preliminary observations from various laboratories have demonstrated that siRNAs specific for CD4 receptor do indeed inhibit HIV-1 replication.(62, 63) After transfection of cultured T cells with siRNA against the mRNA for CD4, HIV production, after exposure of the cells to the virus, decreased substantially. (64) CCR5, an HIV-1 co-receptor for the M-tropic HIV-1 variant, providing an attractive cellular target for siRNAs since homozygous deletions of CCR5 effectively confer protection from HIV-1 without any serious deleterious effects in immune function.(65) At least one group has taken advantage of this target for RNA*i*-mediated gene silencing, demonstrating that in vitro knockdown of CCR5 by siRNAs provided marked protection from HIV-1 infection.(63)

Although suppression of the primary receptor CD4 may be restricted by its normal role in the immune system, CCR5 seems dispensable for normal life.(66) Unfortunately, not all HIV-1 strains require CCR5, and the inhibition of CCR5 may result in the selection of HIV-1 variants that use CXCR4 as a co-receptor. It is critical to identify this particular aspect by studying strain variants.

#### **7. Escape strategies of HIV-1 from siRNA**

One of the hallmarks of RNA*i* is its sequence-specific knockdown of the target transcript, but unfortunately, it also presents a way out for HIV-1, since single nucleotide substitutions

Crippling of HIV at Multiple Stages with

prevent escape strategies observed by HIV-1 by various labs.

**9. Limitations and hurdles of** *in vivo* **delivery of RNA***i*

this approach is not practical for clinical use. (76, 77)

molecule in order to get optimal inhibition.

be administered in a combined fashion to prevent HIV-1 escape strategies.

Recombinant Adeno-Associated Viral Mediated RNA Interference 393

eradicate HIV-1 disease. With this backdrop of HIV-1 drug development research, we propose to develop a cocktail of HIV-1 drug analogous to the current clinical use of combinations of antiviral drugs that target the reverse transcriptase and protease enzymes. These combinatorial approaches attacking multiple targets were designed essentially to

Analogous to the current clinical practice of HAART therapy, RNA*i* approaches should also

Although RNA*i* mediated inhibition through siRNAs to knockdown HIV-1 genes in the laboratory has been successful, transfection of these purchased siRNA from commercial sources is impractical and has little value for translational work. siRNA is highly labile and often requires exceptionally high levels to achieve gene silencing *in vivo*. Further, the genesilencing effect of siRNA is directly dependent on the number of molecules available in the cells, underscoring the need for development of plasmid vectors for the continuous synthesis of siRNA inside the cell. Current challenges facing the *in vivo* application of siRNA are the maintenance of duplex stability to avoid endonuclease degradation, need for improved delivery and the need to minimize immunological responses.(70) Though successful *in vivo* application of siRNA was demonstrated in liver through high-pressure tail vein injection in a murine model, its applicability to humans is limited.(71, 72) The quantity of siRNA necessary for efficient silencing is incompatible with scale-up to larger preclinical models.(73) Liposomal packaging(74) and chemical modification of the RNA and polyethylene glycol (PEG)(75) conjugated methods give stability to siRNA molecules, but still require large amounts of RNA*i* and are financially non-viable techniques. Hydrodynamic transfection of siRNA has been successful in targeting organs in mice, but

One way to address this problem is to construct a siRNA sequence for insertion in a vector for intracellular expression of siRNA. Here the siRNA cassette is driven by RNA polymerase III promoter such as U6 that express sense and antisense strands separated by short "hairpin" RNAs (shRNAs) that are cleaved by the dicer to produce siRNA. In some cases both the sense and anti-sense siRNA strands are transcribed separately, which then hybridize *in vivo* to make the siRNA. Expression from a DNA plasmid or a viral vector such as shRNA enhances stability and safe delivery of siRNA apart from providing continuous production *in vivo*. It has been demonstrated that the transfection of human cells with plasmid encoding shRNA against HIV-1 *rev* drastically inhibited viral replication over a period of several days. Further, the highest degree of inhibition of viral replication was

Selection of highly accessible targets within the HIV-1 RNA genome should be determined first with antisense DNA oligonucleotide arrays before designing shRNA.(78, 79) There is recent evidence that the efficacy of siRNAs is similarly influenced by secondary structure in the target transcript.(80). These selection criteria should be used in the design of the siRNA

One of the important limitations of siRNA-mediated drug delivery is the vehicle to carry the inhibitory molecules to the target cells. Viral vectors are generally more efficient vehicles for

achieved by simultaneously targeting two distinct sites within *rev*.(51, 69)

**10. Efficient delivery and expression of shRNA by viral vectors** 

in the target region can drastically decrease the efficiency of the knockdown. HIV-1 has a high mutation rate, and this is one of the reasons why RNA*i* gene silencing has not yet been shown to protect cells against HIV-1 in long-term virus replication assays although they were successful in the short term. For example, the effects of anti-HIV-1 siRNAs in lymphocytes were progressively dampened by the emergence of viral mutant genes *tat* and *nef* through nucleotide substitution or deletions within the siRNA target sequences.(44, 59) HIV-1 can also escape from RNA*i*-mediated inhibition through mutations that alter the local RNA secondary structure.(67) This emergence of escape mutants occurs even without necessarily changing the encoded protein after prolonged culturing. In order to circumvent the emergence of resistant viruses, targeting of conserved sequences and the simultaneous use of multiple siRNAs have been suggested. Further strategies to prevent this siRNA escape strategy by HIV-1 suggested the use of anti-sense for *tat* and *nef* genes. Unlike siRNA, the anti-sense approach is not a natural phenomenon occurring in the cells and no escape strategies have been developed by HIV-1. By the combination of gene knockout by two approaches, an effective and complete suppression of HIV-1 can be achieved.

#### **8. Multi-targeted knockdown of HIV-1 genes**

Although targeting a single HIV-1 sequence can result in strong inhibition of viral replication, it is likely followed by viral escape. Thus far, studies establishing the utility of siRNAs in suppressing HIV-1 infection failed in the long run because of the high mutation rate of HIV-1 replication. There are considerable challenges in achieving this long-term inhibition, preventing the transient success achieved from translating into clinical advantage. Therefore, approaches that not only target different stages of the viral life cycle but also simultaneously target specific sets of cellular genes that are needed for viral entry should be explored. In fact, it has been clearly demonstrated that the introduction of multiple siRNAs specific for HIV-1 could lead to viral RNA degradation and replication during different stages of the viral life cycle.(59) This multi-frontal RNA*i*-mediated attack on HIV-1 potentially inhibits the mutation escape mechanism. There have been several successful demonstrations of inhibition of HIV-1 replication using siRNA targeting distinct steps of the viral life cycle. HIV RNA in the post entry complex was successfully degraded abolishing the integration of proviral DNA when siRNAs targeted more than one region.(43) Dual-specific short hairpin siRNA constructs containing an intervening bridge, targeted against both receptors were demonstrated to successfully inhibit HIV-1 replication, thus demonstrating the practical utility of an siRNA multi-frontal attack on HIV-1.(60)

It has been previously established that if the length of siRNA exceeds 30 bp, there is an induction of nonspecific antiviral interferon responses.(33) Contrary to this belief, it was shown recently that this phenomenon might not be applicable to all sequences. Chang et al. generated 38 bp siRNAs that can induce targeted gene silencing of more than one gene without nonspecific antiviral responses. This structural flexibility of gene silencing with siRNAs needs to be further explored in order to achieve complete inhibition of HIV-1 by targeting simultaneously several regions.(68) By targeting two separate regions to knockout transcription of the gene *rev*, the highest degree of inhibition of viral replication was achieved.(69)

These newer drug designs had shown initial promise, but fell short of practical utility in providing adequate protection in every case. Since no effective therapy is currently available for prevention, new and innovative therapies are urgently needed to control, prevent and

in the target region can drastically decrease the efficiency of the knockdown. HIV-1 has a high mutation rate, and this is one of the reasons why RNA*i* gene silencing has not yet been shown to protect cells against HIV-1 in long-term virus replication assays although they were successful in the short term. For example, the effects of anti-HIV-1 siRNAs in lymphocytes were progressively dampened by the emergence of viral mutant genes *tat* and *nef* through nucleotide substitution or deletions within the siRNA target sequences.(44, 59) HIV-1 can also escape from RNA*i*-mediated inhibition through mutations that alter the local RNA secondary structure.(67) This emergence of escape mutants occurs even without necessarily changing the encoded protein after prolonged culturing. In order to circumvent the emergence of resistant viruses, targeting of conserved sequences and the simultaneous use of multiple siRNAs have been suggested. Further strategies to prevent this siRNA escape strategy by HIV-1 suggested the use of anti-sense for *tat* and *nef* genes. Unlike siRNA, the anti-sense approach is not a natural phenomenon occurring in the cells and no escape strategies have been developed by HIV-1. By the combination of gene knockout by

two approaches, an effective and complete suppression of HIV-1 can be achieved.

Although targeting a single HIV-1 sequence can result in strong inhibition of viral replication, it is likely followed by viral escape. Thus far, studies establishing the utility of siRNAs in suppressing HIV-1 infection failed in the long run because of the high mutation rate of HIV-1 replication. There are considerable challenges in achieving this long-term inhibition, preventing the transient success achieved from translating into clinical advantage. Therefore, approaches that not only target different stages of the viral life cycle but also simultaneously target specific sets of cellular genes that are needed for viral entry should be explored. In fact, it has been clearly demonstrated that the introduction of multiple siRNAs specific for HIV-1 could lead to viral RNA degradation and replication during different stages of the viral life cycle.(59) This multi-frontal RNA*i*-mediated attack on HIV-1 potentially inhibits the mutation escape mechanism. There have been several successful demonstrations of inhibition of HIV-1 replication using siRNA targeting distinct steps of the viral life cycle. HIV RNA in the post entry complex was successfully degraded abolishing the integration of proviral DNA when siRNAs targeted more than one region.(43) Dual-specific short hairpin siRNA constructs containing an intervening bridge, targeted against both receptors were demonstrated to successfully inhibit HIV-1 replication, thus demonstrating the practical utility of an siRNA multi-frontal attack on HIV-1.(60) It has been previously established that if the length of siRNA exceeds 30 bp, there is an induction of nonspecific antiviral interferon responses.(33) Contrary to this belief, it was shown recently that this phenomenon might not be applicable to all sequences. Chang et al. generated 38 bp siRNAs that can induce targeted gene silencing of more than one gene without nonspecific antiviral responses. This structural flexibility of gene silencing with siRNAs needs to be further explored in order to achieve complete inhibition of HIV-1 by targeting simultaneously several regions.(68) By targeting two separate regions to knockout transcription of the gene *rev*, the highest degree of inhibition of viral replication was

These newer drug designs had shown initial promise, but fell short of practical utility in providing adequate protection in every case. Since no effective therapy is currently available for prevention, new and innovative therapies are urgently needed to control, prevent and

**8. Multi-targeted knockdown of HIV-1 genes** 

achieved.(69)

eradicate HIV-1 disease. With this backdrop of HIV-1 drug development research, we propose to develop a cocktail of HIV-1 drug analogous to the current clinical use of combinations of antiviral drugs that target the reverse transcriptase and protease enzymes. These combinatorial approaches attacking multiple targets were designed essentially to prevent escape strategies observed by HIV-1 by various labs.

Analogous to the current clinical practice of HAART therapy, RNA*i* approaches should also be administered in a combined fashion to prevent HIV-1 escape strategies.

### **9. Limitations and hurdles of** *in vivo* **delivery of RNA***i*

Although RNA*i* mediated inhibition through siRNAs to knockdown HIV-1 genes in the laboratory has been successful, transfection of these purchased siRNA from commercial sources is impractical and has little value for translational work. siRNA is highly labile and often requires exceptionally high levels to achieve gene silencing *in vivo*. Further, the genesilencing effect of siRNA is directly dependent on the number of molecules available in the cells, underscoring the need for development of plasmid vectors for the continuous synthesis of siRNA inside the cell. Current challenges facing the *in vivo* application of siRNA are the maintenance of duplex stability to avoid endonuclease degradation, need for improved delivery and the need to minimize immunological responses.(70) Though successful *in vivo* application of siRNA was demonstrated in liver through high-pressure tail vein injection in a murine model, its applicability to humans is limited.(71, 72) The quantity of siRNA necessary for efficient silencing is incompatible with scale-up to larger preclinical models.(73) Liposomal packaging(74) and chemical modification of the RNA and polyethylene glycol (PEG)(75) conjugated methods give stability to siRNA molecules, but still require large amounts of RNA*i* and are financially non-viable techniques. Hydrodynamic transfection of siRNA has been successful in targeting organs in mice, but this approach is not practical for clinical use. (76, 77)

One way to address this problem is to construct a siRNA sequence for insertion in a vector for intracellular expression of siRNA. Here the siRNA cassette is driven by RNA polymerase III promoter such as U6 that express sense and antisense strands separated by short "hairpin" RNAs (shRNAs) that are cleaved by the dicer to produce siRNA. In some cases both the sense and anti-sense siRNA strands are transcribed separately, which then hybridize *in vivo* to make the siRNA. Expression from a DNA plasmid or a viral vector such as shRNA enhances stability and safe delivery of siRNA apart from providing continuous production *in vivo*. It has been demonstrated that the transfection of human cells with plasmid encoding shRNA against HIV-1 *rev* drastically inhibited viral replication over a period of several days. Further, the highest degree of inhibition of viral replication was achieved by simultaneously targeting two distinct sites within *rev*.(51, 69)

Selection of highly accessible targets within the HIV-1 RNA genome should be determined first with antisense DNA oligonucleotide arrays before designing shRNA.(78, 79) There is recent evidence that the efficacy of siRNAs is similarly influenced by secondary structure in the target transcript.(80). These selection criteria should be used in the design of the siRNA molecule in order to get optimal inhibition.

#### **10. Efficient delivery and expression of shRNA by viral vectors**

One of the important limitations of siRNA-mediated drug delivery is the vehicle to carry the inhibitory molecules to the target cells. Viral vectors are generally more efficient vehicles for

Crippling of HIV at Multiple Stages with

an ideal *in vivo* RNA*i* delivery vehicle.

been demonstrated.(7)

**12. Capsid mutant rAAV for enhanced transduction** 

Recombinant Adeno-Associated Viral Mediated RNA Interference 395

of tissues.(93-95) First generation rAAV vectors were single stranded, but the development of self-complimentary (double stranded) rAAV vectors helped to avoid delay in trans-gene expression.(96) Multiple administration of the rAAV vectors is possible to overcome neutralization by the antibody produced following the initial administration due to the availability of multiple serotypes with significantly higher trans-gene expression levels than that of prototype single stranded-vectors.(97) rAAV-based vectors have the potential for stable long-term trans-gene expression. rAAV is naturally gutless vector, which do not express any viral genes or cause a cytotoxic cellular immune response in the host. Furthermore, rAAV vectors show only a modest frequency of integration into host genome, thus avoiding insertional mutagenesis.(89) Overall, rAAV vectors fulfill the requirements for

One of the shortcomings of the traditional rAAV vectorology is low transduction efficiency, which requires large doses of vectors to achieve the desired effect. This is due to the phosphorylation of AAV capsids at tyrosine residues in the cell, which leads to a ubiquitin-proteasome-mediated destruction of the majority of rAAV particles and a decrease in transduction efficiency.(98) Gene therapy with these traditional rAAV vectors necessitates the delivery of undesirably high doses of the virus in order to achieve therapeutic benefit.(99) Recent advances have lead to the generation of rAAV vectors with mutant capsids protecting them from ubiquitin-proteasome-mediated degradation in the cytosol, eventually leading to an increase in DNA transduction efficiency.(7) We acquired these next generation rAAV vectors from Dr. Srivastava's laboratory consisting of a variant of rAAV-2/8 with a mutated capsid making the vector resistant to degradation in the cytosol.(7) By using triple-capsid mutant, pACG2-3M (Y444F, Y500F & Y730F)(7), along with the self-complimentary rAAV vector, we achieved a significant enhancement in rAAV2-sc green fluorescent protein (GFP) mediated transduction (Fig-1). With pseudotyped rAAV vectors and capsid mutations, even greater *in vivo* transduction efficiency has

Fig. 1. HEK-293 cells transduced with various preparations of rAAV-GFP showing transduction efficiency based on green fluorescence intensity. A. Wild type rAAV-GFP; B.

rAAV double stranded-GFP; C. rAAV-Capsid mutant.

shRNA *in vivo* than nonviral vectors.(81) Adenovirus, retro- or lentivirus, and AAV have been successfully used for this purpose. Retroviral/lentiviral vectors can potentially generate insertional mutagenesis, while adenoviral vectors trigger unacceptable levels of immune responses with concerns of safety.(82) shRNA can be packaged as recombinant viral vectors for better delivery in the whole organism. Retroviral vectors are successfully used for shRNA delivery, derived from moloney murine leukemia virus (MMLV). Lentiviral vectors are derived from HIV itself and can infect all the cells without the need for receptor interaction. Studies with lentiviral vectors silencing CCR5 have been performed but showed that the down regulating effect of CCR5 alone was insufficient. However, combinatorial constructs targeted to both CXCR4 and CCR5 and have shown better efficacy.(61) Retroviral/lentiviral vectors randomly integrate into the genome, generate insertional mutagenesis, and are derived from pathogenic viruses.

Dual-specific short hairpin siRNA constructs containing an intervening spacer, targeting receptor and co-receptor, demonstrated the practical utility of shRNA constructs synthesized as a single transcript. Since the shRNA design will permit tandem assembly of multiple motifs, it is now possible to introduce promising multivalent siRNA constructs into viral vectors for *in vivo* gene therapeutic applications. Based on this rationale, recent work with synthetic siRNAs demonstrated that down regulating either CXCR4 or CCR5 will protect cells from X4 or R5 HIV-1 strains, respectively, at the level of viral entry.(83) As mentioned earlier, CCR5 is a co-receptor, necessary for cellular entry by HIV-1 (R5 tropic viral strain), but is dispensable for normal human physiology. Owing to its crucial role in HIV-1 infection, the CCR5 co-receptor has been the subject of many therapeutic approaches, including RNA*i*-mediated gene silence therapy. siRNA targeting was shown to be effective; however, complete knockdown remained an elusive goal. In one study, transgenic macrophages expressing high levels of CCR5 were used for testing the efficacy of lentiviral vectors carrying CCR5 shRNA. Lentiviral delivery of longer (28-mer) shRNA were shown to be very effective in gene knockdown.(84) Thus, anti-CCR5 shRNA viral delivery is a promising candidate for clinical application.

We have tested retroviral vectors for gene therapy(85, 86); however, retroviral-mediated gene therapy is limited by a variety of practical and theoretical concerns, such as the immunogenicity of viral capsid proteins and insertional mutagenesis, which limit their utility for clinical purposes.(87)

Adenoviral vectors have also been successfully used for the delivery of shRNA(88) but they are well known to trigger unacceptable levels of immune responsiveness due to their large size and thereby limit repeat administration. Stability and efficiency is not the concern with viral vectors, but safety is a primary concern.

#### **11. Recombinant Adeno-Associated Virus (rAAV) – Ideal RNA***i* **gene silence therapy vectors**

AAV belongs to the parvovirus family and is the only viral vector not known to be associated with any human disease(89) and the smallest vector suitable for RNA*i*-mediated gene silencing. Due to the safety(90), efficacy and potency provided by rAAV vectors, they make better alternative to the more commonly-used retroviral, lentiviral and adenovirus based vectors for gene therapy. rAAV vectors are easy to propagate and have many characteristics that make them a better choice for somatic gene therapy with RNA*i*-mediated gene silencing.(91, 92) rAAV vectors have long been established to transduce a wide variety

shRNA *in vivo* than nonviral vectors.(81) Adenovirus, retro- or lentivirus, and AAV have been successfully used for this purpose. Retroviral/lentiviral vectors can potentially generate insertional mutagenesis, while adenoviral vectors trigger unacceptable levels of immune responses with concerns of safety.(82) shRNA can be packaged as recombinant viral vectors for better delivery in the whole organism. Retroviral vectors are successfully used for shRNA delivery, derived from moloney murine leukemia virus (MMLV). Lentiviral vectors are derived from HIV itself and can infect all the cells without the need for receptor interaction. Studies with lentiviral vectors silencing CCR5 have been performed but showed that the down regulating effect of CCR5 alone was insufficient. However, combinatorial constructs targeted to both CXCR4 and CCR5 and have shown better efficacy.(61) Retroviral/lentiviral vectors randomly integrate into the genome, generate insertional

Dual-specific short hairpin siRNA constructs containing an intervening spacer, targeting receptor and co-receptor, demonstrated the practical utility of shRNA constructs synthesized as a single transcript. Since the shRNA design will permit tandem assembly of multiple motifs, it is now possible to introduce promising multivalent siRNA constructs into viral vectors for *in vivo* gene therapeutic applications. Based on this rationale, recent work with synthetic siRNAs demonstrated that down regulating either CXCR4 or CCR5 will protect cells from X4 or R5 HIV-1 strains, respectively, at the level of viral entry.(83) As mentioned earlier, CCR5 is a co-receptor, necessary for cellular entry by HIV-1 (R5 tropic viral strain), but is dispensable for normal human physiology. Owing to its crucial role in HIV-1 infection, the CCR5 co-receptor has been the subject of many therapeutic approaches, including RNA*i*-mediated gene silence therapy. siRNA targeting was shown to be effective; however, complete knockdown remained an elusive goal. In one study, transgenic macrophages expressing high levels of CCR5 were used for testing the efficacy of lentiviral vectors carrying CCR5 shRNA. Lentiviral delivery of longer (28-mer) shRNA were shown to be very effective in gene knockdown.(84) Thus, anti-CCR5 shRNA viral delivery is a

We have tested retroviral vectors for gene therapy(85, 86); however, retroviral-mediated gene therapy is limited by a variety of practical and theoretical concerns, such as the immunogenicity of viral capsid proteins and insertional mutagenesis, which limit their

Adenoviral vectors have also been successfully used for the delivery of shRNA(88) but they are well known to trigger unacceptable levels of immune responsiveness due to their large size and thereby limit repeat administration. Stability and efficiency is not the concern with

**11. Recombinant Adeno-Associated Virus (rAAV) – Ideal RNA***i* **gene silence** 

AAV belongs to the parvovirus family and is the only viral vector not known to be associated with any human disease(89) and the smallest vector suitable for RNA*i*-mediated gene silencing. Due to the safety(90), efficacy and potency provided by rAAV vectors, they make better alternative to the more commonly-used retroviral, lentiviral and adenovirus based vectors for gene therapy. rAAV vectors are easy to propagate and have many characteristics that make them a better choice for somatic gene therapy with RNA*i*-mediated gene silencing.(91, 92) rAAV vectors have long been established to transduce a wide variety

mutagenesis, and are derived from pathogenic viruses.

promising candidate for clinical application.

viral vectors, but safety is a primary concern.

utility for clinical purposes.(87)

**therapy vectors** 

of tissues.(93-95) First generation rAAV vectors were single stranded, but the development of self-complimentary (double stranded) rAAV vectors helped to avoid delay in trans-gene expression.(96) Multiple administration of the rAAV vectors is possible to overcome neutralization by the antibody produced following the initial administration due to the availability of multiple serotypes with significantly higher trans-gene expression levels than that of prototype single stranded-vectors.(97) rAAV-based vectors have the potential for stable long-term trans-gene expression. rAAV is naturally gutless vector, which do not express any viral genes or cause a cytotoxic cellular immune response in the host. Furthermore, rAAV vectors show only a modest frequency of integration into host genome, thus avoiding insertional mutagenesis.(89) Overall, rAAV vectors fulfill the requirements for an ideal *in vivo* RNA*i* delivery vehicle.

#### **12. Capsid mutant rAAV for enhanced transduction**

One of the shortcomings of the traditional rAAV vectorology is low transduction efficiency, which requires large doses of vectors to achieve the desired effect. This is due to the phosphorylation of AAV capsids at tyrosine residues in the cell, which leads to a ubiquitin-proteasome-mediated destruction of the majority of rAAV particles and a decrease in transduction efficiency.(98) Gene therapy with these traditional rAAV vectors necessitates the delivery of undesirably high doses of the virus in order to achieve therapeutic benefit.(99) Recent advances have lead to the generation of rAAV vectors with mutant capsids protecting them from ubiquitin-proteasome-mediated degradation in the cytosol, eventually leading to an increase in DNA transduction efficiency.(7) We acquired these next generation rAAV vectors from Dr. Srivastava's laboratory consisting of a variant of rAAV-2/8 with a mutated capsid making the vector resistant to degradation in the cytosol.(7) By using triple-capsid mutant, pACG2-3M (Y444F, Y500F & Y730F)(7), along with the self-complimentary rAAV vector, we achieved a significant enhancement in rAAV2-sc green fluorescent protein (GFP) mediated transduction (Fig-1). With pseudotyped rAAV vectors and capsid mutations, even greater *in vivo* transduction efficiency has been demonstrated.(7)

Fig. 1. HEK-293 cells transduced with various preparations of rAAV-GFP showing transduction efficiency based on green fluorescence intensity. A. Wild type rAAV-GFP; B. rAAV double stranded-GFP; C. rAAV-Capsid mutant.

Crippling of HIV at Multiple Stages with

**15. Acknowledgments** 

corrections.

**16. References** 

(2006).

Recombinant Adeno-Associated Viral Mediated RNA Interference 397

the hurdles to overcome to proceed to the next stage, and possible solutions. Although RNA*i* molecules can be introduced into cells as double stranded or expressed from a plasmid to inhibit abnormally elevated genes, transfection of these purchased molecules from commercial sources is impractical and has little value for translational work. The main difficulty thus far in extending the power of RNA interference (RNA*i*) to clinical practice has been the development of safe vectors coding for shRNAs to achieve persistent knockdown *in vivo*. rAAV vectors are different from other vectors, since the only gene expressed from recombinant vector is the trans-gene itself, naturally gutless by design and thus avoiding any cytotoxic cellular immune responses in the host. Furthermore, rAAV vectors show only a modest frequency of integration into the host genome, thus avoiding insertional mutagenesis, which has been a stumbling block for the clinical use of retroviral or lentiviral vectors. Development of self-complimentary (also known as double stranded) vectors to avoid delay in trans-gene expression(96, 114) and packaging with capsid mutants(115) to increase transduction efficiency has further contributed to rAAV vectorology. Recent advances in our understanding of RNA*i* make rAAV an especially attractive candidate for anti-HIV-1 gene therapy, and rAAV-based RNA*i* approaches can be combined with other

This work has been conducted at the John D Dingell VA Medical Center, Detroit, MI 48201 and Karmanos Cancer Institute, Detroit, MI 48201. Ariel J. Harden helped with manuscript

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CLINICAL RESEARCH, Vol. 1, 2010.

#### **13. rAAV vectors for siRNA delivery** *in vivo* **as short hairpin RNA (shRNA)**

An ideal vector system for RNA*i* expression should be efficient and allow stable expression of the shRNA cassette without causing insertional mutagenesis or undesired immune responses. AAV is a small virus of 4.7 kb and relatively simple in its organization, comprising only two genes *Rep* and *Cap*. AAV vectors are extremely efficient tools for gene delivery *in vitro* and *in vivo*, as demonstrated by a number of laboratories. rAAV vectors have been shown to efficiently transduce hematopoietic cells.(100-102) Moreover, rAAV only retain inverted terminal repeats but do not express any viral genes and thus are gutless by design and definition more over it has not been associated with any human pathogenic, making it the vector of choice for human gene therapy. Because of their efficient transcription and inability to recombine with HIV-1, rAAV vectors represent a promising form of anti-retroviral gene therapy.(100)

One of the first studies using rAAV vector to deliver shRNA by Tomar et al. provided initial proof of principle that rAAV vector particles can be engineered to express shRNA.(88) They showed efficient knockdown of p53 and caspase 8 proteins. Subsequent studies by several investigators further demonstrated the usefulness of the rAAV vectors to express the shRNA cassette.(103-106) rAAV vectors have also been successfully used for a variety of gene silence experiments.(103-105) Use of rAAV vector encoding an anti-sense RNA against HIV-1 has been well documented by various labs.(23, 27, 100, 107) We studied the effect of anti-sense p53 gene transduction in a multiple myeloma cell line, ARH77, using AAV vector, where we delivered p53 cDNA in an anti-sense orientation.(108) *In vivo* studies in mice showed persistent knockdown of the target tyrosine hydroxylase in a Parkinson's disease model. They further demonstrated that reduction in the target elicited behavioral defects in the treated mice and created a phenotype reminiscent of rodent models of Parkinson's disease.

rAAV-mediated transduction is very efficient, particularly when compared with passive entry of simple siRNA or plasmid DNA.(109-111) rAAV siRNA delivery has been recently tested by several groups and shown to be highly efficient. Specific and efficient inhibition of HIV-1 replication was demonstrated in cultures.(91, 112) Together, this underscores the great promise of pseudo-typing shRNA-expressing AAV vectors to achieve targeted and controlled siRNA induction *in vivo*. Although the targeting of a single HIV-1 sequence can result in strong inhibition of viral replication, it is likely to be followed by viral escape. In fact, in most *in vitro* tests, siRNA did not stand the test of long-term protection against HIV-1. To overcome this escape strategy by HIV-1, we have a multi-pronged attack on HIV-1. First, HIV-1 is targeted at multiple genes for inhibition. Second, HIV-1 entry is inhibited by targeting siRNA to its cellular receptor CCR5 that is resistant to mutation. Third, we are introducing an anti-sense approach to knockdown HIV-1 *tat*, shown to be responsible for siRNA escape. Synthetic bi-specific or combinatorial constructs targeting both CXCR4 and CCR5 receptors have shown to confer resistance to HIV-1 infection much stronger than that conferred by targeting each one alone, giving a clear indication that multiple targeting is better than a single target.(22, 43, 61, 113)

#### **14. Conclusion**

Gene silencing therapy has the potential to inhibit HIV-1 replication and increase patient quality of life as an additional therapeutic class, and may serve as an adjuvant to current HAART treatment. This review gives a brief introduction regarding the emergence of RNAi, the hurdles to overcome to proceed to the next stage, and possible solutions. Although RNA*i* molecules can be introduced into cells as double stranded or expressed from a plasmid to inhibit abnormally elevated genes, transfection of these purchased molecules from commercial sources is impractical and has little value for translational work. The main difficulty thus far in extending the power of RNA interference (RNA*i*) to clinical practice has been the development of safe vectors coding for shRNAs to achieve persistent knockdown *in vivo*. rAAV vectors are different from other vectors, since the only gene expressed from recombinant vector is the trans-gene itself, naturally gutless by design and thus avoiding any cytotoxic cellular immune responses in the host. Furthermore, rAAV vectors show only a modest frequency of integration into the host genome, thus avoiding insertional mutagenesis, which has been a stumbling block for the clinical use of retroviral or lentiviral vectors. Development of self-complimentary (also known as double stranded) vectors to avoid delay in trans-gene expression(96, 114) and packaging with capsid mutants(115) to increase transduction efficiency has further contributed to rAAV vectorology. Recent advances in our understanding of RNA*i* make rAAV an especially attractive candidate for anti-HIV-1 gene therapy, and rAAV-based RNA*i* approaches can be combined with other therapeutic modalities to make a combinatorial therapy akin to HAART.

#### **15. Acknowledgments**

This work has been conducted at the John D Dingell VA Medical Center, Detroit, MI 48201 and Karmanos Cancer Institute, Detroit, MI 48201. Ariel J. Harden helped with manuscript corrections.

#### **16. References**

396 Recent Translational Research in HIV/AIDS

An ideal vector system for RNA*i* expression should be efficient and allow stable expression of the shRNA cassette without causing insertional mutagenesis or undesired immune responses. AAV is a small virus of 4.7 kb and relatively simple in its organization, comprising only two genes *Rep* and *Cap*. AAV vectors are extremely efficient tools for gene delivery *in vitro* and *in vivo*, as demonstrated by a number of laboratories. rAAV vectors have been shown to efficiently transduce hematopoietic cells.(100-102) Moreover, rAAV only retain inverted terminal repeats but do not express any viral genes and thus are gutless by design and definition more over it has not been associated with any human pathogenic, making it the vector of choice for human gene therapy. Because of their efficient transcription and inability to recombine with HIV-1, rAAV vectors represent a promising

One of the first studies using rAAV vector to deliver shRNA by Tomar et al. provided initial proof of principle that rAAV vector particles can be engineered to express shRNA.(88) They showed efficient knockdown of p53 and caspase 8 proteins. Subsequent studies by several investigators further demonstrated the usefulness of the rAAV vectors to express the shRNA cassette.(103-106) rAAV vectors have also been successfully used for a variety of gene silence experiments.(103-105) Use of rAAV vector encoding an anti-sense RNA against HIV-1 has been well documented by various labs.(23, 27, 100, 107) We studied the effect of anti-sense p53 gene transduction in a multiple myeloma cell line, ARH77, using AAV vector, where we delivered p53 cDNA in an anti-sense orientation.(108) *In vivo* studies in mice showed persistent knockdown of the target tyrosine hydroxylase in a Parkinson's disease model. They further demonstrated that reduction in the target elicited behavioral defects in the treated mice

rAAV-mediated transduction is very efficient, particularly when compared with passive entry of simple siRNA or plasmid DNA.(109-111) rAAV siRNA delivery has been recently tested by several groups and shown to be highly efficient. Specific and efficient inhibition of HIV-1 replication was demonstrated in cultures.(91, 112) Together, this underscores the great promise of pseudo-typing shRNA-expressing AAV vectors to achieve targeted and controlled siRNA induction *in vivo*. Although the targeting of a single HIV-1 sequence can result in strong inhibition of viral replication, it is likely to be followed by viral escape. In fact, in most *in vitro* tests, siRNA did not stand the test of long-term protection against HIV-1. To overcome this escape strategy by HIV-1, we have a multi-pronged attack on HIV-1. First, HIV-1 is targeted at multiple genes for inhibition. Second, HIV-1 entry is inhibited by targeting siRNA to its cellular receptor CCR5 that is resistant to mutation. Third, we are introducing an anti-sense approach to knockdown HIV-1 *tat*, shown to be responsible for siRNA escape. Synthetic bi-specific or combinatorial constructs targeting both CXCR4 and CCR5 receptors have shown to confer resistance to HIV-1 infection much stronger than that conferred by targeting each one alone, giving a clear indication that multiple targeting is

Gene silencing therapy has the potential to inhibit HIV-1 replication and increase patient quality of life as an additional therapeutic class, and may serve as an adjuvant to current HAART treatment. This review gives a brief introduction regarding the emergence of RNAi,

and created a phenotype reminiscent of rodent models of Parkinson's disease.

**13. rAAV vectors for siRNA delivery** *in vivo* **as short hairpin RNA (shRNA)** 

form of anti-retroviral gene therapy.(100)

better than a single target.(22, 43, 61, 113)

**14. Conclusion** 


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**20** 

*Australia* 

**Cell-Delivered Gene Therapy for HIV** 

Maureen Boyd3, John M. Murray2,4,5 and Geoff Symonds3,5 *1The Faculty of Medicine, The University of New South Wales, Sydney* 

*4The Kirby Institute, The University of New South Wales, Sydney NSW* 

*3Calimmune Australia, 405 Liverpool St Darlinghurst, NSW* 

*5St Vincent's Institute for Applied Medical Research* 

*405 Liverpool St Darlinghurst, NSW* 

Scott Ledger1, Borislav Savkovic2, Michelle Millington3, Helen Impey3,

*2The School of Mathematics and Statistics, The University of New South Wales, Sydney* 

Gene therapy involves the transfer of genetic material into cells of an individual to treat an underlying illness either through the expression of advantageous genes or the silencing of disadvantageous ones (Flotte 2007; Kohn and Candotti 2009). Gene therapy has been used successfully to treat several diseases, for example SCID-X1 (Cavazzana-Calvo, Hacein-Bey et al. 2000) and SCID-ADA (Aiuti 2004) and holds out promise as a more general treatment regimen (Flotte 2007). One of the driving forces behind the area of research into the treatment of HIV is the resistance to, and side effects of, the current drugs being used. This development of resistance and the need for continuous and ongoing daily medication have been major shortcomings of conventional highly active antiretroviral therapy (HAART) when employed as a treatment against HIV (Perno, Moyle et al. 2008). An additional driving force behind interest in gene therapy is the potential for a one-off treatment that would continue to work for the life of the individual (Symonds, Johnstone et al. 2010). One can envisage gene therapy as a full or partial replacement for HAART, that may help to overcome issues of viral resistance,

co-morbidity and attendant compliance (i.e. daily administration of HAART for life).

While HAART is a systemic form of treatment which provides a substantial level of protection to HIV susceptible cells in the body for many years, it is highly susceptible to the development of a resistant HIV quasispecies, that may selectively expand due to the strong evolutionary pressure exerted by HAART (Perno, Moyle et al. 2008). Whereas HAARTbased treatments bathe each cell in some level of drug, gene-therapy results in a polar population dynamic consisting of gene protected and unprotected cells. This is due to the fact that it is neither practical nor possible to have a protective gene against HIV introduced into all cells of the body, but rather only a subset of the total cell population is afforded protection (Symonds, Johnstone et al. 2010). This polar dynamic is predicted to provide additional pressures to the suriviving HIV population (Applegate, Birkett et al. 2010). Cells that might be afforded protection include CD4+ T cells and macrophages, which are known to be targets of HIV infection, as well as other cell populations susceptible to HIV infection. In this chapter we describe the biological and clinical underpinnings of gene-therapy including the therapeutic genes employed for protection against HIV, delivery methods of

**1. Introduction** 


### **Cell-Delivered Gene Therapy for HIV**

Scott Ledger1, Borislav Savkovic2, Michelle Millington3, Helen Impey3, Maureen Boyd3, John M. Murray2,4,5 and Geoff Symonds3,5 *1The Faculty of Medicine, The University of New South Wales, Sydney 2The School of Mathematics and Statistics, The University of New South Wales, Sydney 3Calimmune Australia, 405 Liverpool St Darlinghurst, NSW 4The Kirby Institute, The University of New South Wales, Sydney NSW 5St Vincent's Institute for Applied Medical Research 405 Liverpool St Darlinghurst, NSW Australia* 

#### **1. Introduction**

404 Recent Translational Research in HIV/AIDS

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p53 transduction leads to overexpression of bcl-2 and dexamethasone resistance in

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and B. Ramratnam. Efficient gene transfer of HIV-1-specific short hairpin RNA into human lymphocytic cells using recombinant adeno-associated virus vectors. Molecular

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of MDR1 small interfering RNA by self-complementary recombinant adenoassociated virus vector. Molecular Therapy: the Journal of the American Society of

R.W. Herzog, K.A. Weigel-Van Aken, J.A. Hobbs, S. Zolotukhin, N. Muzyczka, A. Srivastava, L. Zhong, B. Li, G. Jayandharan, C.S. Mah, L. Govindasamy, M. Agbandje-McKenna, R.W. Herzog, K.A. Weigel-Van Aken, J.A. Hobbs, S. Zolotukhin, N. Muzyczka, and A. Srivastava. Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene Gene therapy involves the transfer of genetic material into cells of an individual to treat an underlying illness either through the expression of advantageous genes or the silencing of disadvantageous ones (Flotte 2007; Kohn and Candotti 2009). Gene therapy has been used successfully to treat several diseases, for example SCID-X1 (Cavazzana-Calvo, Hacein-Bey et al. 2000) and SCID-ADA (Aiuti 2004) and holds out promise as a more general treatment regimen (Flotte 2007). One of the driving forces behind the area of research into the treatment of HIV is the resistance to, and side effects of, the current drugs being used. This development of resistance and the need for continuous and ongoing daily medication have been major shortcomings of conventional highly active antiretroviral therapy (HAART) when employed as a treatment against HIV (Perno, Moyle et al. 2008). An additional driving force behind interest in gene therapy is the potential for a one-off treatment that would continue to work for the life of the individual (Symonds, Johnstone et al. 2010). One can envisage gene therapy as a full or partial replacement for HAART, that may help to overcome issues of viral resistance, co-morbidity and attendant compliance (i.e. daily administration of HAART for life).

While HAART is a systemic form of treatment which provides a substantial level of protection to HIV susceptible cells in the body for many years, it is highly susceptible to the development of a resistant HIV quasispecies, that may selectively expand due to the strong evolutionary pressure exerted by HAART (Perno, Moyle et al. 2008). Whereas HAARTbased treatments bathe each cell in some level of drug, gene-therapy results in a polar population dynamic consisting of gene protected and unprotected cells. This is due to the fact that it is neither practical nor possible to have a protective gene against HIV introduced into all cells of the body, but rather only a subset of the total cell population is afforded protection (Symonds, Johnstone et al. 2010). This polar dynamic is predicted to provide additional pressures to the suriviving HIV population (Applegate, Birkett et al. 2010). Cells that might be afforded protection include CD4+ T cells and macrophages, which are known to be targets of HIV infection, as well as other cell populations susceptible to HIV infection.

In this chapter we describe the biological and clinical underpinnings of gene-therapy including the therapeutic genes employed for protection against HIV, delivery methods of

Cell-Delivered Gene Therapy for HIV 407

absence of a functional CCR5 co-receptor, especially when concerning strains capable of utilizing the CXCR4 coreceptor (Agrawal, Lu et al. 2004; Jin, Agrawal et al. 2008). Further evidence towards the beneficial effect of the CCR5d32 protein comes from evidence that a polymorphism in the promoter region in CCR5-/- individuals can affect the protective capabilities of the d32 mutation. It has been demonstrated that an increase in CCR5d32 protein expression will improve resistance to HIV, while decreased CCR5d32 expression

This CCR5d32 mutation has been successfully utilized in a patient, who suffered from both HIV/AIDS and leukaemia (Hutter, Nowak et al. 2009; Allers, Hutter et al. 2010). This individual, termed the "Berlin patient", had complete ablation of their immune system (to treat the leukemia) before matched allogeneic donor hematopoietic stem cells (HSC) homozygous for the CCR5d32 mutation were transfused into the patient. After one recurrence of leukaemia and a repeat of the treatment (ablation and reconstitution), the patient has had undetectable levels of HIV (and no recurring leukaemia) for more than 3 years without the use of any antiretroviral drugs (Hutter, Nowak et al. 2009; Allers, Hutter et al. 2010). This unique result of "functional" cure of HIV indicates significant potential for

**2.2 Choosing a stage of HIV infection cycle to inhibit: Which therapeutic genes hold** 

Gene-therapy may be aimed to target various stages of the HIV infection cycle as shown in Figure 1. Class 1 therapy inhibits all steps prior to viral integration into the cellular genome, Class 2 inhibits expression of viral genes and Class 3 inhibits production of new virions once integration and expression has taken place (von Laer, Hasselmann et al. 2006). According to predictions from mathematical modelling, as discussed in section 3.1, Class 1 gene therapies are likely to be the most effective as they inhibit HIV at the first steps, and provide a selective advantage to these cells by avoiding any viral or immunological induced death from infection. Hence many gene therapeutics currently under investigation include components that impair attachment or fusion stages of the viral life-cycle (Symonds,

While all these classes are potential HIV gene therapeutics, practically, the use of multiple therapeutics in combination is likely to be the most effective method. This is analogous to the antiretroviral situation where it does not take long for HIV resistance to emerge against single antiretroviral drugs. These antiretroviral drugs have been shown to be far more effective when used in combination. It is for this reason that gene therapy research has often been focused on the use of multiple gene therapuetics used in conjunction with one-another. As well as the variety of targets being investigated, there is additionally a wide range of methods to achieve inhibition of these targets. The most commonly employed methods to-

**Antisense (Class 2):** Antisense RNA is a synthetic nucleotide sequence that binds to mRNA in order to inhibit its function. This method can be used against a wide range of targets,

**Aptamers (Class 2 or 3)**: Aptamers are single-stranded RNAs or DNAs. They disrupt at the protein level by tightly binding to their target ligand (Que-Gewirth and Sullenger 2007). Aptamers can be used to target a wide array of proteins and as such have potential to be

the use of gene-therapy to mimic this result by down-regulation of CCR5.

reduces the protective effect (Jin, Agrawal et al. 2008).

**2.2.1 Classes and methods of HIV inhibition** 

**out promise?** 

Johnstone et al. 2010).

date include the following:

used in multiple settings.

including the HIV envelope (Levine, Humeau et al. 2006).

the vectors carrying these protective genes into the cells, expression cassettes and finally the target cells into which the protective genes are introduced. We then estimate the potential in-vivo protective effects of gene-therapy against HIV.

#### **2. Biological and clinical aspects of gene-therapy**

In this section we look at the biological and clinical aspects of gene-therapy. Observations associated with natural immunity that may be utilized in gene-therapy against HIV are discussed in section 2.1. Stages of the HIV infection cycle that may be inhibited by genetherapy, and the various gene therapeutic that may be employed to this aim, are the subject of section 2.2. Various delivery vectors and promoters that can achieve effective delivery and transcription of the protective gene into the cell to be transduced are the subject of section 2.3. The biological underpinnings of the target cell to be transduced with a protective gene, either CD4+ T cells or Hematopoietic Stem Cells (HSC) are discussed in section 2.4. The clinical aspects of collection of cells for transduction via apheresis and associated preparation regimens are discussed in section 2.5. Finally, Section 2.6 is concerned with clinical trials to-date of anti-HIV gene-therapy and results reported therein.

#### **2.1 CCR5 and the 32-nucleotide deletion mutation: A strong case for gene therapy**

Recent additional impetus for gene-therapy for HIV is based upon the earlier observation that some individuals do not become infected upon repeated exposure to HIV (Zimmerman, Buckler-White et al. 1997). Studies of these individuals led to the discovery of a mutation in CCR5, an important co-receptor for HIV attachment to target cells prior to infection. Such a mutation was found to confer natural immunity against HIV (Zimmerman, Buckler-White et al. 1997).

The mutation discovered was found to be a 32 nucleotide deletion (CCR5d32) within the CCR5 gene (Zimmerman, Buckler-White et al. 1997). This mutation was observed to be very common among individuals of European background and it has subsequently been determined that of Caucasian individuals, approximately 10% are heterozygous and 1-3% homozygous for this mutation (Dean, Carrington et al. 1996; Liu, Paxton et al. 1996; Samson, Libert et al. 1996; Agrawal, Lu et al. 2004), with the mutation being almost non-existent in all other populations. There has been considerable speculation regarding the origin and purpose of the mutation. It has been shown that the percentage of CCR5d32 mutation occurring in today's population is roughly comparable to that found in samples from individuals of the Bronze Age (approximately 3000 years ago) (Hummel, Schmidt et al. 2005; Hedrick and Verrelli 2006). There is evidence suggesting that smallpox provided a selective advantage for CCR5d32 (Galvani and Slatkin 2003), indicating that there may be other selective advantages associated with the mutation. The mutation does not seem to present any significant disadvantages to the individuals other than an increased risk of West-Nile disease (Glass, McDermott et al. 2006). Such observations led to an interest in mimicking this natural mutation for HIV-infected individuals via genetic manipulation (i.e. transduction) of cells vulnerable to HIV infection. (see below)

It has been noted that the 32 nucleotide deletion results in 31 new amino acids being coded for, resulting in an active CCR5d32 protein. This protein instead of presenting as CCR5 receptors on the cell surface like the wild-type counterpart, CCR5d32 actually binds to and interacts with CXCR4 receptors (Agrawal, Lu et al. 2004), the other major coreceptor for HIV attachment. This provides an additional protection against HIV infection beyond the mere

the vectors carrying these protective genes into the cells, expression cassettes and finally the target cells into which the protective genes are introduced. We then estimate the potential

In this section we look at the biological and clinical aspects of gene-therapy. Observations associated with natural immunity that may be utilized in gene-therapy against HIV are discussed in section 2.1. Stages of the HIV infection cycle that may be inhibited by genetherapy, and the various gene therapeutic that may be employed to this aim, are the subject of section 2.2. Various delivery vectors and promoters that can achieve effective delivery and transcription of the protective gene into the cell to be transduced are the subject of section 2.3. The biological underpinnings of the target cell to be transduced with a protective gene, either CD4+ T cells or Hematopoietic Stem Cells (HSC) are discussed in section 2.4. The clinical aspects of collection of cells for transduction via apheresis and associated preparation regimens are discussed in section 2.5. Finally, Section 2.6 is concerned with

clinical trials to-date of anti-HIV gene-therapy and results reported therein.

**2.1 CCR5 and the 32-nucleotide deletion mutation: A strong case for gene therapy**  Recent additional impetus for gene-therapy for HIV is based upon the earlier observation that some individuals do not become infected upon repeated exposure to HIV (Zimmerman, Buckler-White et al. 1997). Studies of these individuals led to the discovery of a mutation in CCR5, an important co-receptor for HIV attachment to target cells prior to infection. Such a mutation was found to confer natural immunity against HIV

The mutation discovered was found to be a 32 nucleotide deletion (CCR5d32) within the CCR5 gene (Zimmerman, Buckler-White et al. 1997). This mutation was observed to be very common among individuals of European background and it has subsequently been determined that of Caucasian individuals, approximately 10% are heterozygous and 1-3% homozygous for this mutation (Dean, Carrington et al. 1996; Liu, Paxton et al. 1996; Samson, Libert et al. 1996; Agrawal, Lu et al. 2004), with the mutation being almost non-existent in all other populations. There has been considerable speculation regarding the origin and purpose of the mutation. It has been shown that the percentage of CCR5d32 mutation occurring in today's population is roughly comparable to that found in samples from individuals of the Bronze Age (approximately 3000 years ago) (Hummel, Schmidt et al. 2005; Hedrick and Verrelli 2006). There is evidence suggesting that smallpox provided a selective advantage for CCR5d32 (Galvani and Slatkin 2003), indicating that there may be other selective advantages associated with the mutation. The mutation does not seem to present any significant disadvantages to the individuals other than an increased risk of West-Nile disease (Glass, McDermott et al. 2006). Such observations led to an interest in mimicking this natural mutation for HIV-infected individuals via genetic manipulation (i.e. transduction) of

It has been noted that the 32 nucleotide deletion results in 31 new amino acids being coded for, resulting in an active CCR5d32 protein. This protein instead of presenting as CCR5 receptors on the cell surface like the wild-type counterpart, CCR5d32 actually binds to and interacts with CXCR4 receptors (Agrawal, Lu et al. 2004), the other major coreceptor for HIV attachment. This provides an additional protection against HIV infection beyond the mere

in-vivo protective effects of gene-therapy against HIV.

(Zimmerman, Buckler-White et al. 1997).

cells vulnerable to HIV infection. (see below)

**2. Biological and clinical aspects of gene-therapy** 

absence of a functional CCR5 co-receptor, especially when concerning strains capable of utilizing the CXCR4 coreceptor (Agrawal, Lu et al. 2004; Jin, Agrawal et al. 2008). Further evidence towards the beneficial effect of the CCR5d32 protein comes from evidence that a polymorphism in the promoter region in CCR5-/- individuals can affect the protective capabilities of the d32 mutation. It has been demonstrated that an increase in CCR5d32 protein expression will improve resistance to HIV, while decreased CCR5d32 expression reduces the protective effect (Jin, Agrawal et al. 2008).

This CCR5d32 mutation has been successfully utilized in a patient, who suffered from both HIV/AIDS and leukaemia (Hutter, Nowak et al. 2009; Allers, Hutter et al. 2010). This individual, termed the "Berlin patient", had complete ablation of their immune system (to treat the leukemia) before matched allogeneic donor hematopoietic stem cells (HSC) homozygous for the CCR5d32 mutation were transfused into the patient. After one recurrence of leukaemia and a repeat of the treatment (ablation and reconstitution), the patient has had undetectable levels of HIV (and no recurring leukaemia) for more than 3 years without the use of any antiretroviral drugs (Hutter, Nowak et al. 2009; Allers, Hutter et al. 2010). This unique result of "functional" cure of HIV indicates significant potential for the use of gene-therapy to mimic this result by down-regulation of CCR5.

#### **2.2 Choosing a stage of HIV infection cycle to inhibit: Which therapeutic genes hold out promise?**

#### **2.2.1 Classes and methods of HIV inhibition**

Gene-therapy may be aimed to target various stages of the HIV infection cycle as shown in Figure 1. Class 1 therapy inhibits all steps prior to viral integration into the cellular genome, Class 2 inhibits expression of viral genes and Class 3 inhibits production of new virions once integration and expression has taken place (von Laer, Hasselmann et al. 2006). According to predictions from mathematical modelling, as discussed in section 3.1, Class 1 gene therapies are likely to be the most effective as they inhibit HIV at the first steps, and provide a selective advantage to these cells by avoiding any viral or immunological induced death from infection. Hence many gene therapeutics currently under investigation include components that impair attachment or fusion stages of the viral life-cycle (Symonds, Johnstone et al. 2010).

While all these classes are potential HIV gene therapeutics, practically, the use of multiple therapeutics in combination is likely to be the most effective method. This is analogous to the antiretroviral situation where it does not take long for HIV resistance to emerge against single antiretroviral drugs. These antiretroviral drugs have been shown to be far more effective when used in combination. It is for this reason that gene therapy research has often been focused on the use of multiple gene therapuetics used in conjunction with one-another. As well as the variety of targets being investigated, there is additionally a wide range of methods to achieve inhibition of these targets. The most commonly employed methods todate include the following:

**Antisense (Class 2):** Antisense RNA is a synthetic nucleotide sequence that binds to mRNA in order to inhibit its function. This method can be used against a wide range of targets, including the HIV envelope (Levine, Humeau et al. 2006).

**Aptamers (Class 2 or 3)**: Aptamers are single-stranded RNAs or DNAs. They disrupt at the protein level by tightly binding to their target ligand (Que-Gewirth and Sullenger 2007). Aptamers can be used to target a wide array of proteins and as such have potential to be used in multiple settings.

Cell-Delivered Gene Therapy for HIV 409

shRNA hairpin structure is cleaved by the cellular machinery into siRNA which is then bound to the RNA-induced silencing complex. This complex binds to and cleaves mRNAs which match the siRNA that is bound to it (Hannon and Rossi 2004). The use of siRNA for gene silencing has become a method of choice and can be potentially applied to many targets, including down-regulation of CCR5 that will decrease target cell infectivity by HIV and other host receptors as the removal or impairment of these receptors will render HIV

> Remove/prevent expression of CCR5

Remove/prevent expression of CD4

Remove/prevent expression of CXCR4

Prevent virion maturation

Prevent entry of HIV through host-cell membrane

Disrupt *tat* gene Tar decoy,

Disrupt *rev* gene siRNA, REV

Disrupt gag gene Ribozyme,

Zinc-finger, siRNA

Zinc-finger, siRNA

Zinc-finger, siRNA, ribozyme

siRNA, ribozyme

Antisense RNA,

siRNA

mutants

siRNA,

siRNA

**Class Target Site Why Goal How** 

co-receptor

co-receptor

Transcription

Important for

virion assembly

Table 1. A list of some HIV gene therapy targets, the goals and the mechanics of how they are being explored. This table shows a variety of Class 1, 2 and 3 therapies and the range of

**Fusion Inhibitors (Class 1):** One fusion inhibitor which has been researched in detail is the maC46 peptide (C46) (Zahn, Hermann et al. 2008). It inhibits viral fusion by interacting with the N-terminal hydrophobic alpha-helix. This prevents changes essential for membrane fusion of the virus and host cell. This fusion inhibitor has been found to be highly effective

**Zinc Finger Nucleases (ZFNs) (Class 1 or 2):** ZFNs bind to targeted open reading frames. Two juxtaposed ZFN's on DNA results in dimerisation of the endonuclease domains, generating a double-stranded break at the targeted DNA (Porteus and Carroll 2005). The

entry

virion Translation

virion maturation

at blocking HIV replication (Zahn, Hermann et al. 2008).

receptor for HIV attachment

Essential for viral

non-infectious (Class 1).

1 Membrane Fusion (HIV heptad repeat)

3 Env,

1 CCR5 Important

1 CD4 Essential

1 CXCR4 Important

2 Tat Important for

2 Rev Important for

3 Gag Important for

Protease, Helicase

approaches against targets.

Fig. 1. The three Classes of gene-therapy according to cycle of HIV infection inhibited as defined by von Laer et al(von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006). Class 1 inhibits all steps in the infection cycle prior to integration of the HIV RNA into the cellular genome. In particular, Class 1 inhibits either the entry of the HIV virion into the cell (i.e. inhibition of attachment/integration of the HIV virion through the CD4, CCR5 and X4 receptors/coreceptors) or inhibits integration into the cellular genome once a virion has entered the cell (i.e. blocks uncoating, reverse transcription or integration). Class 2 inhibits gene expression and the production of structural components required for the assembly of new HIV virions. Class 2 also results in lower susceptitibliy to cell death through the cytotoxic T lymphocyte (CTL) immune response, as a result of reduced recongnition via the Major Histocompatiblity Complex (MHC). Finally, Class 3 inhibits the assembly and export of virions from the infected cell.

**Intracellular Antibodies (Class 1 or 3):** Intracellular antibodies, or "intrabodies", are designed to bind to and inactivate target molecules inside host cells (Chen, Bagley et al. 1994). One target which has been used by intrabodies is CCR5, whereby the intrabodies bind to CCR5 and block surface expression (Rossi, June et al. 2007).

**Ribozymes (Class 2):** Ribozymes are catalytic RNA molecules that have the ability to degrade RNA in a sequence-specific manner (Sun, Wang et al. 1995). When used as anti-HIV agents, they have the potential to target multiple steps, affecting incoming RNA (during infection, in this sense they can act in part as Class1), primary RNA transcripts (from integrated provirus), spliced mRNAs and mature RNA being packaged into virions. These are primarily Class 2 inhibitors and examples are those designed to target the conserved regions of HIV such as the overlapping regions of *vpr* and *tat* reading frames (Mitsuyasu, Merigan et al. 2009). Highly conserved regions are desirable as targets so that sequence specificity is more likely to be maintained.

**Short hairpin RNA (Class 1 or 2):** Short hairpin RNA is a sequence of RNA that folds back upon itself in a hairpin turn; it can be used to initiate RNA interference and consequently silence gene expression (McIntyre and Fanning 2006). shRNA expression vectors utilise a promoter to drive expression of the shRNA. As an integrated vector, this expression cassette will be passed on to daughter cells, allowing the gene silencing to be maintained *in vivo*. The

Fig. 1. The three Classes of gene-therapy according to cycle of HIV infection inhibited as defined by von Laer et al(von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006). Class 1 inhibits all steps in the infection cycle prior to integration of the HIV RNA into the cellular genome. In particular, Class 1 inhibits either the entry of the HIV virion into the cell (i.e. inhibition of attachment/integration of the HIV virion through the CD4, CCR5 and X4 receptors/coreceptors) or inhibits integration into the cellular genome once a virion has entered the cell (i.e. blocks uncoating, reverse transcription or integration). Class 2 inhibits gene expression and the production of structural components required for the assembly of new HIV virions. Class 2 also results in lower susceptitibliy to cell death through the cytotoxic T lymphocyte (CTL) immune response, as a result of reduced recongnition via the Major Histocompatiblity Complex (MHC). Finally, Class 3 inhibits the assembly and export

**Intracellular Antibodies (Class 1 or 3):** Intracellular antibodies, or "intrabodies", are designed to bind to and inactivate target molecules inside host cells (Chen, Bagley et al. 1994). One target which has been used by intrabodies is CCR5, whereby the intrabodies bind

**Ribozymes (Class 2):** Ribozymes are catalytic RNA molecules that have the ability to degrade RNA in a sequence-specific manner (Sun, Wang et al. 1995). When used as anti-HIV agents, they have the potential to target multiple steps, affecting incoming RNA (during infection, in this sense they can act in part as Class1), primary RNA transcripts (from integrated provirus), spliced mRNAs and mature RNA being packaged into virions. These are primarily Class 2 inhibitors and examples are those designed to target the conserved regions of HIV such as the overlapping regions of *vpr* and *tat* reading frames (Mitsuyasu, Merigan et al. 2009). Highly conserved regions are desirable as targets so that sequence

**Short hairpin RNA (Class 1 or 2):** Short hairpin RNA is a sequence of RNA that folds back upon itself in a hairpin turn; it can be used to initiate RNA interference and consequently silence gene expression (McIntyre and Fanning 2006). shRNA expression vectors utilise a promoter to drive expression of the shRNA. As an integrated vector, this expression cassette will be passed on to daughter cells, allowing the gene silencing to be maintained *in vivo*. The

to CCR5 and block surface expression (Rossi, June et al. 2007).

of virions from the infected cell.

specificity is more likely to be maintained.

shRNA hairpin structure is cleaved by the cellular machinery into siRNA which is then bound to the RNA-induced silencing complex. This complex binds to and cleaves mRNAs which match the siRNA that is bound to it (Hannon and Rossi 2004). The use of siRNA for gene silencing has become a method of choice and can be potentially applied to many targets, including down-regulation of CCR5 that will decrease target cell infectivity by HIV and other host receptors as the removal or impairment of these receptors will render HIV non-infectious (Class 1).


Table 1. A list of some HIV gene therapy targets, the goals and the mechanics of how they are being explored. This table shows a variety of Class 1, 2 and 3 therapies and the range of approaches against targets.

**Fusion Inhibitors (Class 1):** One fusion inhibitor which has been researched in detail is the maC46 peptide (C46) (Zahn, Hermann et al. 2008). It inhibits viral fusion by interacting with the N-terminal hydrophobic alpha-helix. This prevents changes essential for membrane fusion of the virus and host cell. This fusion inhibitor has been found to be highly effective at blocking HIV replication (Zahn, Hermann et al. 2008).

**Zinc Finger Nucleases (ZFNs) (Class 1 or 2):** ZFNs bind to targeted open reading frames. Two juxtaposed ZFN's on DNA results in dimerisation of the endonuclease domains, generating a double-stranded break at the targeted DNA (Porteus and Carroll 2005). The

Cell-Delivered Gene Therapy for HIV 411

Potential for insertional

(Holt, Wang et al. 2010)

Slight risk of insertional

Slight increase in apoptosis of HSCs

Can only infect actively replicating cells (Roe, Reynolds et al. 1993) May induce insertional mutagenesis (Symonds, Johnstone et al. 2010)

Innate immune response (Liu and

mutagenesis (Philippe, Sarkis et al.

mutagenesis

Muruve 2003)

2006)

Higher transduction efficiency Risk of mutation/recombination

Table 2. A variety of commonly used delivery vectors and their associated advantages and

Nucleofection of DNA involves directly adding the DNA into the targeted cells by disrupting the cell membrane through electroporation. While this is not an ever-present biological vector as those mentioned above, it is more of an event-based vector which can provide a method of entry either for less entry-capable vectors, or plasmid DNA (Aluigi,

Viral delivery vectors are typically made from the backbone of suitable viruses, whereby pathogenic, and (often) replication-mediating genes are removed, and only the essential genes remain (Kootstra and Verma 2003). The therapeutic gene(s) being used is/are then added to the viral backbone. The virus is then able to infect host cells as would its natural counterpart. However without the ability to replicate or express harmful genes. It is used

One of the main concerns regarding gene therapies is the potential for insertional mutagenesis. This has been shown to occur in SCID-X1 trials (Howe, Mansour et al. 2008) where the insertional mutagenesis led to myeloproliferation/leukemia (Howe, Mansour et al. 2008). While insertional mutagenesis events have occurred in this and a few other gene therapy trials eg CGD (Stein, Ott et al. 2010), they have not occurred in HIV gene therapy trials, and a great deal of effort is undertaken to ensure that this event does not occur. To ensure efficient transcription of the therapeutic gene, a suitable promoter is required. A promoter is a region of DNA that facilitates the transcription of nearby downstream gene(s), and is essential for the efficient expression of the desired gene(s). The choice of the promoter to be used in gene therapies is highly important, and various promoters have been tested in laboratory studies and clinical trials. Promoters currently in use in HIV gene therapy studies

only to integrate into the host genome and allow the therapeutic gene to be active.

**Advantage Disadvantage** 

Transposons Can provide permanent expression of multiple genes

(Holt, Wang et al. 2010)

(Zhang, Sankar et al. 1998)

Boyd et al. 2005)

Adenovirus Can infect non-replicating cells

Lentivirus Can infect non-replicating cells

Treatment has been highly effective

Little/no adverse effects (Amado, Mitsuyasu et al. 2004; Macpherson,

(Zufferey, Dull et al. 1998), Does not effect proliferation or differentiation of HSCs (Gervaix, Schwarz et al. 1997)

**Delivery Vector** 

Plasmid DNA Nucleofection

Conditionally replicating virus

disadvantages.

Fogli et al. 2006).

Murine Leukaemia Virus

mutagenic pathways relied on to repair the DNA breaks result in nucleotide mutations at the break-sites, thus permanently disrupting the gene (Porteus and Carroll 2005). While experiments in mice have shown this method to be effective, there is still a risk of nontarget directed mutagenesis. Another limitation of this technique is the inability to add protective genes, as only the effective deletion/inactivation of genes can be performed, thus limiting the applications for the use of ZFNs to applications such as inactivation of CCR5 (Class 1).

#### **2.2.2 Strong arguments for gene-therapy based entry inhibition**

Class 1 inhibitors generally act at the level of HIV binding to the target cell (von Laer, Hasselmann et al. 2006). It is expected that this would be the most effective as HIV is blocked from entry to the target cell and any subsequent replication steps cannot take place. An important recent contribution to the argument for Class 1 inhibitors is the discovery of the cause of the so-called 'bystander effect' where apparently non HIV infected cells also succumb to HIV pathogenesis (Doitsh, Cavrois et al. 2010). The observation that productively infected cells are not the only contributors to host-cell death has been noted previously, however the cause of this cell death had remained unknown until Doitsh et al discovered abortive/nonproductive HIV infection in host-cells (approximately 95% of infected cells) and the induction of apoptosis in these cells (Doitsh, Cavrois et al. 2010). This "bystander effect" is likely to have contributed to the lack of success of some antiretroviral therapy methods, including a variety of clinical trials whereby HIV infection was only inhibited after HIV entry, as host-induced apoptosis would greatly reduce the effectiveness of treatment. This effect indicates a crucial additional benefit of entry-inhibitors over other classes of antiviral treatment.

One of the resistance mechanisms developed by HIV against the antiretroviral CCR5 antagonists, such as maraviroc, is not just the use of other co-receptors such as CXCR4, it is the use of maraviroc-bound CCR5 receptors (Westby, Smith-Burchnell et al. 2007). This mechanism of resistance would not be available against cells containing a down-regulation, or mutation-mediated deletion of the CCR5 receptor produced by gene therapy. An added bonus of the use of attachment and/or fusion inhibitors is that they do not provide crossresistance with other treatment methods such as protease and integration inhibitors.

#### **2.3 Vectors, delivery methods and promoters: Delivering the protective gene into the cell**

The therapeutic used in gene therapy must be carried within a suitable vector or delivery system; for HIV gene therapy these vectors should generally be capable of integrating into the host cells with minimal risk of generation of replication competent lentivirus or insertional mutagenesis (Wu, Wakefield et al. 2000; Symonds, Johnstone et al. 2010). The vector must also be non-toxic to the host while allowing the expression of the relevant gene(s). There are many techniques and delivery vectors which can be utilized for this purpose. Examples of the most commonly used delivery vectors are shown in Table 2.

Transposon-based delivery systems consist of a synthetic transposon and an associated transposase and work via a cut-and-paste mechanism whereby the transposase recognises the inverted direct sequences in the transposon, and then the transposon is excised and later integrated into a target DNA region (Tamhane and Akkina 2008). They can, for example, be used to carry shRNAs.

mutagenic pathways relied on to repair the DNA breaks result in nucleotide mutations at the break-sites, thus permanently disrupting the gene (Porteus and Carroll 2005). While experiments in mice have shown this method to be effective, there is still a risk of nontarget directed mutagenesis. Another limitation of this technique is the inability to add protective genes, as only the effective deletion/inactivation of genes can be performed, thus limiting the applications for the use of ZFNs to applications such as inactivation of

Class 1 inhibitors generally act at the level of HIV binding to the target cell (von Laer, Hasselmann et al. 2006). It is expected that this would be the most effective as HIV is blocked from entry to the target cell and any subsequent replication steps cannot take place. An important recent contribution to the argument for Class 1 inhibitors is the discovery of the cause of the so-called 'bystander effect' where apparently non HIV infected cells also succumb to HIV pathogenesis (Doitsh, Cavrois et al. 2010). The observation that productively infected cells are not the only contributors to host-cell death has been noted previously, however the cause of this cell death had remained unknown until Doitsh et al discovered abortive/nonproductive HIV infection in host-cells (approximately 95% of infected cells) and the induction of apoptosis in these cells (Doitsh, Cavrois et al. 2010). This "bystander effect" is likely to have contributed to the lack of success of some antiretroviral therapy methods, including a variety of clinical trials whereby HIV infection was only inhibited after HIV entry, as host-induced apoptosis would greatly reduce the effectiveness of treatment. This effect indicates a crucial additional benefit of entry-inhibitors over other

One of the resistance mechanisms developed by HIV against the antiretroviral CCR5 antagonists, such as maraviroc, is not just the use of other co-receptors such as CXCR4, it is the use of maraviroc-bound CCR5 receptors (Westby, Smith-Burchnell et al. 2007). This mechanism of resistance would not be available against cells containing a down-regulation, or mutation-mediated deletion of the CCR5 receptor produced by gene therapy. An added bonus of the use of attachment and/or fusion inhibitors is that they do not provide cross-

resistance with other treatment methods such as protease and integration inhibitors.

**2.3 Vectors, delivery methods and promoters: Delivering the protective gene into the** 

The therapeutic used in gene therapy must be carried within a suitable vector or delivery system; for HIV gene therapy these vectors should generally be capable of integrating into the host cells with minimal risk of generation of replication competent lentivirus or insertional mutagenesis (Wu, Wakefield et al. 2000; Symonds, Johnstone et al. 2010). The vector must also be non-toxic to the host while allowing the expression of the relevant gene(s). There are many techniques and delivery vectors which can be utilized for this purpose. Examples of the most commonly used delivery vectors are shown in Table 2. Transposon-based delivery systems consist of a synthetic transposon and an associated transposase and work via a cut-and-paste mechanism whereby the transposase recognises the inverted direct sequences in the transposon, and then the transposon is excised and later integrated into a target DNA region (Tamhane and Akkina 2008). They can, for example, be

**2.2.2 Strong arguments for gene-therapy based entry inhibition** 

CCR5 (Class 1).

classes of antiviral treatment.

used to carry shRNAs.

**cell** 


Table 2. A variety of commonly used delivery vectors and their associated advantages and disadvantages.

Nucleofection of DNA involves directly adding the DNA into the targeted cells by disrupting the cell membrane through electroporation. While this is not an ever-present biological vector as those mentioned above, it is more of an event-based vector which can provide a method of entry either for less entry-capable vectors, or plasmid DNA (Aluigi, Fogli et al. 2006).

Viral delivery vectors are typically made from the backbone of suitable viruses, whereby pathogenic, and (often) replication-mediating genes are removed, and only the essential genes remain (Kootstra and Verma 2003). The therapeutic gene(s) being used is/are then added to the viral backbone. The virus is then able to infect host cells as would its natural counterpart. However without the ability to replicate or express harmful genes. It is used only to integrate into the host genome and allow the therapeutic gene to be active.

One of the main concerns regarding gene therapies is the potential for insertional mutagenesis. This has been shown to occur in SCID-X1 trials (Howe, Mansour et al. 2008) where the insertional mutagenesis led to myeloproliferation/leukemia (Howe, Mansour et al. 2008). While insertional mutagenesis events have occurred in this and a few other gene therapy trials eg CGD (Stein, Ott et al. 2010), they have not occurred in HIV gene therapy trials, and a great deal of effort is undertaken to ensure that this event does not occur.

To ensure efficient transcription of the therapeutic gene, a suitable promoter is required. A promoter is a region of DNA that facilitates the transcription of nearby downstream gene(s), and is essential for the efficient expression of the desired gene(s). The choice of the promoter to be used in gene therapies is highly important, and various promoters have been tested in laboratory studies and clinical trials. Promoters currently in use in HIV gene therapy studies

Cell-Delivered Gene Therapy for HIV 413

The use of CD4+ T cells as target cells for HIV gene therapy has been explored in several studies (see section relating to clinical trials). Isolation and transduction of CD4+ T cells is relatively simple. The key advantage of targeting CD4+ T cells is the ease with which they may be accessed. As they largely populate and regularly traffic through peripheral blood, no stimulatory factors are required to mobilize them prior to collection. Conceptually it can be envisaged that the introduction of a protected population of CD4+ T lymphocytes should have impact as these are the cells specifically depleted by HIV infection; the greater the

One such study involving the therapy of CD4+ T cells was performed by Levine in 2006 (Levine, Humeau et al. 2006) whereby peripheral blood CD4+ T cells were harvested from each subject by apheresis. The collected samples were then depleted of CD8+ cells and monocytes, transduced with the gene construct *ex vivo*, activated via CD3 and CD28 costimulation and expanded before being re-infused into the patients. This method of therapy was shown to be both safe in treatment, and effective in delivery of the therapeutic

Predicted in-vivo dynamics of CD4+ T cell transduction, based on mathematical modelling,

Due to the range of cells which HIV infects, it is thought to be a significant advantage to transduce HSC, as these cells provide a continuous supply (following differentiation) into a range of immunological cells (monocytes, macrophages, CD4+ T cells, CD8+ cells, dendritic cells, microglial cells) which may thus be protected against HIV infection (Carter and Ehrlich 2008). A delay in the newly 'protected cell' production would be expected, thus delaying the effect of the therapeutic gene(s). However, there can still be a significant production of CD4+ T lymphocytes, the supply of which has been predicted to be a rate of approximately 1.65 cells/µL of blood/day (due to thymic reconstitution) (Murray, Kaufmann et al. 2003). This results in the production of a stable population of protected cells

While CD4+ T cells (and other cell types common in peripheral blood) can be obtained relatively simply prior to transduction by apheresis from peripheral blood, HSC must first be mobilised from the bone marrow (discussed in detail below). This creates an additional component to the treatment process. Currently the most common method for the mobilisation of HSC is the use of granulocyte colony stimulating factor (G-CSF), a treatment that usually spans 4-5 days before the apheresis of peripheral blood can begin. Predicted invivo dynamics of HSC transduced with a protective gene are discussed in section 3.2.2.

Current gene therapy protocols for HIV require the isolation of the relevant cells to be transduced, generally following apheresis (Symonds, Johnstone et al. 2010). Apheresis is the process of removing mononuclear cells from blood and returning neutrophils, platelets, plasma and red blood cells to the donor, in order to collect more of one particular part of the blood than could be separated from a unit of whole blood. Apheresis allows for the collection of large quantities of cells, and in the case of gene therapies for HIV, total

**2.5 Collection of cells for transduction: Apheresis and treatment methods for** 

severity of HIV infection the greater the CD4+ T lymphocyte decline.

gene (Levine, Humeau et al. 2006; Brunstein, Miller et al. 2011).

**2.4.2 Transduction of hematopoietic stem cells (HSC)** 

which could impact on CD4+ T cell number and viral load.

lymphocytes, CD4+ cells, or HSC are the cell types collected.

**optimized and high-volume cell collection** 

**2.4.1 Transduction of CD4+ T cells** 

are discussed in section 3.2.1.

are quite diverse and include U6 (human derived), T7 (bacteriophage derived), and Ubc (Human ubiquitin c) (Anderson, Banerjea et al. 2003; Boden, Pusch et al. 2003; Weber and Cannon 2007):

There have been studies using different promoters in HIV gene therapy work-up and many have been shown to be effective. However, due to the many different therapeutic genes, their delivery vectors, and the cells targeted for transduction, it is difficult to determine which promoters are the most effective and as such, each needs to be tested.

It has been noted that a highly expressive promoter may not be the ideal candidate, as many highly efficient promoters can have other side-effects. As noted above, of key concern is the trans-activation (insertional mutagenesis) of nearby cellular genes (Weber and Cannon 2007), potentially leading to oncogenic effects by over-expression of important proteins.

#### **2.4 Transduction targets: Which cells should be protected against HIV?**

HIV infection is typically characterized by CD4+ T cell infection and depletion. In addition, other cells are also infected by HIV, including macrophages and monocytes and most recently there have been reports of hematopoietic stem cell (HSC) infection (Stanley, Kessler et al. 1992; Carter, Onafuwa-Nuga et al. 2010; Carter, McNamara et al. 2011). In the case of gene therapy for HIV the two most common cell types that have been transduced to date with the therapeutic relevant gene are CD4+ T lymphocytes and HSC. Transduction of these cells is expected to provide the best outcome due to CD4+ T cells being the main targets of HIV infection and the ability of HSC to differentiate into all susceptible cells. In this subsection we discuss the biological aspects of transducing either CD4+ T cells or HSC with a protective gene.

Fig. 2. Two ways of achieving cell populations protected against HIV as a result of either transducing CD4+ T cells or HSC. If the CD4+ T cell population is transduced with a protective gene (left), then protection against HIV is only afforded to CD4+ T cells. If on the other hand HSC are transduced with a protective gene (right), then the protected gene is retained by all cells derived from the HSC via differentiation through the myeloid (e.g macrophage) and lymphoid lineages (e.g CD4+ T cell). The approach of transducing HSC thus provides protection against HIV to a broader class of cells.

are quite diverse and include U6 (human derived), T7 (bacteriophage derived), and Ubc (Human ubiquitin c) (Anderson, Banerjea et al. 2003; Boden, Pusch et al. 2003; Weber and

There have been studies using different promoters in HIV gene therapy work-up and many have been shown to be effective. However, due to the many different therapeutic genes, their delivery vectors, and the cells targeted for transduction, it is difficult to determine

It has been noted that a highly expressive promoter may not be the ideal candidate, as many highly efficient promoters can have other side-effects. As noted above, of key concern is the trans-activation (insertional mutagenesis) of nearby cellular genes (Weber and Cannon 2007), potentially leading to oncogenic effects by over-expression of

HIV infection is typically characterized by CD4+ T cell infection and depletion. In addition, other cells are also infected by HIV, including macrophages and monocytes and most recently there have been reports of hematopoietic stem cell (HSC) infection (Stanley, Kessler et al. 1992; Carter, Onafuwa-Nuga et al. 2010; Carter, McNamara et al. 2011). In the case of gene therapy for HIV the two most common cell types that have been transduced to date with the therapeutic relevant gene are CD4+ T lymphocytes and HSC. Transduction of these cells is expected to provide the best outcome due to CD4+ T cells being the main targets of HIV infection and the ability of HSC to differentiate into all susceptible cells. In this subsection we discuss the biological aspects of transducing either CD4+ T cells or HSC with

Fig. 2. Two ways of achieving cell populations protected against HIV as a result of either transducing CD4+ T cells or HSC. If the CD4+ T cell population is transduced with a protective gene (left), then protection against HIV is only afforded to CD4+ T cells. If on the other hand HSC are transduced with a protective gene (right), then the protected gene is retained by all cells derived from the HSC via differentiation through the myeloid (e.g macrophage) and lymphoid lineages (e.g CD4+ T cell). The approach of transducing HSC

thus provides protection against HIV to a broader class of cells.

which promoters are the most effective and as such, each needs to be tested.

**2.4 Transduction targets: Which cells should be protected against HIV?** 

Cannon 2007):

important proteins.

a protective gene.

#### **2.4.1 Transduction of CD4+ T cells**

The use of CD4+ T cells as target cells for HIV gene therapy has been explored in several studies (see section relating to clinical trials). Isolation and transduction of CD4+ T cells is relatively simple. The key advantage of targeting CD4+ T cells is the ease with which they may be accessed. As they largely populate and regularly traffic through peripheral blood, no stimulatory factors are required to mobilize them prior to collection. Conceptually it can be envisaged that the introduction of a protected population of CD4+ T lymphocytes should have impact as these are the cells specifically depleted by HIV infection; the greater the severity of HIV infection the greater the CD4+ T lymphocyte decline.

One such study involving the therapy of CD4+ T cells was performed by Levine in 2006 (Levine, Humeau et al. 2006) whereby peripheral blood CD4+ T cells were harvested from each subject by apheresis. The collected samples were then depleted of CD8+ cells and monocytes, transduced with the gene construct *ex vivo*, activated via CD3 and CD28 costimulation and expanded before being re-infused into the patients. This method of therapy was shown to be both safe in treatment, and effective in delivery of the therapeutic gene (Levine, Humeau et al. 2006; Brunstein, Miller et al. 2011).

Predicted in-vivo dynamics of CD4+ T cell transduction, based on mathematical modelling, are discussed in section 3.2.1.

#### **2.4.2 Transduction of hematopoietic stem cells (HSC)**

Due to the range of cells which HIV infects, it is thought to be a significant advantage to transduce HSC, as these cells provide a continuous supply (following differentiation) into a range of immunological cells (monocytes, macrophages, CD4+ T cells, CD8+ cells, dendritic cells, microglial cells) which may thus be protected against HIV infection (Carter and Ehrlich 2008). A delay in the newly 'protected cell' production would be expected, thus delaying the effect of the therapeutic gene(s). However, there can still be a significant production of CD4+ T lymphocytes, the supply of which has been predicted to be a rate of approximately 1.65 cells/µL of blood/day (due to thymic reconstitution) (Murray, Kaufmann et al. 2003). This results in the production of a stable population of protected cells which could impact on CD4+ T cell number and viral load.

While CD4+ T cells (and other cell types common in peripheral blood) can be obtained relatively simply prior to transduction by apheresis from peripheral blood, HSC must first be mobilised from the bone marrow (discussed in detail below). This creates an additional component to the treatment process. Currently the most common method for the mobilisation of HSC is the use of granulocyte colony stimulating factor (G-CSF), a treatment that usually spans 4-5 days before the apheresis of peripheral blood can begin. Predicted invivo dynamics of HSC transduced with a protective gene are discussed in section 3.2.2.

#### **2.5 Collection of cells for transduction: Apheresis and treatment methods for optimized and high-volume cell collection**

Current gene therapy protocols for HIV require the isolation of the relevant cells to be transduced, generally following apheresis (Symonds, Johnstone et al. 2010). Apheresis is the process of removing mononuclear cells from blood and returning neutrophils, platelets, plasma and red blood cells to the donor, in order to collect more of one particular part of the blood than could be separated from a unit of whole blood. Apheresis allows for the collection of large quantities of cells, and in the case of gene therapies for HIV, total lymphocytes, CD4+ cells, or HSC are the cell types collected.

Cell-Delivered Gene Therapy for HIV 415

Construct Results Reference

Preferential survival of cells with construct. Limited duration of engraftment.

More persistent engraftment compared with gold particle

delivery. No change

Gene marking in first months, then low or undetectable except in one patient when viral load increased. No serious adverse events.

Long term survival of cells at low level. Preferential survival of gene-containing cells in a patient with high

viral load.

days.

No adverse effects. 2 subjects' cells detected containing both the RRE and LN vectors on the day after cell infusion. All subsequent samples negative for the L-RRE-neo vector. Cells containing the control LN vector detected up to 330

CD4+ counts increased in 4/5 patients, viral loads stable, prolonged

engraftment. Well tolerated.

mobilization. Safe to date.

Trial 1 - 3/5 patients showed low-frequency marking of PBMC with ribozyme and vector backbone. Trial 2 – gene marked cells detected after infusion and to one year, and RNA expression

Transient vector

detected.

(Woffendin, Ranga et al. 1996)

(Ranga, Woffendin et al. 1998)

> (Podsakoff, Engel et al. 2005)

(Morgan, Walker et al. 2005)

(Kohn, Bauer et al. 1999; Bauer,

Selander et al.

2000)

(Levine, Humeau et al.

(Michienzi, Castanotto et al. 2003)

2006)

Target/Mechanism

Rev Inhibitory Rev protein,

Rev Inhibitory Rev protein,

particles

vector

vector

rev/TAR Trans-dominant rev with

RRE decoy Retroviral-mediated

Env antisense Single infusion of

rev/tat ribozyme tat and tat/rev ribozyme

ablation

Rev "Humanized" dominant-

Rev M10, delivered to CD4+ cells by gold

Rev M10, delivered to CD4+ cells by retroviral

negative REV protein

nontranslated marker gene (FX) as an internal control in retroviral

or without antisense TAR and control (neo) gene in CD4+ T lymphocytes

transfer of an RRE decoy gene into bone marrow

VRX496TM, a lentiviral construct encoding an antisense targeting HIV env, in CD4+ T cells

in CD34+cells in autologous CD34+ cells and empty vector backbone in two patient groups with and without

CD34+ cells

(huM10) and

of Action

It is common practice to use a stimulating agent such as G-CSF in order to increase the quantity of HSC in the peripheral blood. The resulting increase in cell numbers in peripheral blood is due to redistribution of cells from other compartments of the body (i.e bone marrow and lymph tissue). The use of G-CSF and other stimulating factors is essential when HSC (largely inhabiting the bone marrow) are to be transduced with the therapeutic gene. Various trials have shown HSC cell counts in peripheral blood increase 20-50-fold over the course of GCSF administration (Lane, Law et al. 1995; Law, Lane et al. 1999; Valgimigli, Rigolin et al. 2005).

Fig. 3. Illustration of the clinical aspects of therapeutic apheresis (for HSC harvesting), and the subsequent processes of transduction and reinfusion. The HIV infected individual is first administered G-CSF in order to effect mobilization of HSC from bone marrow into peripiheral blood. The mobilized HSC are then collected from peripheral blood and subsequently transduced with a protective gene. The transduced HSC are then reinfused into the patient.

A technique known as myeloablation has been utilized in some clinical trials (before the transduced cell infusion) in order to improve engraftment of the gene-containing cells (Strayer, Akkina et al. 2005). This procedure involves the killing of HSC, thereby reducing the endogenous non-transduced cells, thereby creating more space for the transduced cell population.

#### **2.6 Important studies involving gene-therapy: Promising results and insights**

Several mouse studies and clinical trials have been conducted in the area of HIV gene therapy, with several different therapuetic targets.

It is common practice to use a stimulating agent such as G-CSF in order to increase the quantity of HSC in the peripheral blood. The resulting increase in cell numbers in peripheral blood is due to redistribution of cells from other compartments of the body (i.e bone marrow and lymph tissue). The use of G-CSF and other stimulating factors is essential when HSC (largely inhabiting the bone marrow) are to be transduced with the therapeutic gene. Various trials have shown HSC cell counts in peripheral blood increase 20-50-fold over the course of GCSF administration (Lane, Law et al. 1995; Law, Lane et al. 1999;

Fig. 3. Illustration of the clinical aspects of therapeutic apheresis (for HSC harvesting), and the subsequent processes of transduction and reinfusion. The HIV infected individual is first

Transduction with Therapeutic Gene

A technique known as myeloablation has been utilized in some clinical trials (before the transduced cell infusion) in order to improve engraftment of the gene-containing cells (Strayer, Akkina et al. 2005). This procedure involves the killing of HSC, thereby reducing the endogenous non-transduced cells, thereby creating more space for the transduced cell

Several mouse studies and clinical trials have been conducted in the area of HIV gene

administered G-CSF in order to effect mobilization of HSC from bone marrow into peripiheral blood. The mobilized HSC are then collected from peripheral blood and subsequently transduced with a protective gene. The transduced HSC are then reinfused

**2.6 Important studies involving gene-therapy: Promising results and insights** 

therapy, with several different therapuetic targets.

Valgimigli, Rigolin et al. 2005).

into the patient.

population.


Cell-Delivered Gene Therapy for HIV 417

Table 3. (Apapted from Symonds et al (Symonds, Johnstone et al. 2010)): A list of HIV gene therapy clinical trials and their outcomes. Each of these studies vary in their gene therapy

To date, as shown in Table 3, several different gene therapies have entered Phase 1 clinical trials, (and some into Phase 2) indicating the safety of a range of HIV gene therapeutics including

**3. Protective effects of anti-HIV gene-therapy: Predictions from mathematical** 

In gene-therapy research, mathematical modelling has been employed to predict the protective effects of anti-HIV gene-therapy (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006). In this section we review current results on mathematical modelling, with respect to predictions of the *in-vivo* anti-HIV protective effects. Mathematical models deal with the complex interactions between gene-therapy, the immune system and HIV infection (Perelson, Essunger et al. 1997). Given the relative sparsity of current clinical trial data of gene-therapy for HIV, and the long time-spans over which predictions are to be made (i.e. over many years) mathematical modelling can provide predictions on the likely *in-vivo* effectiveness of current and future gene-therapies. Modelling work to-date has led to important insights regarding key design factors as well as parameters that should be optimized in order to maximize the effectiveness of therapy (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006). In this section we review

As discussed previously, three different broad stages of the HIV infection cycle may be targeted for inhibition of HIV infection (Figure 1) with the inhibitors referred to as Class 1, 2, and 3 (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006). It is of interest whether inhibiting earlier stages (via Class 1), intermediate stages (via Class 2) or later stages (via Class 3) of the infection cycle might provide maximum effectiveness of the therapy. Is it more desirable to prevent HIV entry and integration into the cellular genome via Class 1, to inhibit the production of structural components for HIV assembly via Class 2, or to inhibit the assembly/export of new HIV virions via Class 3? This question has been addressed by a number of investigators (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006; Applegate, Birkett et al. 2010), subject to a variety of modelling assumptions reflecting differing levels of complexity of the interaction between HIV, gene-

antisense, ribozymes, decoys, intracellular antibodies and zinc fingers targeting CCR5.

Autologous infusion of CD4+ and CD8+ T cells modified by CD4ζ in a murine moloney leukaemia virus

backbone given +/- IL-2.

**3.1 Why is Class 1 gene-therapy the most promising approach?** 

target, and method of targeting the specific region.

Construct Results Reference

rectal biopsy.

Gene-modified cells

followed and detected to 12 months with no difference due to IL-2. No significant change in plasma viral load. CD4ζ signal detected in

(Mitsuyasu, Anton et al. 2000)

Target/Mechanism

Modified T-cell

of Action

receptor

**modeling** 

current results and key insights.

therapy and the immune system.


Construct Results Reference

cell dose

lymphocytes.

de novo production of myeloid and lymphoid cells. Degree of persistence of gene-containing cells dependent on transduced

Safe and feasible procedure. Long-term survival of genetically modified T-

No significant difference mean plasma viral load at primary end-point but lower

TWAUC. No safety

Increased CD4. No significant change in viral load (except after treatment change). Modified cells detected at one year. Low level marking. No major

Preliminary data on 1 patient only ZFN-modified cells persisted in circulation and observed in GALT. Suggested delay in return of viral load after structured treatment interruption.

Engraftment by 11 days. Low levels of gene marking observed up to 24 months as was expression of siRNA

concerns.

toxicity

and CCR

(Amado, Mitsuyasu et al. 1999; Amado, Mitsuyasu et al. 2004)

(Macpherson, Boyd et al. 2005)

(Mitsuyasu, Merigan et al.

(van Lunzen, Glaunsinger et al. 2007)

(2009)

(DiGiusto, Krishnan et al.

2010)

2009)

Target/Mechanism

tat/vpr ribozyme Phase I study: Moloney

HPSC

lymphocytes

CD34+ cells

cells.

CCR5 CCR5-specific zinc finger

membrane anchored peptide C46 fusion inhibitor delivered by retroviral vector in T

nuclease based product, SB-728-T, in autologous CD4+ T cells. Two phase 1 trials with various dosing regimens in different patient groups.

Tat/rev short hairpin RNA, TAR decoy and CCR5 ribozyme expressed from a selfinactivating lentiviral vector transduced in CD34+ cells, along with standard unmanipulated HPCs in 4 patients with HIV and non-Hodgkin's

lymphoma

tat/vpr ribozyme Phase II study: Moloney

tat/vpr ribozyme Retroviral vector

Fusion inhibitor Gene encoding

Tat/rev, CCR5, TAR decoy

murine leukaemia retroviral vector encoding a ribozyme vs control LNL6 vector in CD34+

encoding a ribozyme vs control LNL6 vector to transduce T lymphocytes, predominantly CD4+ T

murine leukaemia virusbased, replicationincompetent gamma retroviral vector with gene encoding a ribozyme vs placebo in

of Action


Table 3. (Apapted from Symonds et al (Symonds, Johnstone et al. 2010)): A list of HIV gene therapy clinical trials and their outcomes. Each of these studies vary in their gene therapy target, and method of targeting the specific region.

To date, as shown in Table 3, several different gene therapies have entered Phase 1 clinical trials, (and some into Phase 2) indicating the safety of a range of HIV gene therapeutics including antisense, ribozymes, decoys, intracellular antibodies and zinc fingers targeting CCR5.

#### **3. Protective effects of anti-HIV gene-therapy: Predictions from mathematical modeling**

In gene-therapy research, mathematical modelling has been employed to predict the protective effects of anti-HIV gene-therapy (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006). In this section we review current results on mathematical modelling, with respect to predictions of the *in-vivo* anti-HIV protective effects. Mathematical models deal with the complex interactions between gene-therapy, the immune system and HIV infection (Perelson, Essunger et al. 1997). Given the relative sparsity of current clinical trial data of gene-therapy for HIV, and the long time-spans over which predictions are to be made (i.e. over many years) mathematical modelling can provide predictions on the likely *in-vivo* effectiveness of current and future gene-therapies. Modelling work to-date has led to important insights regarding key design factors as well as parameters that should be optimized in order to maximize the effectiveness of therapy (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006). In this section we review current results and key insights.

#### **3.1 Why is Class 1 gene-therapy the most promising approach?**

As discussed previously, three different broad stages of the HIV infection cycle may be targeted for inhibition of HIV infection (Figure 1) with the inhibitors referred to as Class 1, 2, and 3 (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006). It is of interest whether inhibiting earlier stages (via Class 1), intermediate stages (via Class 2) or later stages (via Class 3) of the infection cycle might provide maximum effectiveness of the therapy. Is it more desirable to prevent HIV entry and integration into the cellular genome via Class 1, to inhibit the production of structural components for HIV assembly via Class 2, or to inhibit the assembly/export of new HIV virions via Class 3? This question has been addressed by a number of investigators (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006; Applegate, Birkett et al. 2010), subject to a variety of modelling assumptions reflecting differing levels of complexity of the interaction between HIV, genetherapy and the immune system.

Cell-Delivered Gene Therapy for HIV 419

setting remain to be elucidated. Consequently, investigators have turned to predictions from mathematical modelling in order to shed light on the in-vivo dynamics of the two approaches. In this section, we review the predictions from such modelling work to-date.

**3.2.1 Transducing CD4+ T cells with a protective gene: Can we achieve establishment of a sufficiently large and sufficiently "receptor-diverse" CD4+ T cell population that** 

Expansion of numbers of CD4+ T cells containing a protective gene is subject to the ratelimiting step of homeostatic cell division and proliferation (von Laer, Hasselmann et al. 2006). Thus it is important to determine how quickly a substantial CD4+ T cell population could expand from a small initial fraction of protected cells. Such considerations are motivated by the fact that it is currently feasible and practical to transduce only a portion of the total CD4+ T cell population (Dropulic and June 2006; von Laer, Baum et al. 2009), so that expansion of the protected CD4+ T cell population will have to rely on in vivo

While modelling has shown that a small fraction of initially transduced cells could potentially result in significant expansion of the protected CD4+ T cell population, reductions of viral load, and also a delay in the onset of AIDS (Lund, Lund et al. 1997; Leonard and Schaffer 2006; von Laer, Hasselmann et al. 2006; Aviran, Shah et al. 2010), most of these models have assumed a strong feedback mechanism upregulating cellular proliferation when numbers fall below a normal level. Whereas such homeostatic mechanisms are believed to contribute to the maintenance of T cell numbers in healthy individuals (Khaled and Durum 2002), the speed with which they occur is likely to be significantly slower in practice. Current clinical trials have not produced CD4+ T cell expansions at rates as fast as predicted by mathematical modelling (Dropulic and June 2006;

Current estimates of T lymphocyte division put the normal rate at approximately 1 division every 3.5 years for naive T cells and 1 division every 22 weeks for memory T cells (McLean and Michie 1995). If the transduced CD4+ T cells are to expand in vivo, then such time-scales should provide an indication of the slow nature of any in vivo expansion of the transduced CD4+ T cell population unless driven by strong selective

More realistic upper bounds on rates of CD4+ T cell expansion in-vivo under gene-therapy may be obtained by consideration of CD4+ T cell reconstitution on HAART (Byakwaga, Murray et al. 2009). Reconstitution of the CD4+ T cell population under HAART appears relatively slow with average increases of approximately 300 cells/µL observed after about 6 years (Byakwaga, Murray et al. 2009). Given that reconstitution on HAART usually only takes place under complete viral suppression (as opposed to gene-therapy where a measurable viral population may be present), it appears likely that the expansion rates of the protected CD4+ T cell population under gene-therapy may be substantially slower. Unlike the situation with HAART high viral levels may be preferable in early stages of gene therapy to act as a driving force for the expansion of a protected CD4+ T cell population via

Several additional factors might further inhibit the expansion of the protected CD4+ T cell population in-vivo. First, unless sufficient selective survival advantage is conferred to the protected CD4+ T cell population, the protected cell population might not expand

**is protected against HIV?** 

von Laer, Baum et al. 2009).

pressure by HIV.

a selective mechanism.

mechanisms.

Investigations to-date have demonstrated that Class 1 protection appears to be highly desirable in terms of reducing viral loads and increasing CD4+ T cell counts (von Laer, Hasselmann et al. 2006; Applegate, Birkett et al. 2010; Aviran, Shah et al. 2010). The underlying reason for the superiority of Class 1 therapy (over Class 2 and Class 3) has been attributed to the high selective advantage of the protected cell population conferred by Class 1 inhibition (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006; Applegate, Birkett et al. 2010). Since Class 1 inhibits all steps prior to viral integration into the cellular genome (Figure 1), any cell containing the protective gene is less likely to be infected than a non-protected cell. Consequently, Class 1 promotes the survival and expansion of the protected non-infected cells, whereas the non-protected cells are more prone to infection and selective killing through cytopathic effects associated either with the virus or the CTL immune response (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006).

In contrast to Class 1 agents, Class 2 and Class 3 therapies have been shown to require much higher degrees of inhibition in order to achieve clinically significant effects (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006; Applegate, Birkett et al. 2010). Class 2 inhibits cytopathic effects associated with the viral infection and the CTL immune response (Figure 1). Any infected cell with Class 2 protection is therefore longer-lived and also has a reduced viral production rate compared to an unprotected and infected cell (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006; Applegate, Birkett et al. 2010). Class 2 consequently confers a selective survival advantage to the infected cells containing the protective gene relative to other infected cells, but not to non-infected cells containing the protective gene (as is the case with Class 1). In contrast, Class 3 only inhibits the export of new HIV virions from an infected cell and thus provides minimal selective advantage (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006). Hence Class 1 is the only class that confers a selective survival advantage to non-infected cells containing the protective gene (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006; Applegate, Birkett et al. 2010).

Collectively therefore, modelling work to-date implies that Class 1 is essential due to the selective survival advantage conferred to the protected and non-infected cells. Still, it is important to note that augmenting Class 1 with Class 2 and/or Class 3 protection might further increase the effectiveness of therapy (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006; Applegate, Birkett et al. 2010). Recent findings relating to the "Bystander Effect", as discussed previously in section 2.2.2, have lent further support to arguments relating to Class 1 inhibition (Doitsh, Cavrois et al. 2010), as abortive infections (HIV virion enters the cell, but does not integrate into cellular genome) comprise 95% of all cell death resulting from HIV infection.

#### **3.2 Two different transduction approaches: To transduce CD4+ T cells or HSC with a protective gene?**

As discussed previously in section 2.4, it is possible to either transduce CD4+ T cells with a protective gene (for an immediately protected population of CD4+ T cells) or to transduce HSC, that provide protection to CD4+ T cells following differentiation through the lymphoid line and to monocyte/macrophages following differentiation throught the myeloid line. While the relative merits of each approach have attracted substantial interest, the long-term quantitative advantages and disadvantages of each approach in the clinical

Investigations to-date have demonstrated that Class 1 protection appears to be highly desirable in terms of reducing viral loads and increasing CD4+ T cell counts (von Laer, Hasselmann et al. 2006; Applegate, Birkett et al. 2010; Aviran, Shah et al. 2010). The underlying reason for the superiority of Class 1 therapy (over Class 2 and Class 3) has been attributed to the high selective advantage of the protected cell population conferred by Class 1 inhibition (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006; Applegate, Birkett et al. 2010). Since Class 1 inhibits all steps prior to viral integration into the cellular genome (Figure 1), any cell containing the protective gene is less likely to be infected than a non-protected cell. Consequently, Class 1 promotes the survival and expansion of the protected non-infected cells, whereas the non-protected cells are more prone to infection and selective killing through cytopathic effects associated either with the virus or the CTL immune response (von Laer, Hasselmann et al. 2006; von Laer,

In contrast to Class 1 agents, Class 2 and Class 3 therapies have been shown to require much higher degrees of inhibition in order to achieve clinically significant effects (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006; Applegate, Birkett et al. 2010). Class 2 inhibits cytopathic effects associated with the viral infection and the CTL immune response (Figure 1). Any infected cell with Class 2 protection is therefore longer-lived and also has a reduced viral production rate compared to an unprotected and infected cell (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006; Applegate, Birkett et al. 2010). Class 2 consequently confers a selective survival advantage to the infected cells containing the protective gene relative to other infected cells, but not to non-infected cells containing the protective gene (as is the case with Class 1). In contrast, Class 3 only inhibits the export of new HIV virions from an infected cell and thus provides minimal selective advantage (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006). Hence Class 1 is the only class that confers a selective survival advantage to non-infected cells containing the protective gene (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et

Collectively therefore, modelling work to-date implies that Class 1 is essential due to the selective survival advantage conferred to the protected and non-infected cells. Still, it is important to note that augmenting Class 1 with Class 2 and/or Class 3 protection might further increase the effectiveness of therapy (von Laer, Hasselmann et al. 2006; von Laer, Hasselmann et al. 2006; Applegate, Birkett et al. 2010). Recent findings relating to the "Bystander Effect", as discussed previously in section 2.2.2, have lent further support to arguments relating to Class 1 inhibition (Doitsh, Cavrois et al. 2010), as abortive infections (HIV virion enters the cell, but does not integrate into cellular genome) comprise 95% of all

**3.2 Two different transduction approaches: To transduce CD4+ T cells or HSC with a** 

As discussed previously in section 2.4, it is possible to either transduce CD4+ T cells with a protective gene (for an immediately protected population of CD4+ T cells) or to transduce HSC, that provide protection to CD4+ T cells following differentiation through the lymphoid line and to monocyte/macrophages following differentiation throught the myeloid line. While the relative merits of each approach have attracted substantial interest, the long-term quantitative advantages and disadvantages of each approach in the clinical

Hasselmann et al. 2006).

al. 2006; Applegate, Birkett et al. 2010).

cell death resulting from HIV infection.

**protective gene?** 

setting remain to be elucidated. Consequently, investigators have turned to predictions from mathematical modelling in order to shed light on the in-vivo dynamics of the two approaches. In this section, we review the predictions from such modelling work to-date.

#### **3.2.1 Transducing CD4+ T cells with a protective gene: Can we achieve establishment of a sufficiently large and sufficiently "receptor-diverse" CD4+ T cell population that is protected against HIV?**

Expansion of numbers of CD4+ T cells containing a protective gene is subject to the ratelimiting step of homeostatic cell division and proliferation (von Laer, Hasselmann et al. 2006). Thus it is important to determine how quickly a substantial CD4+ T cell population could expand from a small initial fraction of protected cells. Such considerations are motivated by the fact that it is currently feasible and practical to transduce only a portion of the total CD4+ T cell population (Dropulic and June 2006; von Laer, Baum et al. 2009), so that expansion of the protected CD4+ T cell population will have to rely on in vivo mechanisms.

While modelling has shown that a small fraction of initially transduced cells could potentially result in significant expansion of the protected CD4+ T cell population, reductions of viral load, and also a delay in the onset of AIDS (Lund, Lund et al. 1997; Leonard and Schaffer 2006; von Laer, Hasselmann et al. 2006; Aviran, Shah et al. 2010), most of these models have assumed a strong feedback mechanism upregulating cellular proliferation when numbers fall below a normal level. Whereas such homeostatic mechanisms are believed to contribute to the maintenance of T cell numbers in healthy individuals (Khaled and Durum 2002), the speed with which they occur is likely to be significantly slower in practice. Current clinical trials have not produced CD4+ T cell expansions at rates as fast as predicted by mathematical modelling (Dropulic and June 2006; von Laer, Baum et al. 2009).

Current estimates of T lymphocyte division put the normal rate at approximately 1 division every 3.5 years for naive T cells and 1 division every 22 weeks for memory T cells (McLean and Michie 1995). If the transduced CD4+ T cells are to expand in vivo, then such time-scales should provide an indication of the slow nature of any in vivo expansion of the transduced CD4+ T cell population unless driven by strong selective pressure by HIV.

More realistic upper bounds on rates of CD4+ T cell expansion in-vivo under gene-therapy may be obtained by consideration of CD4+ T cell reconstitution on HAART (Byakwaga, Murray et al. 2009). Reconstitution of the CD4+ T cell population under HAART appears relatively slow with average increases of approximately 300 cells/µL observed after about 6 years (Byakwaga, Murray et al. 2009). Given that reconstitution on HAART usually only takes place under complete viral suppression (as opposed to gene-therapy where a measurable viral population may be present), it appears likely that the expansion rates of the protected CD4+ T cell population under gene-therapy may be substantially slower. Unlike the situation with HAART high viral levels may be preferable in early stages of gene therapy to act as a driving force for the expansion of a protected CD4+ T cell population via a selective mechanism.

Several additional factors might further inhibit the expansion of the protected CD4+ T cell population in-vivo. First, unless sufficient selective survival advantage is conferred to the protected CD4+ T cell population, the protected cell population might not expand

Cell-Delivered Gene Therapy for HIV 421

Transducing HSC with a protective gene has two distinct advantages. First, the export of protected de-novo CD4+ T cells from the thymus results in a diversification of the T cell receptor repertoire (Allen, Turner et al. 2011; Wiegers, Kaufmann et al. 2011). Consequently the expanded CD4+ T cell population containing the protective gene exhibits more "extensive" TCR coverage over time, reducing the risk that pathogens might evade the immune response (Nikolich-Zugich, Slifka et al. 2004; Allen, Turner et al. 2011; Wiegers, Kaufmann et al. 2011). Secondly, HSC differentiate into a broad range of cells (besides CD4+ T cells), including macrophages that are susceptible to HIV infection and that may represent important latent HIV reservoirs (Chun, Carruth et al. 1997; Chun, Stuyver et al. 1997; Crowe and Sonza 2000). Consequently, HSC transduction provides protection against HIV to a

The transduction of HSC does not immediately provide a protected population of CD4+ T cells in the periphery, but rather the protected CD4+ T cell population is established relatively slowly as HSC differentiate and are exported from the thymus (Symonds, Johnstone et al. 2010). Thymic production of CD4+ T cells has been estimated at approximately 1.65 cells/µL/day (Murray, Kaufmann et al. 2003) in peripheral blood. Assuming that a percentage *P* of total HSC in the bone marrow is transduced, then one would correspondingly expect that CD4+ T cells containing the protective gene would be exported at a rate of 1.65 x *P* cells/µL/day from the thymus (Murray, Fanning et al. 2009). Such numbers provide estimates of rates at which the establishment of a protected CD4+ T

Achieving high engraftment efficiencies of HSC in the bone marrow is important. While a number of clinical trials in which HSC were transduced reported indications of clinical effect against HIV (Symonds, Johnstone et al. 2010), engraftment percentages in the bone marrow have been relatively low (Dropulic and June 2006; Mitsuyasu, Merigan et al. 2009; von Laer, Baum et al. 2009; Symonds, Johnstone et al. 2010). Such results underscore the need for more effective methods of cell harvesting, transduction and homing, that achieve higher engraftment efficiencies. Increased engraftment percentages should lead to more substantial clinical effects in terms of protection against HIV (Mitsuyasu, Merigan et al. 2009; Murray,

Despite relatively low engraftment efficiencies to-date, it is of practical interest for future research directions to determine what engraftment percentages might suffice for clinically meaningful effects of the therapy. This question was addressed in recent modelling work (Murray, Fanning et al. 2009), that considered HSC transduction with a *tat-vpr* specific anti-HIV ribozyme (OZ1) employed in a recent phase 2 clinical trial (Mitsuyasu, Merigan et al. 2009). Under the assumption that 20% of all HSC in the bone marrow are transduced (i.e. engraftment percentage *P* = 20%), and that correspondingly 20% of CD4+ T cells exported from the thymus contain the protective gene, the modelling predicted reductions of 0.5 *log10* in viral load for a HAART-naive individual after 1 year (Figure 4 A). Benefits in terms of forestalment of onset of AIDS at 8 years post-infection were also estimated (Figure 4 B). Slighly less prononunced effects were observed for patients that were concurrently enrolled on HAART (Murray, Fanning et al. 2009). Such results are encouraging and indicate that relatively modest engraftment percentages could achieve a clinically relevant effect.

**3.3 Resistance development under gene-therapy: How does it differ from HAART?**  Systemic antiretroviral therapy bathes each cell in some level of the drugs being used depending on the penetration of the individual drugs to that region of the body, their

broader class of cells than just CD4+ T cells.

Fanning et al. 2009).

cell population might take place in-vivo in peripheral blood.

Consequently full bone marrow ablation may be unnecessary.

substantially in-vivo. Second, the transduced CD4+ T-cells might have increased death rates or decreased proliferative ability due to interference of the protective gene with normal cell functionality (Dropulic and June 2006; von Laer, Baum et al. 2009; Tayi, Bowen et al. 2010). Third, the unprotected de-novo CD4+ T-cells exported from the thymus might effectively dilute the transduced CD4+ T-cells in the periphery (Aviran, Shah et al. 2010). This latter problem may potentially be addressed by subsequent "booster" treatments involving repeated infusions of transduced CD4+ T cells or by also using HSC.

An additional disadvantage associated with the direct transduction of CD4+ T cells is that peripheral expansion of their number does not necessarily correspond to an equivalent expansion in the T cell repertoire (Nikolich-Zugich, Slifka et al. 2004; Allen, Turner et al. 2011; Wiegers, Kaufmann et al. 2011). This is important as any resulting "gaps" in the T cell repertoire may result in increased probability of immune system evasion by pathogens and consequently in increased risk of infection or morbidity (Nikolich-Zugich, Slifka et al. 2004; Allen, Turner et al. 2011; Wiegers, Kaufmann et al. 2011).

Hence although direct transduction of CD4+ T cells results in a faster appearance in peripheral blood of a protected component of this susceptible population, there may be disadvantages in that these may not provide a diverse immune response and other cell populations will not protected.

#### **3.2.2 Transducing HSC with a protective gene: Increasing T cell receptor repertoire and broadening class of protected cells**

An alternative to transducing CD4+ T cells directly is to instead transduce HSC. In this case, the production of de-novo CD4+ T cells containing the protective gene occurs as a result of HSC differentiation through the lymphoid line and subsequent export from the thymus (Symonds, Johnstone et al. 2010).

Fig. 4. Modelling predictions by Murray et al.(Murray, Fanning et al. 2009) regarding comparison of the scenario that 20% of all HSC in the bone marrow are transduced with a tat-vpr specific anti-HIV ribozyme (OZ1) versus the scenario that no gene-therapy treatment is received. Reproduced with permission from Murray et al.(Murray, Fanning et al. 2009). The patient was assumed HAART-naive. The time-scale on the horizontal ordinate denotes the time since receiving gene-therapy at year 0. (A) Treatment with OZ1, log10 HIV RNA copies/ml (solid line); No treatment, log10 HIV RNA copies/ml (dashed line). (B) Treatment with OZ1, total CD4+ T lymphocytes/mm3 (solid line), OZ1+CD4+ T lymphocytes/mm3 (dash–dot line); No treatment , total CD4+ T lymphocytes/mm3 (dashed line).

substantially in-vivo. Second, the transduced CD4+ T-cells might have increased death rates or decreased proliferative ability due to interference of the protective gene with normal cell functionality (Dropulic and June 2006; von Laer, Baum et al. 2009; Tayi, Bowen et al. 2010). Third, the unprotected de-novo CD4+ T-cells exported from the thymus might effectively dilute the transduced CD4+ T-cells in the periphery (Aviran, Shah et al. 2010). This latter problem may potentially be addressed by subsequent "booster" treatments involving

An additional disadvantage associated with the direct transduction of CD4+ T cells is that peripheral expansion of their number does not necessarily correspond to an equivalent expansion in the T cell repertoire (Nikolich-Zugich, Slifka et al. 2004; Allen, Turner et al. 2011; Wiegers, Kaufmann et al. 2011). This is important as any resulting "gaps" in the T cell repertoire may result in increased probability of immune system evasion by pathogens and consequently in increased risk of infection or morbidity (Nikolich-Zugich, Slifka et al. 2004;

Hence although direct transduction of CD4+ T cells results in a faster appearance in peripheral blood of a protected component of this susceptible population, there may be disadvantages in that these may not provide a diverse immune response and other cell

**3.2.2 Transducing HSC with a protective gene: Increasing T cell receptor repertoire** 

Fig. 4. Modelling predictions by Murray et al.(Murray, Fanning et al. 2009) regarding comparison of the scenario that 20% of all HSC in the bone marrow are transduced with a tat-vpr specific anti-HIV ribozyme (OZ1) versus the scenario that no gene-therapy treatment is received. Reproduced with permission from Murray et al.(Murray, Fanning et al. 2009). The patient was assumed HAART-naive. The time-scale on the horizontal ordinate denotes the time since receiving gene-therapy at year 0. (A) Treatment with OZ1, log10 HIV RNA copies/ml (solid line); No treatment, log10 HIV RNA copies/ml (dashed line). (B) Treatment with OZ1, total CD4+ T lymphocytes/mm3 (solid line), OZ1+CD4+ T lymphocytes/mm3

(dash–dot line); No treatment , total CD4+ T lymphocytes/mm3 (dashed line).

An alternative to transducing CD4+ T cells directly is to instead transduce HSC. In this case, the production of de-novo CD4+ T cells containing the protective gene occurs as a result of HSC differentiation through the lymphoid line and subsequent export from the thymus

repeated infusions of transduced CD4+ T cells or by also using HSC.

Allen, Turner et al. 2011; Wiegers, Kaufmann et al. 2011).

populations will not protected.

(Symonds, Johnstone et al. 2010).

**and broadening class of protected cells** 

Transducing HSC with a protective gene has two distinct advantages. First, the export of protected de-novo CD4+ T cells from the thymus results in a diversification of the T cell receptor repertoire (Allen, Turner et al. 2011; Wiegers, Kaufmann et al. 2011). Consequently the expanded CD4+ T cell population containing the protective gene exhibits more "extensive" TCR coverage over time, reducing the risk that pathogens might evade the immune response (Nikolich-Zugich, Slifka et al. 2004; Allen, Turner et al. 2011; Wiegers, Kaufmann et al. 2011). Secondly, HSC differentiate into a broad range of cells (besides CD4+ T cells), including macrophages that are susceptible to HIV infection and that may represent important latent HIV reservoirs (Chun, Carruth et al. 1997; Chun, Stuyver et al. 1997; Crowe and Sonza 2000). Consequently, HSC transduction provides protection against HIV to a broader class of cells than just CD4+ T cells.

The transduction of HSC does not immediately provide a protected population of CD4+ T cells in the periphery, but rather the protected CD4+ T cell population is established relatively slowly as HSC differentiate and are exported from the thymus (Symonds, Johnstone et al. 2010). Thymic production of CD4+ T cells has been estimated at approximately 1.65 cells/µL/day (Murray, Kaufmann et al. 2003) in peripheral blood. Assuming that a percentage *P* of total HSC in the bone marrow is transduced, then one would correspondingly expect that CD4+ T cells containing the protective gene would be exported at a rate of 1.65 x *P* cells/µL/day from the thymus (Murray, Fanning et al. 2009). Such numbers provide estimates of rates at which the establishment of a protected CD4+ T cell population might take place in-vivo in peripheral blood.

Achieving high engraftment efficiencies of HSC in the bone marrow is important. While a number of clinical trials in which HSC were transduced reported indications of clinical effect against HIV (Symonds, Johnstone et al. 2010), engraftment percentages in the bone marrow have been relatively low (Dropulic and June 2006; Mitsuyasu, Merigan et al. 2009; von Laer, Baum et al. 2009; Symonds, Johnstone et al. 2010). Such results underscore the need for more effective methods of cell harvesting, transduction and homing, that achieve higher engraftment efficiencies. Increased engraftment percentages should lead to more substantial clinical effects in terms of protection against HIV (Mitsuyasu, Merigan et al. 2009; Murray, Fanning et al. 2009).

Despite relatively low engraftment efficiencies to-date, it is of practical interest for future research directions to determine what engraftment percentages might suffice for clinically meaningful effects of the therapy. This question was addressed in recent modelling work (Murray, Fanning et al. 2009), that considered HSC transduction with a *tat-vpr* specific anti-HIV ribozyme (OZ1) employed in a recent phase 2 clinical trial (Mitsuyasu, Merigan et al. 2009). Under the assumption that 20% of all HSC in the bone marrow are transduced (i.e. engraftment percentage *P* = 20%), and that correspondingly 20% of CD4+ T cells exported from the thymus contain the protective gene, the modelling predicted reductions of 0.5 *log10* in viral load for a HAART-naive individual after 1 year (Figure 4 A). Benefits in terms of forestalment of onset of AIDS at 8 years post-infection were also estimated (Figure 4 B). Slighly less prononunced effects were observed for patients that were concurrently enrolled on HAART (Murray, Fanning et al. 2009). Such results are encouraging and indicate that relatively modest engraftment percentages could achieve a clinically relevant effect. Consequently full bone marrow ablation may be unnecessary.

#### **3.3 Resistance development under gene-therapy: How does it differ from HAART?**

Systemic antiretroviral therapy bathes each cell in some level of the drugs being used depending on the penetration of the individual drugs to that region of the body, their

Cell-Delivered Gene Therapy for HIV 423

this will be even more evident for gene therapy delivered to HSC since it will take some time for the protected CD4+ T cells to mature from the HSC and appear in the periphery (Applegate, Birkett et al. 2010). However there is a considerable difference between gene

Unless myeloablation is conducted to eliminate endogenous non-gene containing HSC and T cells, it is expected that there will always be a sizeable proportion of HSC and CD4+ T cells that do not contain the therapy. Achieving 20% gene transduced HSC without ablation may be an upper bound (this remains to be tested). Similar limitations exist for those trials that instead transduce peripheral CD4+ T cells. At any time there is an estimated 2% of total T cells in peripheral blood and not all T cells are likely to traffic to this compartment. So large-scale apheresis of CD4+ T cells from peripheral blood will remove, and be able to

Hence gene therapy will partition HIV-susceptible cells into a bipartite population: those containing the therapy and those that do not. This 0-1 distribution is very different from the continuous distribution of drug concentration within cells for an individual receiving antiretroviral therapy (Figure 5). In this situation gene therapy is less likely to lead to the development of resistance (Applegate, Birkett et al. 2010). However there is a trade-off in that it is also less suppressive for the same reason. As cells containing gene therapy become more widespread in the body of an infected individual they will exert more pressure on the virus and select for resistance (Leonard and Schaffer 2006). For this reason the same general principles that apply to antiretroviral therapy are also valid in this context. Gene therapy

Mathematical modelling of gene therapy delivered to HSC that targets multiple mechanisms with reasonable efficacy and where the resistant virus is also less fit than wild-type determined that this therapy will reduce virus and maintain a viable T cell population for extended periods without the expansion of resistant virus (Applegate, Birkett et al. 2010). However there were important qualifications to the extent of this success. Primarily the gene therapy needs to be Class 1 and inhibit infection of cells. Additionally the likelihood of resistance to a particular component of the therapy and the fitness cost that incurs will also

**3.4 Future perspectives: What can we expect from gene-therapy against HIV?** 

Gene therapy holds out high promise as an effective therapy against HIV. The definition of success of gene-therapy treatment may vary, depending on a variety of circumstances. As discussed in Section 2.1, an obvious success would be one similar to that of the Berlin Patient whereby an individual would be completely and sustainably cured of HIV and have their immune system restored to "normal" levels. It is however important to provide more practical goals, as it is not likely that a "cure" will be achieved with all patients, and as such, more "modest" goals might be more practical and more realistic. Removing the need for an individual to be on HAART would be defined as success, as this can save the individual from life-long drug regimens often with considerable side-effects (Yeni 2006). Another "successful" outcome might consists in preservation of immune system functionality depite the presence of measurable viral loads, as observed during SIV infection in its natural hosts

As discussed in section 3.2.2 above, predictions from mathematical modelling indicate that full ablation of the immune system need not be necessary in order for clinically significant

therapy and a systemic treatment that is not suitably suppressive.

infuse, only a fraction of their total.

should target multiple viral and cellular mechanisms.

contribute to the speed at which virus overcomes the therapy.

(Liovat, Jacquelin et al. 2009; Pandrea, Silvestri et al. 2009).

concentration, pharmacokinetics and timing between dosages (Abdel-Rahman and Kauffman 2004). The clinical management of the combinations of drugs used in a regimen is an important part of successful treatment through suppressing the development of drug resistance. Early in the development of antiretroviral drugs there were few agents available and by necessity these were applied as monotherapy leading to the failure of these and subsequent drugs from the same class. Current HAART regimens involve three drugs from at least 2 drug classes to limit the likelihood that mutations in the HIV quasispecies will be present prior to the commencement of therapy or will develop subsequently.

Fig. 5. Illustration of principles behind selection pressures driving the development of resistance with antiretroviral therapy and with gene-therapy. Adapted with permission from Applegate et al.(Applegate, Birkett et al. 2010). (A) The horizontal ordinate denotes the concentration of antiretroviral drug received, and the vertical ordinate denotes the frequency of cells receiving the antiretroviral drug concentration. The selection pressure driving resistance in systemic antiretroviral therapy results from bathing each cell in some drug concentration. This provides a "continuous spectrum" for selection of HIV escape mutants, since many cells will receive suboptimal drug concentrations allowing viral replication and the preferential development of drug-resistant strains (as shown by shaded region indicated by the arrow). (B) The horizontal ordinate splits the cell population into two parts of either having a protective gene or not. The vertical ordinate denotes the frequency of cells containing the gene and not containing the gene. The bipartite distribution of protected and unprotected results provides a different selection environment whereby sufficient wild-type replication takes place in the non-protected population (i.e. no gene), thus mitigating the escape of viral mutants.

Similar concerns exist for the development of resistance to HIV gene therapy (Leonard and Schaffer 2006; Applegate, Birkett et al. 2010). The quasispecies nature of HIV and its high mutation rate imply the existence of every single mutation to any agent prior to the start of therapy. If there is sufficient viral replication under therapy, even for a reasonably short period, there is the chance that these singly resistant clones will evolve into variants with additional mutations and that are highly resistant to therapy. HIV gene therapy seems to fall into the classification of approaches that lend themselves to the development of resistance: not all cells will contain the therapy and so there will be considerable viral replication, and

concentration, pharmacokinetics and timing between dosages (Abdel-Rahman and Kauffman 2004). The clinical management of the combinations of drugs used in a regimen is an important part of successful treatment through suppressing the development of drug resistance. Early in the development of antiretroviral drugs there were few agents available and by necessity these were applied as monotherapy leading to the failure of these and subsequent drugs from the same class. Current HAART regimens involve three drugs from at least 2 drug classes to limit the likelihood that mutations in the HIV quasispecies will be

present prior to the commencement of therapy or will develop subsequently.

Fig. 5. Illustration of principles behind selection pressures driving the development of resistance with antiretroviral therapy and with gene-therapy. Adapted with permission from Applegate et al.(Applegate, Birkett et al. 2010). (A) The horizontal ordinate denotes the

frequency of cells containing the gene and not containing the gene. The bipartite distribution of protected and unprotected results provides a different selection environment whereby sufficient wild-type replication takes place in the non-protected population (i.e. no gene),

Similar concerns exist for the development of resistance to HIV gene therapy (Leonard and Schaffer 2006; Applegate, Birkett et al. 2010). The quasispecies nature of HIV and its high mutation rate imply the existence of every single mutation to any agent prior to the start of therapy. If there is sufficient viral replication under therapy, even for a reasonably short period, there is the chance that these singly resistant clones will evolve into variants with additional mutations and that are highly resistant to therapy. HIV gene therapy seems to fall into the classification of approaches that lend themselves to the development of resistance: not all cells will contain the therapy and so there will be considerable viral replication, and

concentration of antiretroviral drug received, and the vertical ordinate denotes the frequency of cells receiving the antiretroviral drug concentration. The selection pressure driving resistance in systemic antiretroviral therapy results from bathing each cell in some drug concentration. This provides a "continuous spectrum" for selection of HIV escape mutants, since many cells will receive suboptimal drug concentrations allowing viral replication and the preferential development of drug-resistant strains (as shown by shaded region indicated by the arrow). (B) The horizontal ordinate splits the cell population into two parts of either having a protective gene or not. The vertical ordinate denotes the

thus mitigating the escape of viral mutants.

this will be even more evident for gene therapy delivered to HSC since it will take some time for the protected CD4+ T cells to mature from the HSC and appear in the periphery (Applegate, Birkett et al. 2010). However there is a considerable difference between gene therapy and a systemic treatment that is not suitably suppressive.

Unless myeloablation is conducted to eliminate endogenous non-gene containing HSC and T cells, it is expected that there will always be a sizeable proportion of HSC and CD4+ T cells that do not contain the therapy. Achieving 20% gene transduced HSC without ablation may be an upper bound (this remains to be tested). Similar limitations exist for those trials that instead transduce peripheral CD4+ T cells. At any time there is an estimated 2% of total T cells in peripheral blood and not all T cells are likely to traffic to this compartment. So large-scale apheresis of CD4+ T cells from peripheral blood will remove, and be able to infuse, only a fraction of their total.

Hence gene therapy will partition HIV-susceptible cells into a bipartite population: those containing the therapy and those that do not. This 0-1 distribution is very different from the continuous distribution of drug concentration within cells for an individual receiving antiretroviral therapy (Figure 5). In this situation gene therapy is less likely to lead to the development of resistance (Applegate, Birkett et al. 2010). However there is a trade-off in that it is also less suppressive for the same reason. As cells containing gene therapy become more widespread in the body of an infected individual they will exert more pressure on the virus and select for resistance (Leonard and Schaffer 2006). For this reason the same general principles that apply to antiretroviral therapy are also valid in this context. Gene therapy should target multiple viral and cellular mechanisms.

Mathematical modelling of gene therapy delivered to HSC that targets multiple mechanisms with reasonable efficacy and where the resistant virus is also less fit than wild-type determined that this therapy will reduce virus and maintain a viable T cell population for extended periods without the expansion of resistant virus (Applegate, Birkett et al. 2010). However there were important qualifications to the extent of this success. Primarily the gene therapy needs to be Class 1 and inhibit infection of cells. Additionally the likelihood of resistance to a particular component of the therapy and the fitness cost that incurs will also contribute to the speed at which virus overcomes the therapy.

#### **3.4 Future perspectives: What can we expect from gene-therapy against HIV?**

Gene therapy holds out high promise as an effective therapy against HIV. The definition of success of gene-therapy treatment may vary, depending on a variety of circumstances. As discussed in Section 2.1, an obvious success would be one similar to that of the Berlin Patient whereby an individual would be completely and sustainably cured of HIV and have their immune system restored to "normal" levels. It is however important to provide more practical goals, as it is not likely that a "cure" will be achieved with all patients, and as such, more "modest" goals might be more practical and more realistic. Removing the need for an individual to be on HAART would be defined as success, as this can save the individual from life-long drug regimens often with considerable side-effects (Yeni 2006). Another "successful" outcome might consists in preservation of immune system functionality depite the presence of measurable viral loads, as observed during SIV infection in its natural hosts (Liovat, Jacquelin et al. 2009; Pandrea, Silvestri et al. 2009).

As discussed in section 3.2.2 above, predictions from mathematical modelling indicate that full ablation of the immune system need not be necessary in order for clinically significant

Cell-Delivered Gene Therapy for HIV 425

induced mobilisation were discussed, indicating their role in treatments. With mobilisation being crucial for the efficient transduction of HSC, and ablation of non-tranduced cells having the potential to provide a significant proportional increase in the amount of protected cells, both are critical when designing treatment regimens. Finally, clinical trials whereby HIV gene therapy has been conducted, and the outcomes of these trials were summarised, highlighting the high safety levels associated with gene therapuetics. Due to the observed high safety in these studies, with the promise of reasonable levels of efficacy and a proof of concept (in the Berlin Patient), HIV gene therapies are a very promising area

In the final sections we discussed predictions obtained from mathematical modelling regarding the in-vivo effectiveness of gene-therapy. We outlined why HIV virion entry inhibition via Class 1 gene-therapy has been shown to be essential in terms of achieving clinically meaningful effects. We explained how the selective survival advantage conferred to non-infected cells containing the Class 1 protective gene is the key factor contributing to the success of Class 1 therapy. We saw that transduction of CD4+ T cells provides an immediately protected CD4+ T cell population, but that in-vivo expansion of the protected cells may be a slow process and does not result in increased T cell receptor diversity in the expanded population. In contrast, transduction of HSC results in higher T cell receptor diversity, and in protection of a broader range of cells than solely CD4+ T cells. We also discussed the differences in viral resistance development under HAART and under genetherapy. While HAART bathes each cell in some drug concentration, resulting in suboptimal dosages for many cells and consequent promotion in escape of viral mutants, gene-therapy partitions the cell population into protected (contains gene) and unprotected (does not contain gene) cell populations. We outlined how this bi-partite distribution promotes the expansion of a cell population protected against HIV, while at the same time mitigating risks of viral mutation escape as a result of sufficient wild-type viral replication in the nonprotected cell population. Finally, we discussed future perspectives outlining how genetherapy promises to achieve sufficient preservation of immune system functionality (without HAART-associated toxicity and non-adherence issues) resulting in forestallement of AIDS and thereby achieving similar effects as observed during SIV infection in its natural hosts. We also outlined how gene-based therapies may be employed in conjunction or disjunction with HAART depending on individual patient circumstances and viral tropism

In conclusion, based on the clinical results and mathematical modeling work to-date, further clinical investigation of gene-therapy is more than justified, as gene-therapy holds high promise in terms of controlling HIV infection, preserving immune system functionality, and

(2009). "Trial watch: novel HIV gene therapy enters Phase I trial." Nat Rev Drug Discov 8(4):

Abdel-Rahman, S. M. and R. E. Kauffman (2004). "The integration of pharmacokinetics and

pharmacodynamics: understanding dose-response." Annu Rev Pharmacol Toxicol

of HIV research.

in the infected individual.

**5. References** 

267.

44: 111-136.

prevention of the onset of AIDS.

effects to be observed (Murray, Fanning et al. 2009). Assuming HSC engraftment percentages of about 20%, it has been predicted that substantial viral control may be achieved and CD4+ T cell counts maintained above the critical limit of 200 cells/µL (Murray, Fanning et al. 2009). The major challenge to achieving substantial clinical effect thus relates to achieving sufficient engraftment percentages.

Gene therapy will have varying degrees of effectiveness depending on the circumstances of the individual. The length of time for which an individual has been infected with HIV is an important factor to consider when providing gene therapy treatment. Due to the tropism of HIV in an infected individual changing over the duration of infection from CCR5-tropic to CXCR4-tropic, any treatment targeting CCR5 would be best used on patients in fairly earlyphase infection (Mosier 2009). For gene-therapy aiming to transduce HSC, it also appears reasonable to expect that the therapeutic effects in younger patients will be more pronounced due to their greater rates of thymic activity (Pido-Lopez, Imami et al. 2001). Futhermore, patients on HAART, patients for whom available antiretroviral therapies have been exhausted and patients suffering severe HAART-associated side-effects should also benefit, given that gene-therapy provides an alternative layer of protection via cell-mediated immunity in addition to antiretroviral therapies (Symonds, Johnstone et al. 2010).

Finally, as discussed in section 3.2, both HSC and CD4+ T cells represent feasible targets for transduction. While CD4+ T cell transduction may suffer from limitations due to a restricted T cell receptor repertoire and not protecting other susceptible cell population, it will however provide an immediate protected population. Conversely HSC are limited by the degree of thymic production and bone marrow engraftment, yet have the potential to generate a long-lasting array of HIV protected immune cells. Thus it appears that most effective therapies might employ a combination of these two approaches in order to provide optimum protection, possibly employing infusions of transduced cells.

#### **4. Conclusion**

In this chapter we discussed the current biological underpinnings of gene-therapy against HIV, as well as predictions from mathematical modelling of the clinical effects achievable through gene-therapy.

We discussed the various biological and clinical aspects relating to HIV gene therapy. An indication of the possible effectiveness of gene therapy was provided in terms of the naturally occurring mutation, CCR5d32, which provides extremely high levels of resistance against HIV infection. Most importantly however, the utilisation of this mutation in a bone marrow transplant, ridding an individual of any measurable HIV levels, indicates the capability of using gene therapy to functionally "cure" people of HIV. An assessment of the target areas of HIV gene therapy was conducted, indicating not only the possibility, but also a clear need to target multiple aspects of HIV infection (favourably entry stage), in order to prevent the emergence of resistance. The various options for delivery methods were discussed, indicating a range of techniques by which to introduce the therapeutic gene. Each of these methods exhibit their own advantages and disadvantages, however all are valid options in a variety of situations, with lentiviral vectors showing some of the most promise. The options for the ideal cell-type to target were discussed, indicating validity of using either CD4+ T cells for their immediate effect or HSC for the more sustained and broad spectrum protection. However it is also critical to consider the combination of these as an option in gene therapy. The aspects and priniciples of apheresis, ablation, and G-CSF-

effects to be observed (Murray, Fanning et al. 2009). Assuming HSC engraftment percentages of about 20%, it has been predicted that substantial viral control may be achieved and CD4+ T cell counts maintained above the critical limit of 200 cells/µL (Murray, Fanning et al. 2009). The major challenge to achieving substantial clinical effect

Gene therapy will have varying degrees of effectiveness depending on the circumstances of the individual. The length of time for which an individual has been infected with HIV is an important factor to consider when providing gene therapy treatment. Due to the tropism of HIV in an infected individual changing over the duration of infection from CCR5-tropic to CXCR4-tropic, any treatment targeting CCR5 would be best used on patients in fairly earlyphase infection (Mosier 2009). For gene-therapy aiming to transduce HSC, it also appears reasonable to expect that the therapeutic effects in younger patients will be more pronounced due to their greater rates of thymic activity (Pido-Lopez, Imami et al. 2001). Futhermore, patients on HAART, patients for whom available antiretroviral therapies have been exhausted and patients suffering severe HAART-associated side-effects should also benefit, given that gene-therapy provides an alternative layer of protection via cell-mediated

immunity in addition to antiretroviral therapies (Symonds, Johnstone et al. 2010).

optimum protection, possibly employing infusions of transduced cells.

**4. Conclusion** 

through gene-therapy.

Finally, as discussed in section 3.2, both HSC and CD4+ T cells represent feasible targets for transduction. While CD4+ T cell transduction may suffer from limitations due to a restricted T cell receptor repertoire and not protecting other susceptible cell population, it will however provide an immediate protected population. Conversely HSC are limited by the degree of thymic production and bone marrow engraftment, yet have the potential to generate a long-lasting array of HIV protected immune cells. Thus it appears that most effective therapies might employ a combination of these two approaches in order to provide

In this chapter we discussed the current biological underpinnings of gene-therapy against HIV, as well as predictions from mathematical modelling of the clinical effects achievable

We discussed the various biological and clinical aspects relating to HIV gene therapy. An indication of the possible effectiveness of gene therapy was provided in terms of the naturally occurring mutation, CCR5d32, which provides extremely high levels of resistance against HIV infection. Most importantly however, the utilisation of this mutation in a bone marrow transplant, ridding an individual of any measurable HIV levels, indicates the capability of using gene therapy to functionally "cure" people of HIV. An assessment of the target areas of HIV gene therapy was conducted, indicating not only the possibility, but also a clear need to target multiple aspects of HIV infection (favourably entry stage), in order to prevent the emergence of resistance. The various options for delivery methods were discussed, indicating a range of techniques by which to introduce the therapeutic gene. Each of these methods exhibit their own advantages and disadvantages, however all are valid options in a variety of situations, with lentiviral vectors showing some of the most promise. The options for the ideal cell-type to target were discussed, indicating validity of using either CD4+ T cells for their immediate effect or HSC for the more sustained and broad spectrum protection. However it is also critical to consider the combination of these as an option in gene therapy. The aspects and priniciples of apheresis, ablation, and G-CSF-

thus relates to achieving sufficient engraftment percentages.

induced mobilisation were discussed, indicating their role in treatments. With mobilisation being crucial for the efficient transduction of HSC, and ablation of non-tranduced cells having the potential to provide a significant proportional increase in the amount of protected cells, both are critical when designing treatment regimens. Finally, clinical trials whereby HIV gene therapy has been conducted, and the outcomes of these trials were summarised, highlighting the high safety levels associated with gene therapuetics. Due to the observed high safety in these studies, with the promise of reasonable levels of efficacy and a proof of concept (in the Berlin Patient), HIV gene therapies are a very promising area of HIV research.

In the final sections we discussed predictions obtained from mathematical modelling regarding the in-vivo effectiveness of gene-therapy. We outlined why HIV virion entry inhibition via Class 1 gene-therapy has been shown to be essential in terms of achieving clinically meaningful effects. We explained how the selective survival advantage conferred to non-infected cells containing the Class 1 protective gene is the key factor contributing to the success of Class 1 therapy. We saw that transduction of CD4+ T cells provides an immediately protected CD4+ T cell population, but that in-vivo expansion of the protected cells may be a slow process and does not result in increased T cell receptor diversity in the expanded population. In contrast, transduction of HSC results in higher T cell receptor diversity, and in protection of a broader range of cells than solely CD4+ T cells. We also discussed the differences in viral resistance development under HAART and under genetherapy. While HAART bathes each cell in some drug concentration, resulting in suboptimal dosages for many cells and consequent promotion in escape of viral mutants, gene-therapy partitions the cell population into protected (contains gene) and unprotected (does not contain gene) cell populations. We outlined how this bi-partite distribution promotes the expansion of a cell population protected against HIV, while at the same time mitigating risks of viral mutation escape as a result of sufficient wild-type viral replication in the nonprotected cell population. Finally, we discussed future perspectives outlining how genetherapy promises to achieve sufficient preservation of immune system functionality (without HAART-associated toxicity and non-adherence issues) resulting in forestallement of AIDS and thereby achieving similar effects as observed during SIV infection in its natural hosts. We also outlined how gene-based therapies may be employed in conjunction or disjunction with HAART depending on individual patient circumstances and viral tropism in the infected individual.

In conclusion, based on the clinical results and mathematical modeling work to-date, further clinical investigation of gene-therapy is more than justified, as gene-therapy holds high promise in terms of controlling HIV infection, preserving immune system functionality, and prevention of the onset of AIDS.

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**21** 

*Austria* 

 **T cells** 

**Gene Therapy for HIV-1 Infection** 

Lisa Egerer, Dorothee von Laer and Janine Kimpel *Innsbruck Medical University, Department of Hygiene,* 

 *Microbiology and Social Medicine, Division of Virology, Innsbruck* 

The introduction of the highly active antiretroviral therapy (HAART) for the treatment of HIV-1-infection has dramatically improved the quality of life and the survival of HIVinfected patients. HAART can effectively suppress virus replication and thereby helps to preserve immune functions. However, as HIV-1 persists in latently infected reservoirs (Finzi et al., 1997), complete eradication of the virus by antiretroviral drugs has never been achieved and life-long treatment is required. Moreover, emerging viral resistance and drug toxicity restrict long-term therapeutic efficacy (Brinkman et al., 1999; Vigouroux et al., 1999). As a consequence, HAART has not had a major impact on the global prevalence of HIV-

In addition to HAART and vaccines, gene therapy approaches for HIV-1 infection have been under investigation for more than two decades. Gene therapy could theoretically overcome the limitations of standard antiretroviral drug therapy and facilitate sustained suppression of virus replication after only few treatment cycles. Moreover, the choice of adequate genes or combinations of genes and expression systems could greatly reduce toxicity and prevent the generation of resistant virus strains. Although gene therapy is an expensive and technically challenging therapy today, future developments could simplify the procedures involved and bring down costs. Two basic gene therapeutic strategies for immune reconstitution of AIDS patients have been developed and the safety and efficacy of different approaches have been examined in preclinical and clinical studies. The first strategy aims to specifically kill HIV-infected cells by enhancing the antiviral host immune responses. The second approach, termed 'intracellular immunization', is based on the expression of antiviral genes that prevent HIV-1 replication in its target cells. Furthermore, therapeutic or prophylactic vaccination strategies that aim to enhance anti-HIV immunity and use DNA or viral vectors to express the viral antigens can formally be classified as gene therapy

infection and there is no vaccine in sight that could prevent further virus spread.

approaches. However, such vaccination strategies are not in the scope of this review.

The striking ability of HIV-1 to evade control by the host immune system is a fundamental problem in AIDS pathogenesis. Although most patients develop natural anti-HIV immune responses, the virus does not possess the immunogenicity to mount long-lasting responses strong enough to entirely suppress replication and to allow complete virus eradication from the body. In fact, most affected individuals initially develop immunodominant CD8+ T cell

**2. Enhancing HIV-specific immunity: Adoptive transfer of CD8<sup>+</sup>**

**1. Introduction** 


### **Gene Therapy for HIV-1 Infection**

Lisa Egerer, Dorothee von Laer and Janine Kimpel

*Innsbruck Medical University, Department of Hygiene, Microbiology and Social Medicine, Division of Virology, Innsbruck Austria* 

#### **1. Introduction**

430 Recent Translational Research in HIV/AIDS

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HIV-infected patients with advanced immunodeficiency and drug-resistant virus."

The introduction of the highly active antiretroviral therapy (HAART) for the treatment of HIV-1-infection has dramatically improved the quality of life and the survival of HIVinfected patients. HAART can effectively suppress virus replication and thereby helps to preserve immune functions. However, as HIV-1 persists in latently infected reservoirs (Finzi et al., 1997), complete eradication of the virus by antiretroviral drugs has never been achieved and life-long treatment is required. Moreover, emerging viral resistance and drug toxicity restrict long-term therapeutic efficacy (Brinkman et al., 1999; Vigouroux et al., 1999). As a consequence, HAART has not had a major impact on the global prevalence of HIVinfection and there is no vaccine in sight that could prevent further virus spread.

In addition to HAART and vaccines, gene therapy approaches for HIV-1 infection have been under investigation for more than two decades. Gene therapy could theoretically overcome the limitations of standard antiretroviral drug therapy and facilitate sustained suppression of virus replication after only few treatment cycles. Moreover, the choice of adequate genes or combinations of genes and expression systems could greatly reduce toxicity and prevent the generation of resistant virus strains. Although gene therapy is an expensive and technically challenging therapy today, future developments could simplify the procedures involved and bring down costs. Two basic gene therapeutic strategies for immune reconstitution of AIDS patients have been developed and the safety and efficacy of different approaches have been examined in preclinical and clinical studies. The first strategy aims to specifically kill HIV-infected cells by enhancing the antiviral host immune responses. The second approach, termed 'intracellular immunization', is based on the expression of antiviral genes that prevent HIV-1 replication in its target cells. Furthermore, therapeutic or prophylactic vaccination strategies that aim to enhance anti-HIV immunity and use DNA or viral vectors to express the viral antigens can formally be classified as gene therapy approaches. However, such vaccination strategies are not in the scope of this review.

#### **2. Enhancing HIV-specific immunity: Adoptive transfer of CD8<sup>+</sup> T cells**

The striking ability of HIV-1 to evade control by the host immune system is a fundamental problem in AIDS pathogenesis. Although most patients develop natural anti-HIV immune responses, the virus does not possess the immunogenicity to mount long-lasting responses strong enough to entirely suppress replication and to allow complete virus eradication from the body. In fact, most affected individuals initially develop immunodominant CD8+ T cell

Gene Therapy for HIV-1 Infection 433

2000; Hufert et al., 1997). However, although the CTLs were capable of lysing HIV-1 infected cells *in vivo*, only a transient effect on virus replication was observed (Brodie et al., 1999; Brodie et al., 2000). A major problem of this therapeutic concept is that the CTLs isolated from patients with advanced disease are often exhausted and terminally differentiated and lack full effector functions. As *in vitro* expansion of these cells is accompanied with a further loss of function, the transferred cell numbers may be insufficient to induce long-term effects. Methods that allow generation of large numbers of fully functional CTLs therefore would be

An alternative to the isolation and expansion of existing antigen-specific T cells from a patient, is the genetic modification of cells resulting in the expression of recombinant HIVspecific receptors. The cell types used for this genetic 'redirection' can be peripheral blood T cells, but also hematopoietic stem cells (HSC), which can afterwards differentiate into immune cells targeting HIV-1. As the isolation and manipulation of T cells is currently easier to perform compared to stem-cell modification, all studies that have been conducted so far used peripheral blood T cells. The receptors used to redirect the immune cells to target HIV-1 can either be natural T cell receptors or artificial chimeric antigen receptors (CARs). For the natural TCRs, CTL clones with high avidity TCRs specific for the target antigens are selected *in vitro.* The alpha and beta chains of these TCRs are then cloned and used to transduce patient T cells. This approach has been used successfully in the clinic to treat patients with melanoma, where in some cases tumor regression has been observed (Morgan et al., 2006). The isolation and cloning of high-avidity HIV-specific TCRs is also feasible. Joseph and colleagues constructed a lentiviral vector expressing a TCR specific for the HIV-1 Gag epitope SL9. Transduction of human primary T cells led to the conversion of peripheral blood CD8+ T cells into HIV-specific CTLs (Joseph et al., 2008). These CTLs exerted anti-HIV activity *in vitro* and *in vivo* in a humanized mouse model. In a similar study, an SL9-specific TCR with enhanced affinity was shown to efficiently mediate control of HIV-1 *in vitro* (Varela-Rohena et al., 2008). Currently this approach is evaluated in a phase I clinical trial (www.ClinicalTrials.gov; identifier: NCT00991224). The use of natural TCRs to generate virus-specific CTLs is limited by the major histocompatibility complex (MHC)-restriction of the TCR. TCRs recognize peptides only if they are presented on a specific type of MHC. Therefore, T cells expressing a given TCR can only be used for treatment of MHC-matched patients. The clinical trial described above, for instance, can only include patients with the HLA-type A\*02. For a broader application of this therapy concept a set of TCRs would be required that allows treatment of patients with various haplotypes. Besides, in gene modified CTLs, the genetically transferred TCR can mispair with the endogenous TCR, which may affect its function and lead to autoimmunity. Another drawback of the approach is the downregulation of MHC molecules in HIV-infected cells, which impedes the presentation of viral peptides and recognition by the CTLs (Sommermeyer et al., 2006). CARs are chimeric antigen receptors composed of an extracellular antigen binding motive connected to an intracellular signal transduction domain via a flexible linker and a transmembrane domain. The antigen binding motive usually is an antigen-specific singlechain variable fragment derived from a monoclonal antibody, while the signal transduction part comes from the CD3 ζ-chain. CARs trigger an MHC-independent antigen recognition (Eshhar et al., 2001). Two CARs with HIV-1 specificity have been developed by Roberts and colleagues (Roberts et al., 1994). The antigen-binding part consists either of the gp120 binding domain from human CD4 or an antibody against gp41. T cells transduced with either of the receptors specifically recognized and killed HIV-infected cells *in vitro*. The

required to facilitate success of this promising therapy approach.

(cytotoxic T lymphocyte, CTL) responses during the acute phase of HIV-1 infection, resulting in transient virus control and a decrease in plasma viremia (Borrow et al., 1994). The importance of CTLs was further confirmed in experiments with SIV-infected nonhuman primates, where acute infection could not be controlled in animals depleted of CD8+ T lymphocytes (Schmitz et al., 1999). There is a strong negative correlation between emergence of virus-specific CTLs and the viral set point, as patients with strong early CTL responses show significantly lower viral set points and a slower disease progression (Streeck et al., 2009). However, later, during the chronic phase of HIV-1 infection generation of CD8+ T cell responses seems to be impaired and dysfunctional CTLs fail to control virus replication in most patients. In contrast, long-term non-progressors – individuals which are HIV-1 seropositive, but do not progress to AIDS – retain high levels of virus-specific T cells, indicating that functional CTL responses are crucial for efficient virus control also in chronic infection (Rinaldo et al., 1995).

One reason for the failure of HIV-specific immunity during the chronic stage of infection may be the high variability of the virus resulting from a high replication rate and errorprone reverse transcription (Phillips et al., 1991). Moreover, HIV-1 attacks the immune system itself, the CD4+ helper T cell being its major target cell. This results in the preferential infection and a massive loss of HIV-specific CD4+ T cells already early upon infection, as the virus-specific helper cells migrate to the site of infection where they become activated thereby becoming more susceptible to HIV-1 infection (Demoustier et al., 2002; Douek et al., 2002). Yet, virus-specific CD4+ T cells are urgently needed to help generating strong, durable immune responses and their absence impairs CTL activation and maturation (Kemball et al., 2007; Matloubian et al., 1994). In addition, progressive exhaustion of virus-specific CD8+ T cells is a hallmark of the chronic immune activation during ongoing HIV-1 infection. Here, the constant antigen persistence prevents contraction of the effector T cell pool and development of long-lived memory cells. Instead, a stepwise exhaustion characterized by metabolic and transcriptional changes, reduced cytokine and chemokine secretion, loss of proliferation capacity and cytolytic activity is observed, resulting in impaired CTL effector functions and finally apoptosis (Shankar et al., 2000; Trimble & Lieberman, 1998).

Boosting of the natural CTL responses against HIV-1 by cell and gene therapeutic strategies may help to overcome these problems. For this purpose two different strategies have been developed; the adoptive transfer of autologous antigen-specific T cells after *ex vivo* selection and expansion, and the infusion of genetically armed CD8+ T cells expressing HIV-specific T cell receptors (TCRs). A therapy combining these approaches with the protection of CD4+ T cells to preserve important T-helper cell functions could potentially impact infection dynamics and ultimately facilitate clearance of the virus.

The adoptive transfer of autologous, antigen-specific CD8+ T cell clones has been used successfully to treat persistent viral infections and cancer. The allogeneic organ transplantation from cytomegalovirus or Epstein-Barr virus seropositive donors, for instance, is associated with risks for the recipient, if untreated. Co-transplantation of virusantigen-specific T cells isolated from the donor and expanded *in vitro*, enhanced T cell immunity to the viruses and prevented adverse effects in the immunosuppressed recipients (Heslop et al., 1996; Walter et al., 1995). For HIV-1, however, the transfer of *ex vivo* expanded virus-specific CTLs had only limited success in patients so far. In a study reported by Brodie et al. autologous Gag-specific CTLs were isolated and reinfused into HIV-positive individuals (Brodie et al., 1999). The CTLs engrafted in the patients and were found to migrate to the lymph nodes, which are the major sites of HIV-1 replication (Brodie et al.,

(cytotoxic T lymphocyte, CTL) responses during the acute phase of HIV-1 infection, resulting in transient virus control and a decrease in plasma viremia (Borrow et al., 1994). The importance of CTLs was further confirmed in experiments with SIV-infected nonhuman primates, where acute infection could not be controlled in animals depleted of CD8+ T lymphocytes (Schmitz et al., 1999). There is a strong negative correlation between emergence of virus-specific CTLs and the viral set point, as patients with strong early CTL responses show significantly lower viral set points and a slower disease progression (Streeck et al., 2009). However, later, during the chronic phase of HIV-1 infection generation of CD8+ T cell responses seems to be impaired and dysfunctional CTLs fail to control virus replication in most patients. In contrast, long-term non-progressors – individuals which are HIV-1 seropositive, but do not progress to AIDS – retain high levels of virus-specific T cells, indicating that functional CTL responses are crucial for efficient virus control also in chronic

One reason for the failure of HIV-specific immunity during the chronic stage of infection may be the high variability of the virus resulting from a high replication rate and errorprone reverse transcription (Phillips et al., 1991). Moreover, HIV-1 attacks the immune system itself, the CD4+ helper T cell being its major target cell. This results in the preferential infection and a massive loss of HIV-specific CD4+ T cells already early upon infection, as the virus-specific helper cells migrate to the site of infection where they become activated thereby becoming more susceptible to HIV-1 infection (Demoustier et al., 2002; Douek et al., 2002). Yet, virus-specific CD4+ T cells are urgently needed to help generating strong, durable immune responses and their absence impairs CTL activation and maturation (Kemball et al., 2007; Matloubian et al., 1994). In addition, progressive exhaustion of virus-specific CD8+ T cells is a hallmark of the chronic immune activation during ongoing HIV-1 infection. Here, the constant antigen persistence prevents contraction of the effector T cell pool and development of long-lived memory cells. Instead, a stepwise exhaustion characterized by metabolic and transcriptional changes, reduced cytokine and chemokine secretion, loss of proliferation capacity and cytolytic activity is observed, resulting in impaired CTL effector

functions and finally apoptosis (Shankar et al., 2000; Trimble & Lieberman, 1998).

dynamics and ultimately facilitate clearance of the virus.

Boosting of the natural CTL responses against HIV-1 by cell and gene therapeutic strategies may help to overcome these problems. For this purpose two different strategies have been developed; the adoptive transfer of autologous antigen-specific T cells after *ex vivo* selection and expansion, and the infusion of genetically armed CD8+ T cells expressing HIV-specific T cell receptors (TCRs). A therapy combining these approaches with the protection of CD4+ T cells to preserve important T-helper cell functions could potentially impact infection

The adoptive transfer of autologous, antigen-specific CD8+ T cell clones has been used successfully to treat persistent viral infections and cancer. The allogeneic organ transplantation from cytomegalovirus or Epstein-Barr virus seropositive donors, for instance, is associated with risks for the recipient, if untreated. Co-transplantation of virusantigen-specific T cells isolated from the donor and expanded *in vitro*, enhanced T cell immunity to the viruses and prevented adverse effects in the immunosuppressed recipients (Heslop et al., 1996; Walter et al., 1995). For HIV-1, however, the transfer of *ex vivo* expanded virus-specific CTLs had only limited success in patients so far. In a study reported by Brodie et al. autologous Gag-specific CTLs were isolated and reinfused into HIV-positive individuals (Brodie et al., 1999). The CTLs engrafted in the patients and were found to migrate to the lymph nodes, which are the major sites of HIV-1 replication (Brodie et al.,

infection (Rinaldo et al., 1995).

2000; Hufert et al., 1997). However, although the CTLs were capable of lysing HIV-1 infected cells *in vivo*, only a transient effect on virus replication was observed (Brodie et al., 1999; Brodie et al., 2000). A major problem of this therapeutic concept is that the CTLs isolated from patients with advanced disease are often exhausted and terminally differentiated and lack full effector functions. As *in vitro* expansion of these cells is accompanied with a further loss of function, the transferred cell numbers may be insufficient to induce long-term effects. Methods that allow generation of large numbers of fully functional CTLs therefore would be required to facilitate success of this promising therapy approach.

An alternative to the isolation and expansion of existing antigen-specific T cells from a patient, is the genetic modification of cells resulting in the expression of recombinant HIVspecific receptors. The cell types used for this genetic 'redirection' can be peripheral blood T cells, but also hematopoietic stem cells (HSC), which can afterwards differentiate into immune cells targeting HIV-1. As the isolation and manipulation of T cells is currently easier to perform compared to stem-cell modification, all studies that have been conducted so far used peripheral blood T cells. The receptors used to redirect the immune cells to target HIV-1 can either be natural T cell receptors or artificial chimeric antigen receptors (CARs). For the natural TCRs, CTL clones with high avidity TCRs specific for the target antigens are selected *in vitro.* The alpha and beta chains of these TCRs are then cloned and used to transduce patient T cells. This approach has been used successfully in the clinic to treat patients with melanoma, where in some cases tumor regression has been observed (Morgan et al., 2006). The isolation and cloning of high-avidity HIV-specific TCRs is also feasible. Joseph and colleagues constructed a lentiviral vector expressing a TCR specific for the HIV-1 Gag epitope SL9. Transduction of human primary T cells led to the conversion of peripheral blood CD8+ T cells into HIV-specific CTLs (Joseph et al., 2008). These CTLs exerted anti-HIV activity *in vitro* and *in vivo* in a humanized mouse model. In a similar study, an SL9-specific TCR with enhanced affinity was shown to efficiently mediate control of HIV-1 *in vitro* (Varela-Rohena et al., 2008). Currently this approach is evaluated in a phase I clinical trial (www.ClinicalTrials.gov; identifier: NCT00991224). The use of natural TCRs to generate virus-specific CTLs is limited by the major histocompatibility complex (MHC)-restriction of the TCR. TCRs recognize peptides only if they are presented on a specific type of MHC. Therefore, T cells expressing a given TCR can only be used for treatment of MHC-matched patients. The clinical trial described above, for instance, can only include patients with the HLA-type A\*02. For a broader application of this therapy concept a set of TCRs would be required that allows treatment of patients with various haplotypes. Besides, in gene modified CTLs, the genetically transferred TCR can mispair with the endogenous TCR, which may affect its function and lead to autoimmunity. Another drawback of the approach is the downregulation of MHC molecules in HIV-infected cells, which impedes the presentation of viral peptides and recognition by the CTLs (Sommermeyer et al., 2006).

CARs are chimeric antigen receptors composed of an extracellular antigen binding motive connected to an intracellular signal transduction domain via a flexible linker and a transmembrane domain. The antigen binding motive usually is an antigen-specific singlechain variable fragment derived from a monoclonal antibody, while the signal transduction part comes from the CD3 ζ-chain. CARs trigger an MHC-independent antigen recognition (Eshhar et al., 2001). Two CARs with HIV-1 specificity have been developed by Roberts and colleagues (Roberts et al., 1994). The antigen-binding part consists either of the gp120 binding domain from human CD4 or an antibody against gp41. T cells transduced with either of the receptors specifically recognized and killed HIV-infected cells *in vitro*. The

Gene Therapy for HIV-1 Infection 435

intravenous infusion to paediatric AIDS patients. Although the therapy was well tolerated, evidence of *in vivo* antiviral activity was not observed and consequently, sCD4 has never

Nevertheless, gene therapeutic strategies targeting early steps in the viral life cycle are expected to be the most promising therapeutics for HIV/AIDS, as discussed below. Proteinbased inhibitors targeting the virus entry process are thought to be especially powerful tools as they can prevent infection of the cell. Our group has previously developed a membraneanchored gp41-derived HIV-1 fusion inhibitor, maC46. This protein is expressed on the surface of T cells after transduction with retroviral or lentiviral vectors (Egelhofer et al., 2004; Hermann et al., 2009b; Perez et al., 2005). The protein binds to the HIV-1 gp41 heptad repeat 1 region thereby interfering with six-helix bundle formation during the viral and cellular membrane fusion process. MaC46 expressing T cells are almost completely protected from HIV-1 entry and have a strong selective survival advantage compared to unmodified cells *in vitro* and in mouse models of HIV-1 infection (Egelhofer et al., 2004; Hermann et al., 2009a; Kimpel et al., 2010). Likewise, maC46 has been shown to be one of the most potent anti-HIV gene products currently available (Kimpel et al., 2010). However, in a clinical trial with 10 HIV-1-infected patients with advanced disease and HAART failure, infusion of autologous CD4+ T cells genetically modified to express maC46 did not achieve sustained success. Although a significant rise in overall CD4+ T cell counts was observed in this study, the gene-protected cells did not accumulate over time and consequently, viral loads were not affected (van Lunzen et al., 2007). Recently, we described a secreted version of the fusion inhibitory C46 molecule. This '*in vivo* secreted antiviral entry inhibitor' (iSAVE) showed promising anti-HIV activity *in vitro* and has the potential to confer an overall

been investigated in gene therapy clinical trials (Husson et al., 1992).

antiviral effect *in vivo* despite low levels of gene marking (Egerer et al., 2011).

A different protein-based approach for HIV-1 gene therapy uses antibodies that bind and inactivate proteins and enzymes required for virus replication. Antibodies can be expressed within gene-modified cells as single-chain fragments (scFv), so-called intrabodies, or they can be secreted into the supernatant as neutralizing antibodies. Various intrabodies against HIV-1 proteins including Tat, Vif, Reverse Transcriptase and Integrase have been shown to inhibit virus replication in gene-modified cells *in vitro* (Goncalves et al., 2002; Kitamura et al., 1999; Levy-Mintz et al., 1996; Mhashilkar et al., 1995; Shaheen et al., 1996). Moreover, intrabodies against the viral co-receptors CXCR4 and CCR5 have been designed that retain these proteins in the ER (BouHamdan et al., 2001; Cordelier et al., 2004; Swan et al., 2006). Although these approaches efficiently inhibited HIV-1 replication in cell culture systems, intrabody techniques have not been further evaluated *in vivo*. The secreted version of the broadly neutralizing anti-gp41 monoclonal antibody 2F5 has been analyzed in a humanized mouse model of HIV-1 infection (Sanhadji et al., 2000). In this study, gene-modified cell lines expressing the antibody were implanted as neo-organs into immunodeficient mice repopulated with human CD4+ T cells. The neo-organs engrafted in the peritoneum and permitted continuous secretion of the antibody. Upon infection of the mice with HIV-1, viral loads were greatly reduced compared to control animals. However, due to safety problems, the implantation of neo-organs is not an option for treatment of patients. Recently, Joseph and colleagues reported the secretion of therapeutic concentrations of the broadly neutralizing anti-gp120 antibody 2G12 in humanized mice. Here, immunodeficient mice were transplanted with human hematopoietic stem cells that had previously been modified with a lentiviral vector encoding 2G12. The transduced stem cells differentiated into human

receptor containing CD4 has been tested in three clinical trials. In these studies local effects on virus replication were observed, but unfortunately there was no overall change in viral load (Deeks et al., 2002; Mitsuyasu et al., 2000; Walker et al., 2000).

#### **3. Intracellular immunization: Protection of CD4<sup>+</sup> T cells**

The second gene therapeutic strategy for HIV-1 infection was termed 'intracellular immunization' (Baltimore, 1988) and involves the expression of an antiviral gene in cells susceptible to HIV-1 infection. The target cells for intracellular immunization strategies therefore are mainly peripheral T cells or hematopoietic stem cells. The gene product can either be a protein or an RNA that inhibits HIV-1 replication by interfering with crucial steps of the viral life cycle or by targeting a cellular factor required for virus replication. Efficient genetic protection of the HIV-1 target cell population, i.e. mainly CD4+ T-helper cells, will deprive the virus of the possibility to produce progeny and is therefore expected to result in a drop of viral load and a regeneration of T cell counts. An additional antiviral effect can be achieved, if sufficient T-helper cell clones specific for HIV-1 antigens are protected against viral infection. These gene-protected CD4+ T cells could support the immunologic control of viral replication, without risking infection facilitated by HIV-1 antigen activation. As mentioned above, previous studies have shown that HIV-specific Thelper cell clones, which are crucial for an effective immune control of HIV-1 replication, are preferentially infected by HIV and lost during the course of the disease (Douek et al., 2002).

#### **3.1 Antiviral proteins**

Various types of anti-HIV proteins have been developed over the past years. Dominantnegative forms of both, viral proteins and cellular proteins required for virus replication have been described. These dominant-negative mutant proteins antagonize the activity of their corresponding wild-type proteins and thus prevent viral replication. A transdominant form of the HIV-1 Rev protein, RevM10, has been extensively studied *in vitro* and *in vivo*. RevM10 prevents the export of genomic viral RNA from the nucleus and as a result inhibits production of progeny virus (Malim et al., 1989). In clinical trials genetic modification of CD4+ T cells and CD34+ hematopoietic stem cells with RevM10 has been shown to be safe and provide some selective survival advantage. However, no sustained absolute accumulation of gene-modified cells and accordingly no antiviral effect was observed (Morgan et al., 2005; Podsakoff et al., 2005; Ranga et al., 1998; Woffendin et al., 1996). Furthermore, transdominant mutants of HIV-1 Tat that prevent Tat transactiviation have been developed (Fraisier et al., 1998; Pearson et al., 1990), but were never tested in clinical trials. The same is true for transdominant forms of the HIV-1 proteins Gag (Trono et al., 1989) and Vif (Morgan et al., 1990; Vallanti et al., 2005).

Cellular proteins required for virus replication have also been targeted by dominantnegative mutants. Membrane expression of chemokine receptor 5 (CCR5), which acts as a co-receptor for HIV-1, has been blocked by transdominant negative CCR5 variants upon retroviral expression in human T cells (Luis Abad et al., 2003). Even though inhibition of virus replication was observed in the gene-modified cells, this concept was not pursued further. A truncated soluble form of the cell surface receptor CD4 (sCD4) has been described to protect T cells from entry of laboratory-adapted strains of HIV-1, however, inhibition was less efficient for primary virus isolates (Daar & Ho, 1991; Morgan et al., 1994; Morgan et al., 1990). In a clinical phase I study recombinant soluble CD4 was administered by continuous

receptor containing CD4 has been tested in three clinical trials. In these studies local effects on virus replication were observed, but unfortunately there was no overall change in viral

The second gene therapeutic strategy for HIV-1 infection was termed 'intracellular immunization' (Baltimore, 1988) and involves the expression of an antiviral gene in cells susceptible to HIV-1 infection. The target cells for intracellular immunization strategies therefore are mainly peripheral T cells or hematopoietic stem cells. The gene product can either be a protein or an RNA that inhibits HIV-1 replication by interfering with crucial steps of the viral life cycle or by targeting a cellular factor required for virus replication. Efficient genetic protection of the HIV-1 target cell population, i.e. mainly CD4+ T-helper cells, will deprive the virus of the possibility to produce progeny and is therefore expected to result in a drop of viral load and a regeneration of T cell counts. An additional antiviral effect can be achieved, if sufficient T-helper cell clones specific for HIV-1 antigens are protected against viral infection. These gene-protected CD4+ T cells could support the immunologic control of viral replication, without risking infection facilitated by HIV-1 antigen activation. As mentioned above, previous studies have shown that HIV-specific Thelper cell clones, which are crucial for an effective immune control of HIV-1 replication, are preferentially infected by HIV and lost during the course of the disease (Douek et al., 2002).

Various types of anti-HIV proteins have been developed over the past years. Dominantnegative forms of both, viral proteins and cellular proteins required for virus replication have been described. These dominant-negative mutant proteins antagonize the activity of their corresponding wild-type proteins and thus prevent viral replication. A transdominant form of the HIV-1 Rev protein, RevM10, has been extensively studied *in vitro* and *in vivo*. RevM10 prevents the export of genomic viral RNA from the nucleus and as a result inhibits production of progeny virus (Malim et al., 1989). In clinical trials genetic modification of CD4+ T cells and CD34+ hematopoietic stem cells with RevM10 has been shown to be safe and provide some selective survival advantage. However, no sustained absolute accumulation of gene-modified cells and accordingly no antiviral effect was observed (Morgan et al., 2005; Podsakoff et al., 2005; Ranga et al., 1998; Woffendin et al., 1996). Furthermore, transdominant mutants of HIV-1 Tat that prevent Tat transactiviation have been developed (Fraisier et al., 1998; Pearson et al., 1990), but were never tested in clinical trials. The same is true for transdominant forms of the HIV-1 proteins Gag (Trono et al.,

Cellular proteins required for virus replication have also been targeted by dominantnegative mutants. Membrane expression of chemokine receptor 5 (CCR5), which acts as a co-receptor for HIV-1, has been blocked by transdominant negative CCR5 variants upon retroviral expression in human T cells (Luis Abad et al., 2003). Even though inhibition of virus replication was observed in the gene-modified cells, this concept was not pursued further. A truncated soluble form of the cell surface receptor CD4 (sCD4) has been described to protect T cells from entry of laboratory-adapted strains of HIV-1, however, inhibition was less efficient for primary virus isolates (Daar & Ho, 1991; Morgan et al., 1994; Morgan et al., 1990). In a clinical phase I study recombinant soluble CD4 was administered by continuous

 **T cells** 

load (Deeks et al., 2002; Mitsuyasu et al., 2000; Walker et al., 2000).

**3. Intracellular immunization: Protection of CD4<sup>+</sup>**

1989) and Vif (Morgan et al., 1990; Vallanti et al., 2005).

**3.1 Antiviral proteins** 

intravenous infusion to paediatric AIDS patients. Although the therapy was well tolerated, evidence of *in vivo* antiviral activity was not observed and consequently, sCD4 has never been investigated in gene therapy clinical trials (Husson et al., 1992).

Nevertheless, gene therapeutic strategies targeting early steps in the viral life cycle are expected to be the most promising therapeutics for HIV/AIDS, as discussed below. Proteinbased inhibitors targeting the virus entry process are thought to be especially powerful tools as they can prevent infection of the cell. Our group has previously developed a membraneanchored gp41-derived HIV-1 fusion inhibitor, maC46. This protein is expressed on the surface of T cells after transduction with retroviral or lentiviral vectors (Egelhofer et al., 2004; Hermann et al., 2009b; Perez et al., 2005). The protein binds to the HIV-1 gp41 heptad repeat 1 region thereby interfering with six-helix bundle formation during the viral and cellular membrane fusion process. MaC46 expressing T cells are almost completely protected from HIV-1 entry and have a strong selective survival advantage compared to unmodified cells *in vitro* and in mouse models of HIV-1 infection (Egelhofer et al., 2004; Hermann et al., 2009a; Kimpel et al., 2010). Likewise, maC46 has been shown to be one of the most potent anti-HIV gene products currently available (Kimpel et al., 2010). However, in a clinical trial with 10 HIV-1-infected patients with advanced disease and HAART failure, infusion of autologous CD4+ T cells genetically modified to express maC46 did not achieve sustained success. Although a significant rise in overall CD4+ T cell counts was observed in this study, the gene-protected cells did not accumulate over time and consequently, viral loads were not affected (van Lunzen et al., 2007). Recently, we described a secreted version of the fusion inhibitory C46 molecule. This '*in vivo* secreted antiviral entry inhibitor' (iSAVE) showed promising anti-HIV activity *in vitro* and has the potential to confer an overall antiviral effect *in vivo* despite low levels of gene marking (Egerer et al., 2011).

A different protein-based approach for HIV-1 gene therapy uses antibodies that bind and inactivate proteins and enzymes required for virus replication. Antibodies can be expressed within gene-modified cells as single-chain fragments (scFv), so-called intrabodies, or they can be secreted into the supernatant as neutralizing antibodies. Various intrabodies against HIV-1 proteins including Tat, Vif, Reverse Transcriptase and Integrase have been shown to inhibit virus replication in gene-modified cells *in vitro* (Goncalves et al., 2002; Kitamura et al., 1999; Levy-Mintz et al., 1996; Mhashilkar et al., 1995; Shaheen et al., 1996). Moreover, intrabodies against the viral co-receptors CXCR4 and CCR5 have been designed that retain these proteins in the ER (BouHamdan et al., 2001; Cordelier et al., 2004; Swan et al., 2006). Although these approaches efficiently inhibited HIV-1 replication in cell culture systems, intrabody techniques have not been further evaluated *in vivo*. The secreted version of the broadly neutralizing anti-gp41 monoclonal antibody 2F5 has been analyzed in a humanized mouse model of HIV-1 infection (Sanhadji et al., 2000). In this study, gene-modified cell lines expressing the antibody were implanted as neo-organs into immunodeficient mice repopulated with human CD4+ T cells. The neo-organs engrafted in the peritoneum and permitted continuous secretion of the antibody. Upon infection of the mice with HIV-1, viral loads were greatly reduced compared to control animals. However, due to safety problems, the implantation of neo-organs is not an option for treatment of patients. Recently, Joseph and colleagues reported the secretion of therapeutic concentrations of the broadly neutralizing anti-gp120 antibody 2G12 in humanized mice. Here, immunodeficient mice were transplanted with human hematopoietic stem cells that had previously been modified with a lentiviral vector encoding 2G12. The transduced stem cells differentiated into human

Gene Therapy for HIV-1 Infection 437

abolish function of small RNAs and escape mutants emerge rapidly (Boden et al., 2003; Das et al., 2004; Sabariegos et al., 2006). This problem can be partly overcome by using a combination of small RNAs targeting several conserved regions of the viral genome and ideally expressed from a single therapeutic vector (ter Brake et al., 2006). Alternatively, cellular genes required for virus replication can be targeted, including CD4, CXCR4, CCR5, nuclear factor κB, or LEDGF/p75, which all have been shown to be susceptible to RNAi silencing, thereby blocking viral entry or replication (Anderson & Akkina, 2005; Cordelier et al., 2003; Novina et al., 2002; Surabhi & Gaynor, 2002; Vandekerckhove et al., 2006). The CCR5 receptor is a particularly promising target, as disruption of the CCR5 gene does not alter immune functions (Huang et al., 1996; Samson et al., 1996). A highly potent and noncytotoxic siRNA directed against CCR5 has been developed by the group of Irvin Chen. Stable long-term expression of the siRNA and silencing of the CCR5 gene was observed after transplantation of gene-modified CD34+ hematopoietic progenitor cells in non-human primates. Gene-modified cells isolated from the animals were resistant to simian immunodeficiency virus infection *ex vivo* (An et al., 2007; Liang et al., 2010). The only RNAi approach that has been examined in patients so far is a tat/rev specific short hairpin RNA, which was tested in combination with a ribozyme targeting CCR5 and a TAR decoy in four patients receiving CD34+ hematopoietic progenitor cell transplantation due to AIDS-related lymphoma. In this recently reported clinical trial, stable, but low-level expression of the antiviral RNAs from gene-modified cells was observed for up to 24 months; however, there

Ribozymes are anti-sense RNA molecules with enzymatic activity that have been designed to target and site-specifically cleave essential viral RNAs or cellular mRNAs leading to gene silencing. Many ribozyme-based strategies for treatment of HIV-1 infection have been developed and show promising antiviral activity *in vitro* (Hotchkiss et al., 2004; Sarver et al., 1990; Zhou et al., 1994). Three ribozymes directed against HIV-1 tat/vrp (Amado et al., 2004; Macpherson et al., 2005; Mitsuyasu et al., 2009), HIV-1 rev/tat (Michienzi et al., 2003) and the viral U5 leader region (Wong-Staal et al., 1998) have already been tested in separate clinical trials. The gene transfer was proven to be safe in all studies, but none showed significant antiviral efficacy. As mentioned above, a CCR5-specific ribozyme has recently been analyzed in the clinic in combination with two other types of anti-HIV RNAs, but did

Antisense RNAs are short or long single-stranded RNA molecules binding to complementary HIV-1 mRNAs resulting in the formation of non-functional duplexes. Antisense molecules directed against the HIV-1 trans-activation response element (TAR) and the viral envelope RNA have been developed (Humeau et al., 2004; Lu et al., 2004; Vickers et al., 1991). The conditionally replicating lentiviral vector VRX496TM encodes a long antisense gene against the HIV-1 envelope. In clinical trials patients received autologous CD4+ T cells transduced with VRX496TM. A stabilization of viral load and slightly increased CD4+ T cell counts were observed, the significance of these results, however, remains

In contrast to the antiviral RNA molecules described above, RNA decoys do not attack the HIV-1 RNA. Instead, these small RNA fragments, which are derived from cis-acting elements in the viral genome, competitively bind and sequester viral proteins, thereby interfering with HIV-1 replication. Anti-HIV decoys are mainly based on the HIV-1 regulatory sequences TAR and Rev-responsive-element (RRE), which are bound by the two HIV-1 regulatory proteins Tat and Rev, respectively. The TAR element is a sequence

were no major effects on viral load (DiGiusto et al., 2010).

unclear (Levine et al., 2006).

not have a major influence on HIV-1 replication (DiGiusto et al., 2010).

progeny cells that secreted the functional antibody into the serum. This genetic immunization clearly reduced viral burden upon HIV-1 infection (Joseph et al., 2010).

Another strategy described by Sarkar et al. involves the expression of a modified version of the Cre recombinase (termed Tre) in HIV-1 infected cells. This recombinase has been engineered in a directed evolution approach to recombine a sequence present in the HIV-1 LTRs resulting in site-specific excision of the integrated provirus from an HIV-1 infected host cell genome (Sarkar et al., 2007). Even though proof-of-concept was provided *in vitro*, this approach is far from clinical application as the Tre recombinase is specific for one exclusive LTR sequence and does not recognize the LTRs of other virus strains.

Finally, zinc finger nucleases (ZFNs) are a novel tool in protein-based HIV-1 gene therapy. ZFNs are artificial fusion proteins composed of a DNA-binding and a DNA-cleavage domain. They can be engineered to bind any desired genome sequence and induce doublestrand breaks in the targeted DNA. Repair of the damaged DNA is associated with the introduction of high-frequency deletions and insertions at the site of cleavage. Individuals with a naturally occurring 32 bp deletion mutant of the CCR5 receptor (CCR5∆32) are perfectly healthy, but resistant to infection with R5-tropic strains of HIV-1 (Huang et al., 1996; Samson et al., 1996). Consequently, disruption of the CCR5 locus using ZFNs is not expected to alter immune functions, making it an ideal target for ZFN-based gene therapy. CCR5-specific ZFNs have been studied *in vitro* and in animal models and were shown to render the treated cells resistant to HIV-1 infection (Holt et al., 2010; Perez et al., 2008). Currently three clinical trials are recruiting patients to test this promising approach *in vivo* (www.ClinicalTrials.gov; identifier: NCT01044654, NCT01252641 and NCT00842634).

#### **3.2 Antiviral RNAs**

Antiviral RNAs for intracellular immunization can be grouped into four major categories: RNA interference (RNAi), ribozymes, anti-sense RNAs and RNA decoys. Several RNAibased gene therapy regimens for treatment of HIV-1 infection have proven to be effective in blocking viral replication by selective degradation of either viral RNAs or mRNAs of host factors that are essential for HIV-1 replication. Basically all HIV-1 RNAs have been successfully downregulated by RNAi *in vitro* (Chang et al., 2005; Coburn & Cullen, 2002; Jacque et al., 2002; N. S. Lee et al., 2002; Novina et al., 2002). However, systemic delivery of siRNAs to the relevant cell types *in vivo* is difficult. Kumar and colleagues administered antiviral siRNAs conjugated to a T cell-specific single-chain antibody that undergoes internalization upon binding to T cell surface receptors to humanized mice. This approach allowed targeted delivery of the siRNAs to T cells, which resulted in effective virus inhibition and preserved CD4+ T cell numbers (Kumar et al., 2008). An alternative to the regular injection of exogenous siRNAs is the expression of shRNAs directly in the HIV-1 target cells, but achieving stable transgene expression in the gene-modified cells is a challenge. Yet, constant endogenous expression would be required to obtain efficient suppression of HIV-1 replication and prevent viral escape mutants. At the same time, expression levels have to be tightly regulated in order to avoid cellular toxicity, as saturation of the cellular small RNA-processing pathway due to overexpression of shRNAs can lead to downregulation of cellular microRNAs (miRNAs) and cause severe toxicity (Grimm et al., 2006). Insertion of shRNAs into a natural miRNA backbone has been shown to reduce such toxic effects (McBride et al., 2008).

The high mutation rate of HIV-1 is an additional challenge in developing RNAi-based therapeutics, as a single point mutation within the targeted HIV-1 RNA sequence can

progeny cells that secreted the functional antibody into the serum. This genetic immunization clearly reduced viral burden upon HIV-1 infection (Joseph et al., 2010). Another strategy described by Sarkar et al. involves the expression of a modified version of the Cre recombinase (termed Tre) in HIV-1 infected cells. This recombinase has been engineered in a directed evolution approach to recombine a sequence present in the HIV-1 LTRs resulting in site-specific excision of the integrated provirus from an HIV-1 infected host cell genome (Sarkar et al., 2007). Even though proof-of-concept was provided *in vitro*, this approach is far from clinical application as the Tre recombinase is specific for one

Finally, zinc finger nucleases (ZFNs) are a novel tool in protein-based HIV-1 gene therapy. ZFNs are artificial fusion proteins composed of a DNA-binding and a DNA-cleavage domain. They can be engineered to bind any desired genome sequence and induce doublestrand breaks in the targeted DNA. Repair of the damaged DNA is associated with the introduction of high-frequency deletions and insertions at the site of cleavage. Individuals with a naturally occurring 32 bp deletion mutant of the CCR5 receptor (CCR5∆32) are perfectly healthy, but resistant to infection with R5-tropic strains of HIV-1 (Huang et al., 1996; Samson et al., 1996). Consequently, disruption of the CCR5 locus using ZFNs is not expected to alter immune functions, making it an ideal target for ZFN-based gene therapy. CCR5-specific ZFNs have been studied *in vitro* and in animal models and were shown to render the treated cells resistant to HIV-1 infection (Holt et al., 2010; Perez et al., 2008). Currently three clinical trials are recruiting patients to test this promising approach *in vivo* (www.ClinicalTrials.gov; identifier: NCT01044654, NCT01252641 and NCT00842634).

Antiviral RNAs for intracellular immunization can be grouped into four major categories: RNA interference (RNAi), ribozymes, anti-sense RNAs and RNA decoys. Several RNAibased gene therapy regimens for treatment of HIV-1 infection have proven to be effective in blocking viral replication by selective degradation of either viral RNAs or mRNAs of host factors that are essential for HIV-1 replication. Basically all HIV-1 RNAs have been successfully downregulated by RNAi *in vitro* (Chang et al., 2005; Coburn & Cullen, 2002; Jacque et al., 2002; N. S. Lee et al., 2002; Novina et al., 2002). However, systemic delivery of siRNAs to the relevant cell types *in vivo* is difficult. Kumar and colleagues administered antiviral siRNAs conjugated to a T cell-specific single-chain antibody that undergoes internalization upon binding to T cell surface receptors to humanized mice. This approach allowed targeted delivery of the siRNAs to T cells, which resulted in effective virus inhibition and preserved CD4+ T cell numbers (Kumar et al., 2008). An alternative to the regular injection of exogenous siRNAs is the expression of shRNAs directly in the HIV-1 target cells, but achieving stable transgene expression in the gene-modified cells is a challenge. Yet, constant endogenous expression would be required to obtain efficient suppression of HIV-1 replication and prevent viral escape mutants. At the same time, expression levels have to be tightly regulated in order to avoid cellular toxicity, as saturation of the cellular small RNA-processing pathway due to overexpression of shRNAs can lead to downregulation of cellular microRNAs (miRNAs) and cause severe toxicity (Grimm et al., 2006). Insertion of shRNAs into a natural miRNA backbone has been shown to reduce such

The high mutation rate of HIV-1 is an additional challenge in developing RNAi-based therapeutics, as a single point mutation within the targeted HIV-1 RNA sequence can

exclusive LTR sequence and does not recognize the LTRs of other virus strains.

**3.2 Antiviral RNAs** 

toxic effects (McBride et al., 2008).

abolish function of small RNAs and escape mutants emerge rapidly (Boden et al., 2003; Das et al., 2004; Sabariegos et al., 2006). This problem can be partly overcome by using a combination of small RNAs targeting several conserved regions of the viral genome and ideally expressed from a single therapeutic vector (ter Brake et al., 2006). Alternatively, cellular genes required for virus replication can be targeted, including CD4, CXCR4, CCR5, nuclear factor κB, or LEDGF/p75, which all have been shown to be susceptible to RNAi silencing, thereby blocking viral entry or replication (Anderson & Akkina, 2005; Cordelier et al., 2003; Novina et al., 2002; Surabhi & Gaynor, 2002; Vandekerckhove et al., 2006). The CCR5 receptor is a particularly promising target, as disruption of the CCR5 gene does not alter immune functions (Huang et al., 1996; Samson et al., 1996). A highly potent and noncytotoxic siRNA directed against CCR5 has been developed by the group of Irvin Chen. Stable long-term expression of the siRNA and silencing of the CCR5 gene was observed after transplantation of gene-modified CD34+ hematopoietic progenitor cells in non-human primates. Gene-modified cells isolated from the animals were resistant to simian immunodeficiency virus infection *ex vivo* (An et al., 2007; Liang et al., 2010). The only RNAi approach that has been examined in patients so far is a tat/rev specific short hairpin RNA, which was tested in combination with a ribozyme targeting CCR5 and a TAR decoy in four patients receiving CD34+ hematopoietic progenitor cell transplantation due to AIDS-related lymphoma. In this recently reported clinical trial, stable, but low-level expression of the antiviral RNAs from gene-modified cells was observed for up to 24 months; however, there were no major effects on viral load (DiGiusto et al., 2010).

Ribozymes are anti-sense RNA molecules with enzymatic activity that have been designed to target and site-specifically cleave essential viral RNAs or cellular mRNAs leading to gene silencing. Many ribozyme-based strategies for treatment of HIV-1 infection have been developed and show promising antiviral activity *in vitro* (Hotchkiss et al., 2004; Sarver et al., 1990; Zhou et al., 1994). Three ribozymes directed against HIV-1 tat/vrp (Amado et al., 2004; Macpherson et al., 2005; Mitsuyasu et al., 2009), HIV-1 rev/tat (Michienzi et al., 2003) and the viral U5 leader region (Wong-Staal et al., 1998) have already been tested in separate clinical trials. The gene transfer was proven to be safe in all studies, but none showed significant antiviral efficacy. As mentioned above, a CCR5-specific ribozyme has recently been analyzed in the clinic in combination with two other types of anti-HIV RNAs, but did not have a major influence on HIV-1 replication (DiGiusto et al., 2010).

Antisense RNAs are short or long single-stranded RNA molecules binding to complementary HIV-1 mRNAs resulting in the formation of non-functional duplexes. Antisense molecules directed against the HIV-1 trans-activation response element (TAR) and the viral envelope RNA have been developed (Humeau et al., 2004; Lu et al., 2004; Vickers et al., 1991). The conditionally replicating lentiviral vector VRX496TM encodes a long antisense gene against the HIV-1 envelope. In clinical trials patients received autologous CD4+ T cells transduced with VRX496TM. A stabilization of viral load and slightly increased CD4+ T cell counts were observed, the significance of these results, however, remains unclear (Levine et al., 2006).

In contrast to the antiviral RNA molecules described above, RNA decoys do not attack the HIV-1 RNA. Instead, these small RNA fragments, which are derived from cis-acting elements in the viral genome, competitively bind and sequester viral proteins, thereby interfering with HIV-1 replication. Anti-HIV decoys are mainly based on the HIV-1 regulatory sequences TAR and Rev-responsive-element (RRE), which are bound by the two HIV-1 regulatory proteins Tat and Rev, respectively. The TAR element is a sequence

Gene Therapy for HIV-1 Infection 439

that very early inhibitors are even more favorable, as they can prevent the massive "bystander" killing observed in HIV-1 infection. However, combination of several antiviral genes, targeting different steps in the viral life cycle might synergize most efficiently and

The major drawback of intracellular immunization approaches is the huge number of HIV-1 target cells in the human body (> 1011) that cannot be genetically modified with the available technologies. The frequencies of gene-modified CD4+ T cells achieved *in vivo*, whether by T cell or stem cell targeting, have been disappointingly low, in the range of 0,01% to 1%, or less (Amado et al., 2004; Levine et al., 2006; Macpherson et al., 2005; Morgan et al., 2005; van Lunzen et al., 2007). A significant impact of these few genetically HIV-resistant cells is neither expected on overall HIV-1 infection dynamics nor on immune reconstitution. However, if the gene-protected cells are able to proliferate and preferentially survive compared with unmodified cells, they could accumulate with time and progressively repopulate the immune system (Lund et al., 1997; von Laer et al., 2006). A number of gene products that have been developed could theoretically mediate such a selective survival advantage of the transduced cells as they have been shown to efficiently suppress virus replication and protect the cells from the viral cytopathic effect *in vitro*. However, selective accumulation of gene-protected cells has never been observed in clinical trials so far. In a comparative study, we recently evaluated three intracellular immunization strategies that had previously been used in the clinic, with respect to antiviral activity and survival advantage (Kimpel et al., 2010): (1) the viral entry inhibitor maC46 (class I gene); (2) an HIV-1 tat/rev-specific small hairpin (sh) RNA (class II gene); and (3) an RNA antisense gene specific for the HIV-1 envelope (class II gene). We found robust inhibition of HIV-1 replication with the fusion inhibitor maC46 and the antisense envelope inhibitor. Interestingly, and importantly, a survival advantage was merely demonstrated for cells expressing the maC46 fusion inhibitor both *in vitro* and *in vivo* in a humanized xenotransplant mouse model (Kimpel et al., 2010). This finding confirms *in silico* predictions stating that only class I genes can confer a sufficient selective advantage to allow preferential survival and accumulation of gene-protected, non-infected cells *in vivo* (von Laer et al., 2006). However, even this highly active fusion inhibitor failed to show a clear accumulation of gene-protected cells to therapeutic levels in a previous clinical trial in 10 AIDS patients (van Lunzen et al., 2007). These data show that efficient engraftment and proliferation of the

could be the only way to achieve sustained suppression of HIV-1 replication.

gene-protected cells remain a major challenge in gene therapy for HIV-1 infection.

Yet, such a strong selection and accumulation may not be required for a secreted antiviral gene product. Secreted antiviral proteins or peptides are expected to produce a bystander effect on unmodified neighboring cells, thereby suppressing virus replication and protecting the overall T cell pool even at low levels of gene modification. Such a bystander effect can only be conferred by antiviral proteins, but not by RNAs, as secretion is limited to proteins and peptides. However, the number of reports on secreted antiviral proteins in HIV-1 gene therapy is still very limited. Examples are neutralizing antibodies (Sanhadji et al., 2000), truncated soluble CD4 (Morgan et al., 1994; Morgan et al., 1990) and interferon β (Gay et al., 2004). We have recently reported the generation of an *in vivo* secreted antiviral entry inhibitor (iSAVE), which exerted a strong bystander effect in cell culture (Egerer et al., 2011). Lymphatic tissue is the major site of HIV-1 replication and thus T or B cells could be an ideal target cell for gene therapy approaches based on secreted gene products. Secretion of

**3.4 Selective survival advantage and bystander effect** 

contained in the 5' region of all HIV-1 mRNAs, which forms a stable stem-loop structure (Baudin et al., 1993; Muesing et al., 1987). Binding of the HIV-1 Tat (trans-activator) protein to the TAR element mediates increased viral gene expression (Keen et al., 1996); moreover, the TAR region is required for initiation of reverse transcription (Harrich et al., 1996). Disruption of the Tat/TAR interaction by TAR decoy RNA was shown to effectively prevent HIV-1 replication *in vitro* (Sullenger et al., 1990, 1991). A combination of three antiviral RNAs including a TAR decoy was successfully tested in humanized mouse models of HIV-1 infection (Anderson et al., 2007) and, as mentioned above, was shown to be safe in a clinical trial, although only minor effects on HIV-1 infection could be observed (DiGiusto et al., 2010). Interaction of the viral Rev protein with the RRE is critically required for transport of the unspliced genomic viral mRNA to the cytoplasm (Olsen et al., 1990). RRE decoys provide strong inhibition of HIV-1 replication by blocking the nuclear export of genomic HIV-1 RNA (T. C. Lee et al., 1992; Michienzi et al., 2006). Genetic modification of CD34+ hematopoietic stem cells by retroviral transfer of an RRE decoy gene followed by infusion of the gene-modified cells, was shown to be safe in a clinical trial with paediatric AIDS patients. However, transduction and engraftment levels were very low and no antiviral effect was observed (Kohn et al., 1999).

#### **3.3 The mode of action: Classification of antiviral genes**

The impact of gene-modified cells on systemic HIV-1 kinetics depends critically on the stage of the viral replication cycle at which the inhibition occurs. The antiviral genes can accordingly be grouped into three classes, depending on their effect on the viral life cycle (von Laer et al., 2006). Class I genes inhibit the first steps of the replication cycle prior to integration of the proviral DNA into the host cell genome and thus prevent infection of the cell. Hence, class I includes genes encoding entry inhibitors, as well as inhibitors of reverse transcription and integration. Class II genes have no effect on early steps of the viral replication cycle, but prevent the expression of viral RNA or proteins. Thus, they inhibit the production of infectious virus progeny and the viral cytopathic effect, however, integration of the proviral genome into the host cell chromosomes is not hindered. Cells expressing a class II gene and infected with HIV-1 resemble latently infected cells and according to computer simulations accumulate with time, counteracting the antiviral effect (von Laer et al., 2006). Furthermore, reverse transcription, which can give rise to resistant virus variants, is not inhibited by class II genes. Class III genes interfere with late steps in the viral life cycle such as virion assembly and budding. Consequently, they neither protect the infected cell from recognition by the immune system, as viral protein production is not inhibited, nor from the viral cytopathic effect. Therefore, class III genes alone are not expected to have an overall antiviral effect unless high percentages of cells are genetically modified.

Mathematical modelling predicts that only genes inhibiting early steps in the viral replication cycle provide a selective advantage strong enough to allow for the selection and accumulation of the gene modified cells (Lund et al., 1997; von Laer et al., 2006). Consequently, class I genes are the most promising candidates for successful intracellular immunization strategies. The group of Warner Greene recently found that in *ex vivo* cultures of human tonsils infected with HIV-1 the vast majority of CD4+ T cells died due to nonproductive abortive infection. In these non-permissive cells DNA reverse transcription intermediates elicited proapoptotic responses resulting in release of proinflammatory cytokines and caspase-mediated cell death (Doitsh et al., 2010). Inhibition of HIV-1 entry or early steps of reverse transcription could protect the cells from these fatal effects, indicating

contained in the 5' region of all HIV-1 mRNAs, which forms a stable stem-loop structure (Baudin et al., 1993; Muesing et al., 1987). Binding of the HIV-1 Tat (trans-activator) protein to the TAR element mediates increased viral gene expression (Keen et al., 1996); moreover, the TAR region is required for initiation of reverse transcription (Harrich et al., 1996). Disruption of the Tat/TAR interaction by TAR decoy RNA was shown to effectively prevent HIV-1 replication *in vitro* (Sullenger et al., 1990, 1991). A combination of three antiviral RNAs including a TAR decoy was successfully tested in humanized mouse models of HIV-1 infection (Anderson et al., 2007) and, as mentioned above, was shown to be safe in a clinical trial, although only minor effects on HIV-1 infection could be observed (DiGiusto et al., 2010). Interaction of the viral Rev protein with the RRE is critically required for transport of the unspliced genomic viral mRNA to the cytoplasm (Olsen et al., 1990). RRE decoys provide strong inhibition of HIV-1 replication by blocking the nuclear export of genomic HIV-1 RNA (T. C. Lee et al., 1992; Michienzi et al., 2006). Genetic modification of CD34+ hematopoietic stem cells by retroviral transfer of an RRE decoy gene followed by infusion of the gene-modified cells, was shown to be safe in a clinical trial with paediatric AIDS patients. However, transduction and engraftment levels were very low and no antiviral

The impact of gene-modified cells on systemic HIV-1 kinetics depends critically on the stage of the viral replication cycle at which the inhibition occurs. The antiviral genes can accordingly be grouped into three classes, depending on their effect on the viral life cycle (von Laer et al., 2006). Class I genes inhibit the first steps of the replication cycle prior to integration of the proviral DNA into the host cell genome and thus prevent infection of the cell. Hence, class I includes genes encoding entry inhibitors, as well as inhibitors of reverse transcription and integration. Class II genes have no effect on early steps of the viral replication cycle, but prevent the expression of viral RNA or proteins. Thus, they inhibit the production of infectious virus progeny and the viral cytopathic effect, however, integration of the proviral genome into the host cell chromosomes is not hindered. Cells expressing a class II gene and infected with HIV-1 resemble latently infected cells and according to computer simulations accumulate with time, counteracting the antiviral effect (von Laer et al., 2006). Furthermore, reverse transcription, which can give rise to resistant virus variants, is not inhibited by class II genes. Class III genes interfere with late steps in the viral life cycle such as virion assembly and budding. Consequently, they neither protect the infected cell from recognition by the immune system, as viral protein production is not inhibited, nor from the viral cytopathic effect. Therefore, class III genes alone are not expected to have an

overall antiviral effect unless high percentages of cells are genetically modified.

Mathematical modelling predicts that only genes inhibiting early steps in the viral replication cycle provide a selective advantage strong enough to allow for the selection and accumulation of the gene modified cells (Lund et al., 1997; von Laer et al., 2006). Consequently, class I genes are the most promising candidates for successful intracellular immunization strategies. The group of Warner Greene recently found that in *ex vivo* cultures of human tonsils infected with HIV-1 the vast majority of CD4+ T cells died due to nonproductive abortive infection. In these non-permissive cells DNA reverse transcription intermediates elicited proapoptotic responses resulting in release of proinflammatory cytokines and caspase-mediated cell death (Doitsh et al., 2010). Inhibition of HIV-1 entry or early steps of reverse transcription could protect the cells from these fatal effects, indicating

effect was observed (Kohn et al., 1999).

**3.3 The mode of action: Classification of antiviral genes** 

that very early inhibitors are even more favorable, as they can prevent the massive "bystander" killing observed in HIV-1 infection. However, combination of several antiviral genes, targeting different steps in the viral life cycle might synergize most efficiently and could be the only way to achieve sustained suppression of HIV-1 replication.

#### **3.4 Selective survival advantage and bystander effect**

The major drawback of intracellular immunization approaches is the huge number of HIV-1 target cells in the human body (> 1011) that cannot be genetically modified with the available technologies. The frequencies of gene-modified CD4+ T cells achieved *in vivo*, whether by T cell or stem cell targeting, have been disappointingly low, in the range of 0,01% to 1%, or less (Amado et al., 2004; Levine et al., 2006; Macpherson et al., 2005; Morgan et al., 2005; van Lunzen et al., 2007). A significant impact of these few genetically HIV-resistant cells is neither expected on overall HIV-1 infection dynamics nor on immune reconstitution. However, if the gene-protected cells are able to proliferate and preferentially survive compared with unmodified cells, they could accumulate with time and progressively repopulate the immune system (Lund et al., 1997; von Laer et al., 2006). A number of gene products that have been developed could theoretically mediate such a selective survival advantage of the transduced cells as they have been shown to efficiently suppress virus replication and protect the cells from the viral cytopathic effect *in vitro*. However, selective accumulation of gene-protected cells has never been observed in clinical trials so far. In a comparative study, we recently evaluated three intracellular immunization strategies that had previously been used in the clinic, with respect to antiviral activity and survival advantage (Kimpel et al., 2010): (1) the viral entry inhibitor maC46 (class I gene); (2) an HIV-1 tat/rev-specific small hairpin (sh) RNA (class II gene); and (3) an RNA antisense gene specific for the HIV-1 envelope (class II gene). We found robust inhibition of HIV-1 replication with the fusion inhibitor maC46 and the antisense envelope inhibitor. Interestingly, and importantly, a survival advantage was merely demonstrated for cells expressing the maC46 fusion inhibitor both *in vitro* and *in vivo* in a humanized xenotransplant mouse model (Kimpel et al., 2010). This finding confirms *in silico* predictions stating that only class I genes can confer a sufficient selective advantage to allow preferential survival and accumulation of gene-protected, non-infected cells *in vivo* (von Laer et al., 2006). However, even this highly active fusion inhibitor failed to show a clear accumulation of gene-protected cells to therapeutic levels in a previous clinical trial in 10 AIDS patients (van Lunzen et al., 2007). These data show that efficient engraftment and proliferation of the gene-protected cells remain a major challenge in gene therapy for HIV-1 infection.

Yet, such a strong selection and accumulation may not be required for a secreted antiviral gene product. Secreted antiviral proteins or peptides are expected to produce a bystander effect on unmodified neighboring cells, thereby suppressing virus replication and protecting the overall T cell pool even at low levels of gene modification. Such a bystander effect can only be conferred by antiviral proteins, but not by RNAs, as secretion is limited to proteins and peptides. However, the number of reports on secreted antiviral proteins in HIV-1 gene therapy is still very limited. Examples are neutralizing antibodies (Sanhadji et al., 2000), truncated soluble CD4 (Morgan et al., 1994; Morgan et al., 1990) and interferon β (Gay et al., 2004). We have recently reported the generation of an *in vivo* secreted antiviral entry inhibitor (iSAVE), which exerted a strong bystander effect in cell culture (Egerer et al., 2011). Lymphatic tissue is the major site of HIV-1 replication and thus T or B cells could be an ideal target cell for gene therapy approaches based on secreted gene products. Secretion of

Gene Therapy for HIV-1 Infection 441

The choice of the vector system may have a major impact on the efficacy of HIV-1 gene therapy approaches. There is no ideal vector suitable for all purposes, but the pros and cons have to be balanced for each application. Table 2 summarizes advantages and disadvantages

retroviral *ex vivo* 105-107 TU/ml 8-10 kb + - +

SV-40 *ex vivo* 1012 VP/ml up to 5 kb + - -

AAV, Adeno-Associated Virus; kb, kilo bases; SV-40, Simian Virus-40; TU, transducing

Basic questions to be asked are, whether *ex vivo* or *in vivo* gene transfer is preferred and if long-term expression of the gene product is required. To our knowledge, a vector system that allows efficient direct *in vivo* gene transfer specifically into CD4+ T cells or HSC has not been developed so far. Therefore, approaches based on modification of these cell types (arming of T cells with TCRs or CARs, most intracellular immunizations) always rely on *ex vivo* gene transfer. In contrast, genes encoding secreted antiviral molecules may also be delivered to distinct production sites (e.g. liver, muscle) directly *in vivo* using adenovirus (Ad) or adeno-associated virus (AAV)-derived vectors. The second basic question deals with the long-term-expression of the transferred antiviral gene. Strategies involving arming of T

107 TU/ml (109-1010 TU/ml)\*

**capacity** 

*vivo* 1013 VP/ml up to 36 kb - ++ +

**Integration**

> only with Rep

up to 10 kb + - +

(107 TU/ml)\* >9 kb + - -

**Immunogenicity** 

**Clinical trials** 

+ +

**Target cell T cell HSC**  Easy to obtain ++ + Conditioning required - + Cell dose >1010 108-109 Regeneration of T cell repertoire - + Protection of all HIV-1 target cells - + Insertional mutagenesis Limited +

Table 1. The target cell: T cells versus hematopoietic stem cells (HSC).

of several vector systems commonly used for gene transfer.

**Vector type Application Titer Packaging** 

AAV *in vivo* 1013 VP/ml 3-5 kb

Table 2. Vector systems for gene transfer in HIV-1 gene therapy.

**4. Vector systems for gene transfer** 

Adenoviral *ex vivo* + *in* 

Lentiviral *ex vivo* 

Foamyviral *ex vivo* 105-106 TU/ml

units; VP, vector particles; \* after concentration.

Gamma-

antiviral proteins directly in the lymphoid tissues is likely to lead to high and stable local peptide concentrations and to substantially suppress virus replication. On the other hand, secreted antiviral gene products no longer depend on expression in HIV-1 target cells, but instead several other cell types could serve as producer cells in the body. This facilitates the development of direct *in vivo* gene transfer approaches (e.g. by the use of AAV-vectors), making gene therapy less complex and practicable also for treatment of patients in the developing world. Furthermore, such strategies have the potential for application as a gene transfer vaccine in a prophylactic setting. In this regard, a secreted antiviral gene product could for instance be used as a genetic topical microbicide that aims at the prevention of HIV-1 mucosal transmission. High-level secretion of the antiviral molecules from genemodified target cells in the vagina or rectum has the potential to confer local sterilizing immunity, thus preventing HIV-1 genital transmission.

#### **3.5 Immunogenicity of the antiviral gene product**

Gene therapeutic strategies based on the expression of antiviral proteins are limited by the potential immunogenicity of the antiviral gene product, which can severely impair survival of the transduced cells. Antiviral RNAs have an advantage here, as they generally lack immunogenicity. Also, natural or only slightly altered variants of human proteins are not expected to mount significant immune responses. However, many antiviral proteins are non-self and bear the risk of eliciting a cellular immune response. Thus, to prevent selective deletion of the gene-modified cells by transgene-specific CTLs, it is necessary to minimize or eliminate immunogenicity of the antiviral gene product. The fusion of a Glycine-Alanine repeat derived from the Epstein-Barr virus nuclear antigen 1 (EBNA-1) to immunogenic proteins, such as transdominant HIV-1 Gag, has been shown to significantly reduce immunogenicity and prolong survival of transduced cells *in vivo* (Hammer et al., 2008). The Glycine-Alanine repeat protects the fusion protein from proteasomal degradation and prevents subsequent presentation of potentially immunogenic peptides on MHC class I molecules (Levitskaya et al., 1997). This frustrates the induction of CTLs directed against the transgene product and conceals it from CTL-mediated immune attack.

Another possibility to facilitate immune-evasion, which is feasible for small antiviral peptides only, is the generation of peptides which are devoid of MHC class I epitopes. Our group recently developed antiviral C peptides with potentially reduced immunogenicity, by mutating *in silico*-predicted immunodominant CTL epitopes within the peptide sequence. The mutated peptides retained excellent anti-HIV activity, while no immune responses could be detected in ELIspot assays (unpublished data).

#### **3.6 The target cell for intracellular immunization strategies**

Target cells for intracellular immunization are usually cells that can become infected with HIV-1 (mainly CD4+ T cells) or their progenitors (hematopoietic stem cells). For gene therapeutic approaches based on secreted antiviral molecules, the modification of non-HIVtarget cells is also feasible. So far, mature T cells and hematopoietic stem cells have been used in clinical trials. Advantages and disadvantages of both cell types are summarized in Table 1. Both have in common that they are relatively easy to obtain, and there are protocols for efficient *ex vivo* cultivation and transduction available. Gene modification of HSC has greater therapeutic potential, as it could restore a normal T cell repertoire, allow regeneration of HIV-specific T-helper cells and also protect monocytes/macrophages. However, current stem-cell based therapies are associated with greater risks and toxicity.

antiviral proteins directly in the lymphoid tissues is likely to lead to high and stable local peptide concentrations and to substantially suppress virus replication. On the other hand, secreted antiviral gene products no longer depend on expression in HIV-1 target cells, but instead several other cell types could serve as producer cells in the body. This facilitates the development of direct *in vivo* gene transfer approaches (e.g. by the use of AAV-vectors), making gene therapy less complex and practicable also for treatment of patients in the developing world. Furthermore, such strategies have the potential for application as a gene transfer vaccine in a prophylactic setting. In this regard, a secreted antiviral gene product could for instance be used as a genetic topical microbicide that aims at the prevention of HIV-1 mucosal transmission. High-level secretion of the antiviral molecules from genemodified target cells in the vagina or rectum has the potential to confer local sterilizing

Gene therapeutic strategies based on the expression of antiviral proteins are limited by the potential immunogenicity of the antiviral gene product, which can severely impair survival of the transduced cells. Antiviral RNAs have an advantage here, as they generally lack immunogenicity. Also, natural or only slightly altered variants of human proteins are not expected to mount significant immune responses. However, many antiviral proteins are non-self and bear the risk of eliciting a cellular immune response. Thus, to prevent selective deletion of the gene-modified cells by transgene-specific CTLs, it is necessary to minimize or eliminate immunogenicity of the antiviral gene product. The fusion of a Glycine-Alanine repeat derived from the Epstein-Barr virus nuclear antigen 1 (EBNA-1) to immunogenic proteins, such as transdominant HIV-1 Gag, has been shown to significantly reduce immunogenicity and prolong survival of transduced cells *in vivo* (Hammer et al., 2008). The Glycine-Alanine repeat protects the fusion protein from proteasomal degradation and prevents subsequent presentation of potentially immunogenic peptides on MHC class I molecules (Levitskaya et al., 1997). This frustrates the induction of CTLs directed against the

Another possibility to facilitate immune-evasion, which is feasible for small antiviral peptides only, is the generation of peptides which are devoid of MHC class I epitopes. Our group recently developed antiviral C peptides with potentially reduced immunogenicity, by mutating *in silico*-predicted immunodominant CTL epitopes within the peptide sequence. The mutated peptides retained excellent anti-HIV activity, while no immune responses

Target cells for intracellular immunization are usually cells that can become infected with HIV-1 (mainly CD4+ T cells) or their progenitors (hematopoietic stem cells). For gene therapeutic approaches based on secreted antiviral molecules, the modification of non-HIVtarget cells is also feasible. So far, mature T cells and hematopoietic stem cells have been used in clinical trials. Advantages and disadvantages of both cell types are summarized in Table 1. Both have in common that they are relatively easy to obtain, and there are protocols for efficient *ex vivo* cultivation and transduction available. Gene modification of HSC has greater therapeutic potential, as it could restore a normal T cell repertoire, allow regeneration of HIV-specific T-helper cells and also protect monocytes/macrophages. However, current stem-cell based therapies are associated with greater risks and toxicity.

immunity, thus preventing HIV-1 genital transmission.

transgene product and conceals it from CTL-mediated immune attack.

could be detected in ELIspot assays (unpublished data).

**3.6 The target cell for intracellular immunization strategies** 

**3.5 Immunogenicity of the antiviral gene product** 


Table 1. The target cell: T cells versus hematopoietic stem cells (HSC).

#### **4. Vector systems for gene transfer**

The choice of the vector system may have a major impact on the efficacy of HIV-1 gene therapy approaches. There is no ideal vector suitable for all purposes, but the pros and cons have to be balanced for each application. Table 2 summarizes advantages and disadvantages of several vector systems commonly used for gene transfer.


Table 2. Vector systems for gene transfer in HIV-1 gene therapy.

AAV, Adeno-Associated Virus; kb, kilo bases; SV-40, Simian Virus-40; TU, transducing units; VP, vector particles; \* after concentration.

Basic questions to be asked are, whether *ex vivo* or *in vivo* gene transfer is preferred and if long-term expression of the gene product is required. To our knowledge, a vector system that allows efficient direct *in vivo* gene transfer specifically into CD4+ T cells or HSC has not been developed so far. Therefore, approaches based on modification of these cell types (arming of T cells with TCRs or CARs, most intracellular immunizations) always rely on *ex vivo* gene transfer. In contrast, genes encoding secreted antiviral molecules may also be delivered to distinct production sites (e.g. liver, muscle) directly *in vivo* using adenovirus (Ad) or adeno-associated virus (AAV)-derived vectors. The second basic question deals with the long-term-expression of the transferred antiviral gene. Strategies involving arming of T

Gene Therapy for HIV-1 Infection 443

Vectors derived from gamma-retroviruses (mostly murine leukaemia virus) and lentiviruses (HIV-1) have been used in numerous clinical trials, including *ex vivo* gene transfer trials for HIV-1 infection (DiGiusto et al., 2010; van Lunzen et al., 2007). Replication incompetent viral vectors are made from these viruses by deletion of all genes encoding enzymes and structural proteins (Gag, Pol, Env) from the viral genome. These genes have to be added *in trans* to produce infectious, but replication incompetent, vector particles. The tropism of the vector particles can be altered by modification of the envelope glycoprotein or by pseudotyping with the envelope protein from a different virus (Frecha et al., 2008; Funke et al., 2008). A major difference between gamma-retroviral and lentiviral vectors is that lentiviruses can infect dividing as well as non-dividing cells. In contrast, gammaretroviruses can only transduce dividing cells, as they rely on the collapse of the nuclear membrane during mitosis to enter the nucleus (Roe et al., 1993). Lentiviral transduction protocols therefore usually require a shorter period of pre-activation of the cells. As prolonged *in vitro* culture is associated with differentiation and a loss of *in vivo* repopulation potential, especially for HSC, lentiviral vectors have an advantage here. However, largescale production of lentiviral vectors is more difficult than gamma-retroviral vector

Both, gamma-retroviral and lentiviral vectors integrate randomly into the host cell genome. While gamma-retroviruses usually integrate near transcriptional start sites, lentiviruses have a preference for transcribed regions (Mitchell et al., 2004). As a consequence, transduction with these vectors is always associated with a risk of transformation due to insertional mutagenesis. Indeed, severe side effects caused by vector integration have been reported in gene therapy clinical trials (Hacein-Bey-Abina et al., 2008; Howe et al., 2008). Experiments in animal models showed that vector genotoxicity is higher for transduction of hematopoietic stem cells than for mature T cells (Newrzela et al., 2008) and lower for self inactivating (SIN) vectors compared to conventional long terminal repeat (LTR)-driven vectors (Modlich et al., 2009). SIN vectors have deletions in the promotor and enhancer elements of the 3'LTR, thereby reducing the genotoxic risks, as transactivation of neighboring protooncogenes is less likely. In these vectors, expression of the transgene

Foamyviruses also belong to the family of retroviruses. Foamyviral vectors are generated by deleting enzymes and structural genes from the viral genome and adding these *in trans* during vector production (Rethwilm, 2007). Foamyvirus-derived vectors efficiently transduce resting cells, which makes them an ideal tool to transduce hematopoietic stem cells *ex vivo* (Hirata et al., 1996; Leurs et al., 2003). However, just like SV-40 vectors,

Simian virus-40 (SV-40) belongs to the family of Polyomaviridae. It has been one of the first viruses used as a gene transfer vehicle (Gething & Sambrook, 1981). For the construction of gene transfer vectors, all coding sequences except the origin of replication and the packaging signal can be deleted from wild type SV-40 (Strayer et al., 2002). The resulting vectors efficiently transduce hematopoietic stem cells and lymphocytes *in vitro*, but have

Past clinical trials have shown that random integration of a transgene delivered by an integrating vector bears the risk of severe side effects due to insertional mutagenesis.

**4.2 Integrating viral vectors** 

production due to a lack of stable packaging cell lines.

cassette is driven from an internal promoter.

foamyviral vectors have not yet been tested in clinical trials.

never been tested in clinical trials (Strayer et al., 2005).

**4.3 Targeted integration** 

cells with antigen-specific receptors and intracellular immunization require stable and longlasting production of the antiviral molecules in proliferating cells (T cells or HSC). Consequently, for such approaches, integrating vectors are favorable. These include SV-40 vectors and vectors derived from retroviruses. Stable expression of secreted gene products from slowly dividing cells like liver or muscle cells may also be achieved using nonintegrating vectors like adenoviral or AAV vectors.

#### **4.1 Non-integrating viral vectors**

The only vectors systems that currently allow direct *in vivo* gene transfer are non-integrating viral vectors. As their genome is not stably incorporated into the host cell chromosomes, these vectors have an improved safety profile compared to integrating vectors. For a direct *in vivo* application of integrating vectors, efficient systems for targeted vector delivery would be required, which are not yet available for use in man. Despite the lack of integration, non-integrating vector systems still allow sustained long-term transgene expression, if cells or tissues with a slow turnover are targeted, where the vector genome can stably persist. Moreover, non-integrating vectors can also be used to deliver zinc finger nucleases, which require only transient expression, to diving cells. A number of nonintegrating viral vectors have been evaluated for gene transfer. Currently, adenoviral vectors and vectors derived from the adeno-associated virus are in the focus of interest. Accordingly, Ad vectors are currently used in the above mentioned clinical trials to deliver CCR5-specific zinc finger nucleases to T cells *ex vivo*.

Recombinant adenoviral vectors have been utilized as a gene transfer and vaccine platform for a long time. Ad vectors provide a huge packaging capacity (36 kb), allowing the transfer also of multiple therapeutic genes. Moreover, high-titer production is possible, facilitating direct *in vivo* application with high transduction efficacies. The major obstacle of adenoviral vectors is pre-existing immunity in the general human population. Vector-mediated immune responses cause rapid clearance of Ad vectors, moreover, severe side effects have been observed. This can be partly overcome by using engineered adenovirus serotypes (Dharmapuri et al., 2009). Moreover, production of Ad vectors is prone to contamination with replication competent adenovirus, which complicates clinical grade vector production.

AAV is a non-pathogenic virus that belongs to the family of Parvoviridae. AAV-derived vectors have recently gained particular interest as gene transfer vehicles due to their apathogenicity and very low immunogenicity. Moreover, they can be used for direct *in vivo* gene delivery to both dividing and non-dividing cells. AAV can infect a variety of cell types *in vivo* and different serotypes of AAV have been shown to have varying preferences in their target cell type of choice (Chao et al., 2000; Halbert et al., 2000). However, AAV variants with a preference for T cells or hematopoietic stem cells have not been described. In the absence of the viral Rep protein, AAV vectors do not integrate into the host cell genome, but are maintained in episomal form in the nucleus. This allows very stable transgene expression without causing genotoxicity. The major disadvantage of AAV vectors is their small packaging capacity. In addition, vector production used to be laborious in the past and large-scale manufacturing for clinical trials was complicated. However, novel production systems facilitate faster and simpler high-titer production of AAV vectors in scaleable processes (Clement et al., 2009; Lock et al., 2010).

cells with antigen-specific receptors and intracellular immunization require stable and longlasting production of the antiviral molecules in proliferating cells (T cells or HSC). Consequently, for such approaches, integrating vectors are favorable. These include SV-40 vectors and vectors derived from retroviruses. Stable expression of secreted gene products from slowly dividing cells like liver or muscle cells may also be achieved using non-

The only vectors systems that currently allow direct *in vivo* gene transfer are non-integrating viral vectors. As their genome is not stably incorporated into the host cell chromosomes, these vectors have an improved safety profile compared to integrating vectors. For a direct *in vivo* application of integrating vectors, efficient systems for targeted vector delivery would be required, which are not yet available for use in man. Despite the lack of integration, non-integrating vector systems still allow sustained long-term transgene expression, if cells or tissues with a slow turnover are targeted, where the vector genome can stably persist. Moreover, non-integrating vectors can also be used to deliver zinc finger nucleases, which require only transient expression, to diving cells. A number of nonintegrating viral vectors have been evaluated for gene transfer. Currently, adenoviral vectors and vectors derived from the adeno-associated virus are in the focus of interest. Accordingly, Ad vectors are currently used in the above mentioned clinical trials to deliver

Recombinant adenoviral vectors have been utilized as a gene transfer and vaccine platform for a long time. Ad vectors provide a huge packaging capacity (36 kb), allowing the transfer also of multiple therapeutic genes. Moreover, high-titer production is possible, facilitating direct *in vivo* application with high transduction efficacies. The major obstacle of adenoviral vectors is pre-existing immunity in the general human population. Vector-mediated immune responses cause rapid clearance of Ad vectors, moreover, severe side effects have been observed. This can be partly overcome by using engineered adenovirus serotypes (Dharmapuri et al., 2009). Moreover, production of Ad vectors is prone to contamination with replication competent adenovirus, which complicates clinical

AAV is a non-pathogenic virus that belongs to the family of Parvoviridae. AAV-derived vectors have recently gained particular interest as gene transfer vehicles due to their apathogenicity and very low immunogenicity. Moreover, they can be used for direct *in vivo* gene delivery to both dividing and non-dividing cells. AAV can infect a variety of cell types *in vivo* and different serotypes of AAV have been shown to have varying preferences in their target cell type of choice (Chao et al., 2000; Halbert et al., 2000). However, AAV variants with a preference for T cells or hematopoietic stem cells have not been described. In the absence of the viral Rep protein, AAV vectors do not integrate into the host cell genome, but are maintained in episomal form in the nucleus. This allows very stable transgene expression without causing genotoxicity. The major disadvantage of AAV vectors is their small packaging capacity. In addition, vector production used to be laborious in the past and large-scale manufacturing for clinical trials was complicated. However, novel production systems facilitate faster and simpler high-titer production of AAV vectors in

integrating vectors like adenoviral or AAV vectors.

CCR5-specific zinc finger nucleases to T cells *ex vivo*.

scaleable processes (Clement et al., 2009; Lock et al., 2010).

**4.1 Non-integrating viral vectors** 

grade vector production.

#### **4.2 Integrating viral vectors**

Vectors derived from gamma-retroviruses (mostly murine leukaemia virus) and lentiviruses (HIV-1) have been used in numerous clinical trials, including *ex vivo* gene transfer trials for HIV-1 infection (DiGiusto et al., 2010; van Lunzen et al., 2007). Replication incompetent viral vectors are made from these viruses by deletion of all genes encoding enzymes and structural proteins (Gag, Pol, Env) from the viral genome. These genes have to be added *in trans* to produce infectious, but replication incompetent, vector particles. The tropism of the vector particles can be altered by modification of the envelope glycoprotein or by pseudotyping with the envelope protein from a different virus (Frecha et al., 2008; Funke et al., 2008). A major difference between gamma-retroviral and lentiviral vectors is that lentiviruses can infect dividing as well as non-dividing cells. In contrast, gammaretroviruses can only transduce dividing cells, as they rely on the collapse of the nuclear membrane during mitosis to enter the nucleus (Roe et al., 1993). Lentiviral transduction protocols therefore usually require a shorter period of pre-activation of the cells. As prolonged *in vitro* culture is associated with differentiation and a loss of *in vivo* repopulation potential, especially for HSC, lentiviral vectors have an advantage here. However, largescale production of lentiviral vectors is more difficult than gamma-retroviral vector production due to a lack of stable packaging cell lines.

Both, gamma-retroviral and lentiviral vectors integrate randomly into the host cell genome. While gamma-retroviruses usually integrate near transcriptional start sites, lentiviruses have a preference for transcribed regions (Mitchell et al., 2004). As a consequence, transduction with these vectors is always associated with a risk of transformation due to insertional mutagenesis. Indeed, severe side effects caused by vector integration have been reported in gene therapy clinical trials (Hacein-Bey-Abina et al., 2008; Howe et al., 2008). Experiments in animal models showed that vector genotoxicity is higher for transduction of hematopoietic stem cells than for mature T cells (Newrzela et al., 2008) and lower for self inactivating (SIN) vectors compared to conventional long terminal repeat (LTR)-driven vectors (Modlich et al., 2009). SIN vectors have deletions in the promotor and enhancer elements of the 3'LTR, thereby reducing the genotoxic risks, as transactivation of neighboring protooncogenes is less likely. In these vectors, expression of the transgene cassette is driven from an internal promoter.

Foamyviruses also belong to the family of retroviruses. Foamyviral vectors are generated by deleting enzymes and structural genes from the viral genome and adding these *in trans* during vector production (Rethwilm, 2007). Foamyvirus-derived vectors efficiently transduce resting cells, which makes them an ideal tool to transduce hematopoietic stem cells *ex vivo* (Hirata et al., 1996; Leurs et al., 2003). However, just like SV-40 vectors, foamyviral vectors have not yet been tested in clinical trials.

Simian virus-40 (SV-40) belongs to the family of Polyomaviridae. It has been one of the first viruses used as a gene transfer vehicle (Gething & Sambrook, 1981). For the construction of gene transfer vectors, all coding sequences except the origin of replication and the packaging signal can be deleted from wild type SV-40 (Strayer et al., 2002). The resulting vectors efficiently transduce hematopoietic stem cells and lymphocytes *in vitro*, but have never been tested in clinical trials (Strayer et al., 2005).

#### **4.3 Targeted integration**

Past clinical trials have shown that random integration of a transgene delivered by an integrating vector bears the risk of severe side effects due to insertional mutagenesis.

Gene Therapy for HIV-1 Infection 445

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Targeted integration of transgenes into the host cell genome is therefore expected to massively increase safety. The CCR5 locus is considered to be a safe harbor for transgene integration, as a naturally occurring deletion of 32 bp in the coding sequence for CCR5 causes no clinical symptoms. Moreover, this deletion is associated with a reduced susceptibility for HIV-1 infection (Huang et al., 1996; Samson et al., 1996). Therefore, targeted integration of an anti-HIV transgene into the CCR5 locus could even provide an additional antiviral effect, due to disruption of the CCR5 gene. Zinc finger nucleases binding to CCR5 have been used *in vitro* and in animal models to destroy the CCR5 locus, rendering the treated cells resistant to HIV-1 infection (Holt et al., 2010; Perez et al., 2008). A combination of this approach with targeted integration of antiviral genes holds especially great promise. For such a strategy a donor DNA encoding the desired antiviral gene and containing sequences homologous to the target site has to be present in the cells during the repair of ZFN-induced double-strand breaks by cellular enzymes. This results in the incorporation of the foreign DNA into the targeted region of the host genome by nonhomologous end joining mediated by the cellular DNA repair machinery (Cathomen & Joung, 2008). Such approaches require only transient expression of the zinc finger nuclease and the transgene to achieve stable integration into the host cell genome, which allows use of non-integrating vector systems for gene transfer.

As an alternative to zinc finger nucleases, AAV vectors that contain the viral Rep protein in *cis* or in *trans* can also be used for targeted integration, as in the presence of Rep, AAV vectors target their genome preferentially to a locus on the human chromosome 19, termed AAVS1, without causing any apparent adverse effects (Surosky et al., 1997). As the CCR5 locus, AAVS1 is thus considered a safe harbor for vector integration.

#### **5. Conclusions**

Gene therapeutic approaches for the treatment and possibly prevention of HIV-1 infection hold considerable promise. Although the final breakthrough has not yet been achieved in clinical trials, there has been substantial progress over the last years and future developments might leverage this technology. The major reason for the limited efficacy seen in all HIV-1 gene therapy clinical trials up to now has been the insufficient level of gene modification. It will therefore be particularly important to develop optimized therapeutic regimen and gene transfer technologies that allow therapeutically effective engraftment levels of functional, gene modified cells. Efficient protection of CD4+ T cells could possibly be achieved by using a combination of antiviral genes targeting different steps of the viral life cycle, conferring a substantial *in vivo* selective survival advantage and ideally also a therapeutic bystander effect on unmodified cells. This review describes the potent gene therapeutic tools that have been developed in the past years and it will be exciting to see if these can be integrated into an effective treatment regimen in the near future.

#### **6. References**

Amado, R. G.; Mitsuyasu, R. T.; Rosenblatt, J. D.; Ngok, F. K.; Bakker, A.; Cole, S.; Chorn, N.; Lin, L. S.; Bristol, G.; Boyd, M. P.; MacPherson, J. L.; Fanning, G. C.; Todd, A. V.; Ely, J. A.; Zack, J. A. & Symonds, G. P. (2004). Anti-human immunodeficiency virus hematopoietic progenitor cell-delivered ribozyme in a phase I study: myeloid and

Targeted integration of transgenes into the host cell genome is therefore expected to massively increase safety. The CCR5 locus is considered to be a safe harbor for transgene integration, as a naturally occurring deletion of 32 bp in the coding sequence for CCR5 causes no clinical symptoms. Moreover, this deletion is associated with a reduced susceptibility for HIV-1 infection (Huang et al., 1996; Samson et al., 1996). Therefore, targeted integration of an anti-HIV transgene into the CCR5 locus could even provide an additional antiviral effect, due to disruption of the CCR5 gene. Zinc finger nucleases binding to CCR5 have been used *in vitro* and in animal models to destroy the CCR5 locus, rendering the treated cells resistant to HIV-1 infection (Holt et al., 2010; Perez et al., 2008). A combination of this approach with targeted integration of antiviral genes holds especially great promise. For such a strategy a donor DNA encoding the desired antiviral gene and containing sequences homologous to the target site has to be present in the cells during the repair of ZFN-induced double-strand breaks by cellular enzymes. This results in the incorporation of the foreign DNA into the targeted region of the host genome by nonhomologous end joining mediated by the cellular DNA repair machinery (Cathomen & Joung, 2008). Such approaches require only transient expression of the zinc finger nuclease and the transgene to achieve stable integration into the host cell genome, which allows use

As an alternative to zinc finger nucleases, AAV vectors that contain the viral Rep protein in *cis* or in *trans* can also be used for targeted integration, as in the presence of Rep, AAV vectors target their genome preferentially to a locus on the human chromosome 19, termed AAVS1, without causing any apparent adverse effects (Surosky et al., 1997). As the CCR5

Gene therapeutic approaches for the treatment and possibly prevention of HIV-1 infection hold considerable promise. Although the final breakthrough has not yet been achieved in clinical trials, there has been substantial progress over the last years and future developments might leverage this technology. The major reason for the limited efficacy seen in all HIV-1 gene therapy clinical trials up to now has been the insufficient level of gene modification. It will therefore be particularly important to develop optimized therapeutic regimen and gene transfer technologies that allow therapeutically effective engraftment levels of functional, gene modified cells. Efficient protection of CD4+ T cells could possibly be achieved by using a combination of antiviral genes targeting different steps of the viral life cycle, conferring a substantial *in vivo* selective survival advantage and ideally also a therapeutic bystander effect on unmodified cells. This review describes the potent gene therapeutic tools that have been developed in the past years and it will be exciting to see if

Amado, R. G.; Mitsuyasu, R. T.; Rosenblatt, J. D.; Ngok, F. K.; Bakker, A.; Cole, S.; Chorn, N.;

Lin, L. S.; Bristol, G.; Boyd, M. P.; MacPherson, J. L.; Fanning, G. C.; Todd, A. V.; Ely, J. A.; Zack, J. A. & Symonds, G. P. (2004). Anti-human immunodeficiency virus hematopoietic progenitor cell-delivered ribozyme in a phase I study: myeloid and

of non-integrating vector systems for gene transfer.

**5. Conclusions** 

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**22** 

*Spain* 

**HIV-Screening Strategies for** 

*Ciudad Universitaria s/n, 28040, Madrid* 

**the Discovery of Novel HIV-Inhibitors** 

María José Abad, Luis Miguel Bedoya and Paulina Bermejo *Department of Pharmacology, Faculty of Pharmacy, University Complutense,* 

Since acquired immunodeficiency syndrome (AIDS) was recognized 27 years ago, 25 million people have died of human immunodeficiency virus (HIV)-related causes. On a global scale, although the HIV epidemic has stabilized since 2000, unacceptably high levels of new HIV infection and AIDS death still occur each year. In 2007, there were an estimated 33 million (30-36 million) people living with HIV, and 2 million (1.8-2.3 million) people died due to

There are two main types of HIV: type 1 (HIV-1) and type 2 (HIV-2) (Buonaguro et al., 2007). HIV-1 is the most prevalent in the worldwide pandemic. HIV-2 is present mainly in West Africa, where it was discovered in 1986, and infects about one million people worldwide. HIV-2 is slowly but continuously spreading throughout Europe, Asia and the Americas, and has reached a significant prevalence in countries such as Portugal and India. After more than 20 years of research, HIV remains a difficult target for a vaccine; thus the treatment of

A working knowledge of the HIV replication cycle is essential for understanding the mechanism of action of antiviral drugs. The HIV is an enveloped virus that contains two copies of viral genomic RNA in its core. In addition to the copies of RNA, the viral core also contains the enzymes required for HIV replication. The first step in the HIV replication cycle is the interaction between the envelope proteins of the virus (gp120) and specific host-cell surface receptors (e.g. the T-cell receptor CD4 on the cellular membrane) of the host cell. In the second step, the virus binds to the chemokine coreceptors CXC-chemokine receptor 4 (CXCR4) and CC-chemokine receptor 5 (CCR5). This induces a conformational change in gp120 that opens up a high affinity binding site located within the third variable loop (V3) and surrounding surfaces for the chemokine coreceptors CXCR4 and CCR5. This gives rise to further conformational rearrangements of gp120 that expose the transmembrane glycoprotein gp41, and the heptad repeat (HR) regions of the three subunits of gp41, HR1 and HR2, fold into a six-helical bundle. This ultimately results in the "fusion" of the viral envelope and the cytoplasmic membrane. Fusion creates a pore through which the viral

HIV encodes three enzymes required for replication: HIV-1 reverse transcriptase (RT), HIVintegrase (IN) and HIV-protease (PT). Following entry into the cell, the viral RT enzyme catalyzes the conversion of viral RNA into DNA. This viral DNA enters the nucleus and

AIDS, compared with an estimated 1.7 million (1.5-2.3 million) in 2001.

AIDS continues to focus on the search for chemical anti-HIV agents.

**1. Introduction** 

capsid enters the cells.

Roberts, M. R.; Masur, H. & Lane, H. C. (2000). Long-term in vivo survival of receptor-modified syngeneic T cells in patients with human immunodeficiency virus infection, *Blood*, Vol.96, No.2, pp. 467-474


### **HIV-Screening Strategies for the Discovery of Novel HIV-Inhibitors**

María José Abad, Luis Miguel Bedoya and Paulina Bermejo *Department of Pharmacology, Faculty of Pharmacy, University Complutense, Ciudad Universitaria s/n, 28040, Madrid Spain* 

#### **1. Introduction**

456 Recent Translational Research in HIV/AIDS

Walter, E. A.; Greenberg, P. D.; Gilbert, M. J.; Finch, R. J.; Watanabe, K. S.; Thomas, E. D. &

Woffendin, C.; Ranga, U.; Yang, Z.; Xu, L. & Nabel, G. J. (1996). Expression of a protective

Wong-Staal, F.; Poeschla, E. M. & Looney, D. J. (1998). A controlled, Phase 1 clinical trial to

Zhou, C.; Bahner, I. C.; Larson, G. P.; Zaia, J. A.; Rossi, J. J. & Kohn, E. B. (1994). Inhibition of

patients, *Proc Natl Acad Sci U S A*, Vol.93, No.7, pp. 2889-2894

hammerhead ribozymes, *Gene*, Vol.149, No.1, pp. 33-39

virus infection, *Blood*, Vol.96, No.2, pp. 467-474

*N Engl J Med*, Vol.333, No.16, pp. 1038-1044

Vol.9, No.16, pp. 2407-2425

Roberts, M. R.; Masur, H. & Lane, H. C. (2000). Long-term in vivo survival of receptor-modified syngeneic T cells in patients with human immunodeficiency

Riddell, S. R. (1995). Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor,

gene-prolongs survival of T cells in human immunodeficiency virus-infected

evaluate the safety and effects in HIV-1 infected humans of autologous lymphocytes transduced with a ribozyme that cleaves HIV-1 RNA, *Hum Gene Ther*,

HIV-1 in human T-lymphocytes by retrovirally transduced anti-tat and rev

Since acquired immunodeficiency syndrome (AIDS) was recognized 27 years ago, 25 million people have died of human immunodeficiency virus (HIV)-related causes. On a global scale, although the HIV epidemic has stabilized since 2000, unacceptably high levels of new HIV infection and AIDS death still occur each year. In 2007, there were an estimated 33 million (30-36 million) people living with HIV, and 2 million (1.8-2.3 million) people died due to AIDS, compared with an estimated 1.7 million (1.5-2.3 million) in 2001.

There are two main types of HIV: type 1 (HIV-1) and type 2 (HIV-2) (Buonaguro et al., 2007). HIV-1 is the most prevalent in the worldwide pandemic. HIV-2 is present mainly in West Africa, where it was discovered in 1986, and infects about one million people worldwide. HIV-2 is slowly but continuously spreading throughout Europe, Asia and the Americas, and has reached a significant prevalence in countries such as Portugal and India. After more than 20 years of research, HIV remains a difficult target for a vaccine; thus the treatment of AIDS continues to focus on the search for chemical anti-HIV agents.

A working knowledge of the HIV replication cycle is essential for understanding the mechanism of action of antiviral drugs. The HIV is an enveloped virus that contains two copies of viral genomic RNA in its core. In addition to the copies of RNA, the viral core also contains the enzymes required for HIV replication. The first step in the HIV replication cycle is the interaction between the envelope proteins of the virus (gp120) and specific host-cell surface receptors (e.g. the T-cell receptor CD4 on the cellular membrane) of the host cell. In the second step, the virus binds to the chemokine coreceptors CXC-chemokine receptor 4 (CXCR4) and CC-chemokine receptor 5 (CCR5). This induces a conformational change in gp120 that opens up a high affinity binding site located within the third variable loop (V3) and surrounding surfaces for the chemokine coreceptors CXCR4 and CCR5. This gives rise to further conformational rearrangements of gp120 that expose the transmembrane glycoprotein gp41, and the heptad repeat (HR) regions of the three subunits of gp41, HR1 and HR2, fold into a six-helical bundle. This ultimately results in the "fusion" of the viral envelope and the cytoplasmic membrane. Fusion creates a pore through which the viral capsid enters the cells.

HIV encodes three enzymes required for replication: HIV-1 reverse transcriptase (RT), HIVintegrase (IN) and HIV-protease (PT). Following entry into the cell, the viral RT enzyme catalyzes the conversion of viral RNA into DNA. This viral DNA enters the nucleus and

HIV-Screening Strategies for the Discovery of Novel HIV-Inhibitors 459

Rash, hepatotoxicity

Rash

Rash

Raltegravir (MK-0518) Diarrhea, injection-site reactions, headache

Table 1. Approved antiretroviral drugs for the treatment of HIV infection

The multiple steps of the HIV replication cycle present novel therapeutic targets other than the viral enzyme RT and PT for drug developement. Continued efforts have been made to discover new inhibitors that target not only RT and PT but also other viral targets, achievements that have been reviewed comprehensively in the literature. Several recent novel target inhibitors were discovered using virus-based screening approaches (Table 2). Alternatively, PIs, the next generation of NNRTIs, CCR5 antagonist and IN inhibitors were identified by structure-based drug design, receptor pharmacology and biochemical screening approaches (Westby et al., 2005, Menéndez-Arias & Tözser, 2008, Greene et al., 2008, Liang, 2008, Pang et al., 2009, Marchand et al., 2009, Tan et al., 2010). Historical precedent therefore suggests that diverse screening strategies should be employed for the discovery of new HIV-1 agents. In this review we present a brief overview of various HIV-1 screening strategies and highlight novel approaches and/or significant advances in HIV-1

As mentioned above, HIV cellular entry is a multistep process that requires the interaction of a viral envelope glycoprotein (gp120) and a host receptor (CD4), followed by binding to a coreceptor (CCR5 and CXCR4). The proteins involved in the entry process have become attractive targets for drug design, and HIV-1 replication screens have successfully identified compounds with antiviral activity that act at each of these three steps of HIV entry (Grande

The chemokine receptors CCR5 and CXCR4, membrane proteins belonging to the G-protein coupled receptor super-family, have been identified as essential coreceptors for HIV entry into the cells, and molecules that inhibit HIV entry by targeting CCR5 and CXCR4 have been

Headache, anorexia, leukopenia

Dizziness, hallucinations, insomnio

Stomach upset, diarrhea, nausea Rash, elevated bilirubin, depression Rash, hypertriglyceridemia, diarrhea Stomach upset, diarrhea, nausea Kidney stones, vomiting, headache Diarrhea, headache, fatigue Diarrhea, nausea, rash

Stomach upset, vomiting, taste disturbance Stomach upset, headache, abdominal pain Hypercholesterolemia, diarrhea, nausea

**GENERIC NAME ADVERSE REACTIONS** 

Zidovudine (AZT) *Non-nucleoside inhibitors*  Delavirdine (DLV) Efavirenz (EFV) Etravirine (THC125) Nevirapine (NVP)

**Integrase inhibitors** 

**Protease inhibitors**  Amprenavir (AMP) Atazanavir (ATZ) Darunavir (TMC-114) Fosamprenavir (GW-433908)

Indinavir (IDV) Lopinavir (ABT-378) Nelfinavir (NFV) Ritonavir (RTV) Saquinavir (SQV) Tripanavir (TPV)

screening technology.

et al., 2008, Wang & Duan, 2009).

**2. HIV-1 Entry** 

becomes inserted into the chromosomal DNA of the host cell (integration). This process is facilitated by the viral enzyme IN. Expression of the viral genes leads to production of precursor viral proteins. These proteins and viral RNA are assembled at the cell surface into new viral particles and leave the host cell by a process called budding. During the budding process, they acquire the outer layer and envelope. At this stage, the PT enzyme cleaves the precursor viral proteins into their mature products. If this final phase of the replication cycle does not take place, the released viral particles are non-infectious and not competent to initiate the replication cycle in other susceptible cells.

Once HIV has entered the cell, it must disarm and hijack the intracellular machinery for its own benefit. Normal cell functionality of viral hosts is altered by invading virus proteins to the benefit of the virus. Viral proteins are known to compete with the host proteins, thus disrupting the normal host protein-protein interaction network. HIV-1 encodes the regulatory proteins, Tat and Rev, and four accessory proteins: viral infectivity factor (Vif), viral protein R (Vpr), viral protein U (Vpu) and negative factor (Nef) (Romani & Engelbrecht, 2009, Romani et al., 2010). The regulatory proteins are essential for virus replication by controlling HIV gene expression in host cells. In contrast, accessory proteins are often dispensable for virus replication *in vitro*. The Vif directly binds to and inactivates cellular deoxycytidine deaminase APOBEC3G, a natural antiviral factor that promotes G- to A-hypermutation of viral DNA during reverse transcription. The Vpu has been shown to down-regulate the CD4 receptor, and is also required for effective release of newly formed viral particles.

Anti-HIV drugs are classified into different groups according to their activity on the replicative cycle of HIV. These are virus-cell adsorption, virus-cell fusion, uncoating, reverse transcription, integration, DNA replication, transcription, translation, budding (assembly/release) and maturation. There are currently 25 compounds approved for the treatment of HIV, and most of these are nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) or protease inhibitors (PIs) (Warnke & Barreto, 2007, Zhan et al., 2009) (Table 1). Highly active antiretroviral therapy (HAART), which combines several such drugs (typically three or four), has dramatically improved patients' lives. The therapeutic effects are limited, however, by adverse effects and toxicities caused by long-term use and the emergence of drug resistance.


becomes inserted into the chromosomal DNA of the host cell (integration). This process is facilitated by the viral enzyme IN. Expression of the viral genes leads to production of precursor viral proteins. These proteins and viral RNA are assembled at the cell surface into new viral particles and leave the host cell by a process called budding. During the budding process, they acquire the outer layer and envelope. At this stage, the PT enzyme cleaves the precursor viral proteins into their mature products. If this final phase of the replication cycle does not take place, the released viral particles are non-infectious and not competent to

Once HIV has entered the cell, it must disarm and hijack the intracellular machinery for its own benefit. Normal cell functionality of viral hosts is altered by invading virus proteins to the benefit of the virus. Viral proteins are known to compete with the host proteins, thus disrupting the normal host protein-protein interaction network. HIV-1 encodes the regulatory proteins, Tat and Rev, and four accessory proteins: viral infectivity factor (Vif), viral protein R (Vpr), viral protein U (Vpu) and negative factor (Nef) (Romani & Engelbrecht, 2009, Romani et al., 2010). The regulatory proteins are essential for virus replication by controlling HIV gene expression in host cells. In contrast, accessory proteins are often dispensable for virus replication *in vitro*. The Vif directly binds to and inactivates cellular deoxycytidine deaminase APOBEC3G, a natural antiviral factor that promotes G- to A-hypermutation of viral DNA during reverse transcription. The Vpu has been shown to down-regulate the CD4 receptor, and is also required for effective release of newly formed

Anti-HIV drugs are classified into different groups according to their activity on the replicative cycle of HIV. These are virus-cell adsorption, virus-cell fusion, uncoating, reverse transcription, integration, DNA replication, transcription, translation, budding (assembly/release) and maturation. There are currently 25 compounds approved for the treatment of HIV, and most of these are nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) or protease inhibitors (PIs) (Warnke & Barreto, 2007, Zhan et al., 2009) (Table 1). Highly active antiretroviral therapy (HAART), which combines several such drugs (typically three or four), has dramatically improved patients' lives. The therapeutic effects are limited, however, by adverse effects

and toxicities caused by long-term use and the emergence of drug resistance.

Maraviroc (UK-427) Upper respiratory tract infection, cough, pyrexia

Diarrhea, nausea, headache

Rash, nausea, lipoatrophy

Diarrhea, nausea osteopenia

Rash, abdominal pain, peripheral neuropathy Hyperpigmentation of skin, rash, diarrhea

Decrease in appetite, headache, fatigue

Hepatic steatosis, peripheral neuropathy

**GENERIC NAME ADVERSE REACTIONS** 

Enfuvirtide (T20) Pruritus, pain, discomfort

initiate the replication cycle in other susceptible cells.

viral particles.

**Entry inhibitors** 

**Fusion inhibitors** 

*Nucleoside inhibitors*  Abacavir (ABC) Didanosine (ddl) Emtricitabine (FTC) Stavudine (d4T) Lamivudine (3TC) Tenofovir (DF) Zalcitabine (ddC)

**Reverse transcriptase inhibitors** 


Table 1. Approved antiretroviral drugs for the treatment of HIV infection

The multiple steps of the HIV replication cycle present novel therapeutic targets other than the viral enzyme RT and PT for drug developement. Continued efforts have been made to discover new inhibitors that target not only RT and PT but also other viral targets, achievements that have been reviewed comprehensively in the literature. Several recent novel target inhibitors were discovered using virus-based screening approaches (Table 2). Alternatively, PIs, the next generation of NNRTIs, CCR5 antagonist and IN inhibitors were identified by structure-based drug design, receptor pharmacology and biochemical screening approaches (Westby et al., 2005, Menéndez-Arias & Tözser, 2008, Greene et al., 2008, Liang, 2008, Pang et al., 2009, Marchand et al., 2009, Tan et al., 2010). Historical precedent therefore suggests that diverse screening strategies should be employed for the discovery of new HIV-1 agents. In this review we present a brief overview of various HIV-1 screening strategies and highlight novel approaches and/or significant advances in HIV-1 screening technology.

#### **2. HIV-1 Entry**

As mentioned above, HIV cellular entry is a multistep process that requires the interaction of a viral envelope glycoprotein (gp120) and a host receptor (CD4), followed by binding to a coreceptor (CCR5 and CXCR4). The proteins involved in the entry process have become attractive targets for drug design, and HIV-1 replication screens have successfully identified compounds with antiviral activity that act at each of these three steps of HIV entry (Grande et al., 2008, Wang & Duan, 2009).

The chemokine receptors CCR5 and CXCR4, membrane proteins belonging to the G-protein coupled receptor super-family, have been identified as essential coreceptors for HIV entry into the cells, and molecules that inhibit HIV entry by targeting CCR5 and CXCR4 have been

HIV-Screening Strategies for the Discovery of Novel HIV-Inhibitors 461

High-throughput screening technologies designed to identify compounds that inhibit binding of natural ligands to their cognate G-protein-coupled receptor have been used successfully by the pharmaceutical industry for many years. The disadvantage of this approach is the dependence upon a radiolabeled ligand, which involved a high cost and arouses significant environmental concern when screening large chemical libraries. It is therefore unlikely that radiolabeled ligand binding assays will be widely used in the future. More recently, assays have been developed which identify compounds that inhibit receptor function rather than ligand binding (and thus avoid the need for radiolabeled chemokines). HIV is an enveloped virus, and its envelope proteins complex (Env) controls the key process of viral entry. Env is a complex composed of a transmembrane gp41 subunit and a noncovalently-associated surface gp120 subunit. Infection is initiated by the binding of the virion gp120 Env protein to the CD4 molecule present on some T-cells, macrophages and microglial cells. The interaction induces a conformational change that promotes secondary gp120 binding to the coreceptor CCR5 and CXCR4. Both coreceptors are members of the chemokine receptor family, but CCR5 is the coreceptor for HIV-1 strains that infect macrophages (M-tropic or R5 strains), while CXCR4 is the coreceptor for HIV-1 strains that infect T-cells (T-tropic or X4 strains). Ochsenbauer-Jambor et al. (2006) introduced a T-cell based receptor reporter cell line (JLTRG-RS) that expresses both HIV-1 coreceptors, CXCR4 and CCR5, and offers the convenience of using enhanced green fluorescent protein (EGFP) as a direct and quantitative marker. Unlike previous EGFP-based reporter cell lines, JLTRG-RS cells have an unusually high dynamic signal range, sufficient for plate reader detection using a 384-well format. Because EGFP can be directly and continuously quantified in cell culture, the reporter cell line requires no manipulation during assay preparation or analysis. These characteristics make the system extremely flexible, rapid and inexpensive. Due to its intrinsic flexibility, the JLTRG-RS cell-based reporter system provides a powerful tool which

Immortalized cell lines, transfected with the HIV-1 Env gene, express gp120/gp41 on their surface and can fuse to cells co-expressing CD4 and either CCR5 and CXCR4. Screens based on this approach have been described by a number of laboratories. A cell-based enzymelinked immunosorbent assay (ELISA) was developed using and anti-CXCR4 monoclonal antibody, 12G5, and cells expressing CD4 and CXCR5, the U373-MAGI-CXCR4 (CEM) cell line (Zhao et al., 2003). The assay was sensitive to the well-characterized CXCR4 antagonists, T22, T14012 (a downsized analog of T22) and AMD3100, which effectively inhibited 12G5 binding to CXCR4-expressing cells whereas HIV-1 entry inhibitors targeting CD4 and gp41 in addition to HIV-1 RT and PT inhibitors, did not block the binding of 12G5 to U373-MAGI-CXCR4 (CEM) cells. This suggests that the cell-based ELISA is specific, sensitive,

More recently, two new T-cell-based reporter cell lines were established to measure HIV-1 infectivity (Chilba-Mizutani et al., 2007). One cell naturally expresses CD4 and CXCR4, making it susceptible to X4-tropic viruses, and the other cell line, in which a CCR5 expression vector was introduced, is susceptible to both X4- and R5-tropic viruses. Reporter cells were constructed by transfecting the human T-cell line HPB-Ma, which demonstrated high susceptibility to HIV-1, with genomes expressing two different luciferase reporters: HIV-1 long terminal repeat (LTR)-driven firefly luciferase and cytomegalovirus promoterdriven renilla luciferase. The cell lines were also beneficial for screening new antiretroviral agents, as false inhibition caused by the cytotoxicity of test compounds was easily detected

will considerably facilitate future screening for HIV inhibitors.

convenient, rapid and economical.

by monitoring renilla luciferase activity.


Table 2. Selected novel target inhibitors with potential application for the treatment of HIV infection

in rapid development as antiviral agents. Additionally, the envelope glycoprotein gp120 exists in its native form as a homopolymeric trimer, held on the outer surface of the virion by non-covalent interactions with a fusion glycoprotein gp41 trimer. The crystal structure of gp120 core bound to CD4 reveals specific targets for developing anti-HIV drugs.

**Attachment inhibitors**  CD4 binding peptides

Cyclotriazadisulfonamide Sulfated polysaccharides **CCR5 antagonists** 

1,3,4-trisubstituted pyrrolidines

Prostatin (12-deoxyphorbol ester)

Prostatin (12-deoxyphorbol ester)

Cyclic penta- and tetrapeptides Diketopiperazine mimetics Tetrahydroquinolines

Thiazolylisothiourea derivatives

**Reverse transcriptase inhibitors** 

Tetrazole thioacetinilide derivatives

Peptidomimetic protease inhibitors

Fusion inhibitors peptides Pyrrole derivatives

Nevirapine analogs Efavirenz analogs

**Protease inhibitors**  Cyclic urea derivatives

Calanolide A **Integrase inhibitors**  β-diketo acids GS-9137 Chalcones

infection

**CXCR4 inhibitors** 

Benzodiazepines Alkylamine analogs Non-peptide derivatives **Fusion inhibitors** 

Bicyclams

Ureas

Aminoglycoside-arginine conjugates Poly-arginine aminoglycoside conjugates

1-(3,3-diphenylpropyl)-piperidinyl amides

Non-cyclam polynitrogenated compounds

1-amino-2-phenyl-4-(piperidin-1-yl) butane analogs

3-(4-benzylpiperidin-1-yl)-N-phenylpropylamine derivatives

Tetrahydroimidazo-[4,5,1-jk][1,4]-benzodiazepinone derivatives

Table 2. Selected novel target inhibitors with potential application for the treatment of HIV

in rapid development as antiviral agents. Additionally, the envelope glycoprotein gp120 exists in its native form as a homopolymeric trimer, held on the outer surface of the virion by non-covalent interactions with a fusion glycoprotein gp41 trimer. The crystal structure of

gp120 core bound to CD4 reveals specific targets for developing anti-HIV drugs.

High-throughput screening technologies designed to identify compounds that inhibit binding of natural ligands to their cognate G-protein-coupled receptor have been used successfully by the pharmaceutical industry for many years. The disadvantage of this approach is the dependence upon a radiolabeled ligand, which involved a high cost and arouses significant environmental concern when screening large chemical libraries. It is therefore unlikely that radiolabeled ligand binding assays will be widely used in the future. More recently, assays have been developed which identify compounds that inhibit receptor function rather than ligand binding (and thus avoid the need for radiolabeled chemokines). HIV is an enveloped virus, and its envelope proteins complex (Env) controls the key process of viral entry. Env is a complex composed of a transmembrane gp41 subunit and a noncovalently-associated surface gp120 subunit. Infection is initiated by the binding of the virion gp120 Env protein to the CD4 molecule present on some T-cells, macrophages and microglial cells. The interaction induces a conformational change that promotes secondary gp120 binding to the coreceptor CCR5 and CXCR4. Both coreceptors are members of the chemokine receptor family, but CCR5 is the coreceptor for HIV-1 strains that infect macrophages (M-tropic or R5 strains), while CXCR4 is the coreceptor for HIV-1 strains that infect T-cells (T-tropic or X4 strains). Ochsenbauer-Jambor et al. (2006) introduced a T-cell based receptor reporter cell line (JLTRG-RS) that expresses both HIV-1 coreceptors, CXCR4 and CCR5, and offers the convenience of using enhanced green fluorescent protein (EGFP) as a direct and quantitative marker. Unlike previous EGFP-based reporter cell lines, JLTRG-RS cells have an unusually high dynamic signal range, sufficient for plate reader detection using a 384-well format. Because EGFP can be directly and continuously quantified in cell culture, the reporter cell line requires no manipulation during assay preparation or analysis. These characteristics make the system extremely flexible, rapid and inexpensive. Due to its intrinsic flexibility, the JLTRG-RS cell-based reporter system provides a powerful tool which will considerably facilitate future screening for HIV inhibitors.

Immortalized cell lines, transfected with the HIV-1 Env gene, express gp120/gp41 on their surface and can fuse to cells co-expressing CD4 and either CCR5 and CXCR4. Screens based on this approach have been described by a number of laboratories. A cell-based enzymelinked immunosorbent assay (ELISA) was developed using and anti-CXCR4 monoclonal antibody, 12G5, and cells expressing CD4 and CXCR5, the U373-MAGI-CXCR4 (CEM) cell line (Zhao et al., 2003). The assay was sensitive to the well-characterized CXCR4 antagonists, T22, T14012 (a downsized analog of T22) and AMD3100, which effectively inhibited 12G5 binding to CXCR4-expressing cells whereas HIV-1 entry inhibitors targeting CD4 and gp41 in addition to HIV-1 RT and PT inhibitors, did not block the binding of 12G5 to U373-MAGI-CXCR4 (CEM) cells. This suggests that the cell-based ELISA is specific, sensitive, convenient, rapid and economical.

More recently, two new T-cell-based reporter cell lines were established to measure HIV-1 infectivity (Chilba-Mizutani et al., 2007). One cell naturally expresses CD4 and CXCR4, making it susceptible to X4-tropic viruses, and the other cell line, in which a CCR5 expression vector was introduced, is susceptible to both X4- and R5-tropic viruses. Reporter cells were constructed by transfecting the human T-cell line HPB-Ma, which demonstrated high susceptibility to HIV-1, with genomes expressing two different luciferase reporters: HIV-1 long terminal repeat (LTR)-driven firefly luciferase and cytomegalovirus promoterdriven renilla luciferase. The cell lines were also beneficial for screening new antiretroviral agents, as false inhibition caused by the cytotoxicity of test compounds was easily detected by monitoring renilla luciferase activity.

HIV-Screening Strategies for the Discovery of Novel HIV-Inhibitors 463

HIV-1 RT RNase, and rapidly reported cleavage of the RNA strand with a 14-fold increase in fluorescence intensity. The PhpC-based assay for RNase H was superior to the traditional

HIV-IN represents a potential target for the development of new anti-HIV chemotherapeutic agents. This viral enzyme is required for the integration of viral DNA into the host DNA, which catalyzes two reactions known as processing and strand transfer. The viral DNA is first cleaved by HIV IN at a CA dinucleotide at the 3'-end to leave the two-nucleotide overhanging. This step is known as processing. Then, the protein-DNA complex is transported into the nucleus. The host DNA is cleaved to leave a 5' overhang of five bases, and the 3'-ends of the viral DNA are convalently linked to the 5'-end of the host DNA. Finally, the 5-bases gap between the 5'-end of the viral DNA and the 3'-end of the host DNA is filled in by host cell enzymes. Since IN-negative mutants of HIV do not produce infectious virus particles, and no cellular homologue of HIV IN has been described, IN is considered to be an attractive target. However, in contrast to RT and PT, not a single IN inhibitor has yet entered the anti-HIV drug market. However, using *in vitro* assay systems and the

Most currently used assays for HIV-1 IN target the strand transfer process and follow a similar premise. HIV IN is combined with donor dsDNA, which has been immobilized onto a solid support, to form an enzyme/DNA complex. The reaction is then initiated by the addition of target dsDNA labelled in some manner, and after an incubation period, the ligated product is quantified. John et al. (2005) reported a highly efficient and sensitive high-throughput screen, HIV IN Target SRI Assay for HIV-1 IN activity, using 5' biotin-labelled DNA (5' BIO donor) and 3' digoxygenin-labelled DNA (3' DIG target). Following 3' processing of the 5' BIO donor, strand transfer proceeds with integration of the 5' BIO donor into the 3' DIG target. The assay was used to screen drugs in a high-throughput format, and the assay was also adapted to study mechanistic questions regarding the integration process. For example, using variations of the assay format, it showed a high preference of the E strand of the LTR viral DNA as a target strand compared with its complementary A strand. Wang et al. (2005) described two homogeneous time-resolved FRET-based assays for the measurement of HIV-1 IN 3' processing and strand transfer activities. These assays have also proven their utility for the identification of mechanistically interesting and biologically active inhibitors of HIV-1 IN that

In addition to recombinant enzyme screens, biochemical assays have been developed that measure HIV-1 IN activity in the context of the preintegration complex (PIC), which mediates the integration of the retroviral genome into host cell DNA. The HIV PIC is a large nucleoprotein complex containing the viral CDNA and IN as well as matrix Vpr, RT and a number of host proteins including histones and members of the non-homologous end joining pathway. It is possible that screening for PIC activity, analogous to that in a true infection, may offer an expanded set of targets and yield more biologically relevant compounds. A polymerase chain reaction-based assay for integration has been reported which employs HIV-

In a later stage of the HIV-1 life cycle, HIV PT hydrolyzes precursor polyproteins into functional proteins that are essential for viral assembly and subsequent activity. HIV-1 Gag and Pol polypeptide precursors are cleaved by the viral encoded aspartyl protease to form

molecular beacon approach in terms of responsiveness, speed and ease.

recombinant HIV-1 IN, a variety of HIN IN inhibitors have been identified.

hold potential for further development into potential antiviral drugs.

1 PICs derived from cells infected with single-cycle HIV-1 reporter viruses.

**4. HIV-1 protease** 

#### **3. HIV-1 enzyme targets**

HIV-1 encodes three enzymes required for replication: HIV-1 RT, HIV-1 IN and HIV-1 PT. A number of assays have been developed for screening test compounds against thyese wellknown targetrs fir drug discovery. Utilization in screening campaigns of RT or PT enzymes that contain drug resistant mutations is a common strategy for identifying next-generation HIV-1 inhibitors against these targets.

HIV-1 RT is a multifunctional enzyme involved in several essential activities for viral replication (Sarafianos et al., 2009, Herschhorn & Hiz, 2010). These activities include DNAand RNA-dependent DNA polymerase, ribonuclease H (RNase H), strand transfer and strand displacement activities. RT has been the main target of current antiviral therapies against AIDS. NRTIs have been widely used in HAART, combined with PIs and/or NNRTIs. The high error rates characteristic of HIV-1 RT, however, are a presumed source of the viral hypermutability that contributes mainly to the emergence of resistant variants, although the significant toxicity associated with current anti-HIV drugs also results in treatment failure. These factors in combination drive pharmacologists to develop more potent and less toxic RT inhibitors against the native and drug-resistant variants, which will most certainly remain critical components of future drug regimens.

Although currently marketed agents inhibit the DNA polymerase activity of HIV-1 RT, inhibition of any of the step in the reverse transcription process would result in inhibition of viral replication. Therefore various assays suitable for testing compounds in a highthroughput screening format have been described for measuring the DNA polymerase, RNase H and DNA strand transfer activities of HIV-1 RT.

Examples of isotopic assays for measuring DNA polymerase activity include "microarray compound screening technology", and "scintillation proximity assay technology" (Xuei et al., 2003). Inhibition reverse transcription by targeting the RNase H activity of HIV-1 RT is another approach of interest, since mutations in the NNRTI allosteric domain or the RT active site are not expected to affect inhibitors that bind to the RNase H domain. Although RNase H-mediated cleavage of hybrid RNA/DNA duplex occurs either concurrently with DNA polymerization or independently, most RNase H assays target the latter. Parniak et al. (2003) described a homogeneous "fluorescence resonance energy transfer" (FRET) assay for measuring RNase H activity. The duplex substrate contains a fluorescein label on the 3'-end of the RNA, which is quenched by a Dabcyl label on the 5'-end of the DNA strand. When the substrate is cleaved by RNase H, the interaction between the fluorescein and Dabcyl is removed, resulting in an increase in the fluorescence signal.

A fluorescence polarization (FP) microplate assay for screening compounds against the RNase H activity of HIV-1 RT has also been developed (Nakayama et al., 2006). This homogeneous assay uses a hybrid 18-mer DNA/RNA duplex substrate composed of an RNA oligonucleotide labelled with 6-carboxytetramethyl rhodamine at the 3'-end, thast is annealed to a complementary unlabeled DNA strand substrate cleavage by RNase H to produce small RNA fragments (1-4 mer), resulting in a significant change in the measured FP value.

More recently, a 6-phenylpyrrolocytidine (PhpC)-based assay has been incorporated into highthroughput microplate assay format, and may form the basis for a new screen for inhibitors of HIV-1 RNase H (Wahba et al., 2010). The PhpC-containing RNA formed native-like duplex structures with complementary DNA or RNA. The PhpC-modification was found to act as a sensitive reporter group, and was non-disruptive to structure and the enzymatic activity of RNase H. A RNA/DNA hybrid possessing a single PhpC insert was an excellent substrate for

HIV-1 encodes three enzymes required for replication: HIV-1 RT, HIV-1 IN and HIV-1 PT. A number of assays have been developed for screening test compounds against thyese wellknown targetrs fir drug discovery. Utilization in screening campaigns of RT or PT enzymes that contain drug resistant mutations is a common strategy for identifying next-generation

HIV-1 RT is a multifunctional enzyme involved in several essential activities for viral replication (Sarafianos et al., 2009, Herschhorn & Hiz, 2010). These activities include DNAand RNA-dependent DNA polymerase, ribonuclease H (RNase H), strand transfer and strand displacement activities. RT has been the main target of current antiviral therapies against AIDS. NRTIs have been widely used in HAART, combined with PIs and/or NNRTIs. The high error rates characteristic of HIV-1 RT, however, are a presumed source of the viral hypermutability that contributes mainly to the emergence of resistant variants, although the significant toxicity associated with current anti-HIV drugs also results in treatment failure. These factors in combination drive pharmacologists to develop more potent and less toxic RT inhibitors against the native and drug-resistant variants, which will

Although currently marketed agents inhibit the DNA polymerase activity of HIV-1 RT, inhibition of any of the step in the reverse transcription process would result in inhibition of viral replication. Therefore various assays suitable for testing compounds in a highthroughput screening format have been described for measuring the DNA polymerase,

Examples of isotopic assays for measuring DNA polymerase activity include "microarray compound screening technology", and "scintillation proximity assay technology" (Xuei et al., 2003). Inhibition reverse transcription by targeting the RNase H activity of HIV-1 RT is another approach of interest, since mutations in the NNRTI allosteric domain or the RT active site are not expected to affect inhibitors that bind to the RNase H domain. Although RNase H-mediated cleavage of hybrid RNA/DNA duplex occurs either concurrently with DNA polymerization or independently, most RNase H assays target the latter. Parniak et al. (2003) described a homogeneous "fluorescence resonance energy transfer" (FRET) assay for measuring RNase H activity. The duplex substrate contains a fluorescein label on the 3'-end of the RNA, which is quenched by a Dabcyl label on the 5'-end of the DNA strand. When the substrate is cleaved by RNase H, the interaction between the fluorescein and Dabcyl is

A fluorescence polarization (FP) microplate assay for screening compounds against the RNase H activity of HIV-1 RT has also been developed (Nakayama et al., 2006). This homogeneous assay uses a hybrid 18-mer DNA/RNA duplex substrate composed of an RNA oligonucleotide labelled with 6-carboxytetramethyl rhodamine at the 3'-end, thast is annealed to a complementary unlabeled DNA strand substrate cleavage by RNase H to produce small

More recently, a 6-phenylpyrrolocytidine (PhpC)-based assay has been incorporated into highthroughput microplate assay format, and may form the basis for a new screen for inhibitors of HIV-1 RNase H (Wahba et al., 2010). The PhpC-containing RNA formed native-like duplex structures with complementary DNA or RNA. The PhpC-modification was found to act as a sensitive reporter group, and was non-disruptive to structure and the enzymatic activity of RNase H. A RNA/DNA hybrid possessing a single PhpC insert was an excellent substrate for

RNA fragments (1-4 mer), resulting in a significant change in the measured FP value.

most certainly remain critical components of future drug regimens.

RNase H and DNA strand transfer activities of HIV-1 RT.

removed, resulting in an increase in the fluorescence signal.

**3. HIV-1 enzyme targets** 

HIV-1 inhibitors against these targets.

HIV-1 RT RNase, and rapidly reported cleavage of the RNA strand with a 14-fold increase in fluorescence intensity. The PhpC-based assay for RNase H was superior to the traditional molecular beacon approach in terms of responsiveness, speed and ease.

HIV-IN represents a potential target for the development of new anti-HIV chemotherapeutic agents. This viral enzyme is required for the integration of viral DNA into the host DNA, which catalyzes two reactions known as processing and strand transfer. The viral DNA is first cleaved by HIV IN at a CA dinucleotide at the 3'-end to leave the two-nucleotide overhanging. This step is known as processing. Then, the protein-DNA complex is transported into the nucleus. The host DNA is cleaved to leave a 5' overhang of five bases, and the 3'-ends of the viral DNA are convalently linked to the 5'-end of the host DNA. Finally, the 5-bases gap between the 5'-end of the viral DNA and the 3'-end of the host DNA is filled in by host cell enzymes. Since IN-negative mutants of HIV do not produce infectious virus particles, and no cellular homologue of HIV IN has been described, IN is considered to be an attractive target. However, in contrast to RT and PT, not a single IN inhibitor has yet entered the anti-HIV drug market. However, using *in vitro* assay systems and the recombinant HIV-1 IN, a variety of HIN IN inhibitors have been identified.

Most currently used assays for HIV-1 IN target the strand transfer process and follow a similar premise. HIV IN is combined with donor dsDNA, which has been immobilized onto a solid support, to form an enzyme/DNA complex. The reaction is then initiated by the addition of target dsDNA labelled in some manner, and after an incubation period, the ligated product is quantified. John et al. (2005) reported a highly efficient and sensitive high-throughput screen, HIV IN Target SRI Assay for HIV-1 IN activity, using 5' biotin-labelled DNA (5' BIO donor) and 3' digoxygenin-labelled DNA (3' DIG target). Following 3' processing of the 5' BIO donor, strand transfer proceeds with integration of the 5' BIO donor into the 3' DIG target. The assay was used to screen drugs in a high-throughput format, and the assay was also adapted to study mechanistic questions regarding the integration process. For example, using variations of the assay format, it showed a high preference of the E strand of the LTR viral DNA as a target strand compared with its complementary A strand. Wang et al. (2005) described two homogeneous time-resolved FRET-based assays for the measurement of HIV-1 IN 3' processing and strand transfer activities. These assays have also proven their utility for the identification of mechanistically interesting and biologically active inhibitors of HIV-1 IN that hold potential for further development into potential antiviral drugs.

In addition to recombinant enzyme screens, biochemical assays have been developed that measure HIV-1 IN activity in the context of the preintegration complex (PIC), which mediates the integration of the retroviral genome into host cell DNA. The HIV PIC is a large nucleoprotein complex containing the viral CDNA and IN as well as matrix Vpr, RT and a number of host proteins including histones and members of the non-homologous end joining pathway. It is possible that screening for PIC activity, analogous to that in a true infection, may offer an expanded set of targets and yield more biologically relevant compounds. A polymerase chain reaction-based assay for integration has been reported which employs HIV-1 PICs derived from cells infected with single-cycle HIV-1 reporter viruses.

#### **4. HIV-1 protease**

In a later stage of the HIV-1 life cycle, HIV PT hydrolyzes precursor polyproteins into functional proteins that are essential for viral assembly and subsequent activity. HIV-1 Gag and Pol polypeptide precursors are cleaved by the viral encoded aspartyl protease to form

HIV-Screening Strategies for the Discovery of Novel HIV-Inhibitors 465

historically been used to identify antiviral compounds. HIV-1 replication assays offer the

As the methodology used in the determination of the antiviral activity and the interpretation of the results have been virtually specific to each laboratory and are thus not comparable to one another, simple procedures and guidelines for evaluating antiviral and/or virucidal activities of compounds are needed. Various cell culture-based assays are currently available and can be successfully applied for the antiviral or virucidal determination of substances. Antiviral agents interfere with one or more dynamic processes during virus biosynthesis, making them candidates for clinically useful antiviral drugs; whereas virucidal substances inactivate virus infectivity extracellularly and are therefore better candidates for antiseptics,

Cost, simplicity, accuracy and reproducibility are the key factors determining the selection of the assay system, but selectivity, specificity and sensitivity also need to be taken into account. The methods commonly used for evaluation of *in vitro* antiviral activities are based on the different abilities of viruses to replicate in cultured cells. Some viruses can cause cytopathic effect (CPE) or form plaques. Others are capable of producing specialized functions or cell transformation. Virus replications in cell culture may also be monitored by the detection of viral products such as viral DNA, RNA or polypeptides. Thus, the antiviral test selected may be based on inhibition of CPE, reduction or inhibition of plaque formation. Several different HIV-1 replication assays have been described that could be adapted for medium-to-high-throughput screening. Such assays can generally be subdivided into one of

three categories: reporter virus assays, reporter cell assays or cell protection assays.

of the virally encoded reporter gene (Adelson et al., 2003; Dey & Berger, 2003).

In reporter virus assays, a reporter gene is introduced into the virus genome, usually in place of a viral gene not required for replication, in the target cells of interest. The concept of using HIV-1 reporter viruses to monitor HIV-1 replication was first introduced using a replication competent HIV-1 reporter virus containing the chloramphenicol acetyltransferase gene in place of HIV-1 Nef sequences. Cells are then infected with the recombinant reporter virus and virus replication is quantified by measuring the expression

For reporter cell assays, the target cells of interest are engineered to contain a reporter gene, which is activated upon viral infection. Virus replication is measured by monitoring induction of the reporter gene in the infected target cells. These assays have been used for some time to monitor HIV-1 infection and measure the activity of HIV-1 inhibitors. Kremb et al. (2010) presented a full HIV-replication system for the identification and analysis of HIV inhibitors. This technology is based on adherently growing HIV-susceptible cells, with a stable fluorescent reporter gene activated by HIV Tat and Rev. A fluorescence-based assay was designed to measure HIV infection through two parameters relating to the early and the late phases of HIV replication respectively. These results concluded that this technology is a versatile tool for the discovery and characterization of HIV inhibitors. Reporter cell assays have also been adapted to allow analysis of CCR5 as well as CXCR4 tropic HIV strains

In cell protection assays, CPE resulting from virus replication are measured by determining cell viability using a dye reduction method. These assays represent a more conventional approach to antiviral screening and have been used successfully to execute antiviral screens and identify new HIV-1 inhibitors. Although cell protection assay formats have been available for some time, they continue to be the cornerstone of many HIV-1 drug discovery

advantage of screening for multiple targets in the context of a natural infection.

exhibiting a broad spectrum of germicidal activities.

(Miyake et al., 2003).

programs.

the mature structural and enzymatic gene products. During virus assembly, the viral Gag polyprotein must be effectively processed and transported to the cell membrane. Cofactors such as the phospholipid phosphatidylinositol (4,5) biphosphate, the ADP ribosylation factor binding proteins or tumour susceptibility gene 101, are required for the intracellular transport and budding of HIV particles. While these are just a few examples of virus-host cell interactions, each one represents a potential new target under rigorous research with their validation being actively pursued.

The functional structure of HIV-1 PT is a homodimer containing an active site created in the cleft between the monomers as part of a four-stranded β turn. The active site region is capped by two identical β-hairpin loops (the flaps, residues 45-55 in each monomer), which undergo significant conformational changes upon substrate binding. All PIs currently licensed for the treatment of HIV infections mimic the substrate and block the active site. Another strategy is to develop compounds that bind to the subunit interface and thus block dimerization. As a result, drug discovery efforts continue to focus on the identification of new inhibitors against this validated target that are active against HIV-1 variants which are resistant to the currently available HIV-1 PIs. In line with these efforts, the assays described here may be conducted with wild-type proteins or variants that contain mutations conferring resistance to current HIV-1 PIs.

FRET assays are more commonly used for HIV-1 PT. Synthetic peptide substrates typically consist of a cleavage sequence flanked with fluorescent donor and acceptor labels. The fluorescence signal is low in the intact peptide because the donor is quenched by the nearby acceptor. Once the substrate is cleaved by HIV-1 PT, the FRET interaction is removed, and the fluorescence increases. Hamilton et al. (2003) described a biochemical detection method for peptide products of enzymatic reactions, based on the formation of PSD95/Disc-large/ZO-1 (PDZ) domain\* peptide ligand complexes. The product sensor involves using masked or cryptic PDZ domain peptide ligands as enzyme substrates. The practical applicability of this PDZ-based detection method is determined by the affinity of the PDZ\* peptide ligand interaction, and the efficiency of the enzyme to process the masked peptide ligand. These results showed that the Na+/H+ exchanger regulatory factor, which binds to the consensus sequence Thr/Ser-X-Leu-COOH, can be used to extend the flexibility in the recognition of the carboxy-terminal amino acid of the ligand, and monitor the enzymatic activity of HIV PT.

In addition to enzyme assays, a number of cell-based assays have been reported for HIV-1 PT. A green fluorescent protein (GFP)-PT chimera was developed that can be expressed in mammalian cells, causing minimal toxicity until autocatalytic cleavage occurs (Lindsten et al., 2001). The precursor is activated *in vivo* by autocatalytic cleavage, resulting in rapid elimination of PT-expressing cells. Treatment with therapeutic doses of HIV-1 PIs results in a dose-dependent accumulation of the fluorescent precursor that can be easily detected and quantified by flow cytometric and fluorimetric assays. More recently, Majerova-Uhlikova et al. (2006) described a new assay that might serve as a non-infectious, rapid, cheap and reliable alternative to the currently used phenotypic assays. These investigations showed that in the GFP-PT reporter, the HIV wild-type PT can be replaced by a drug-resistant HIV PT mutant, yielding a simple and biologically relevant tool for the quantitative analysis of drug-resistant HIV PT mutant susceptibility to HIV PTs.

#### **5. HIV-1 replication screens**

Although biochemical high-throughput screening and structure-based drug design approaches are currently preferred over holistic approaches, HIV-1 replication screens have

the mature structural and enzymatic gene products. During virus assembly, the viral Gag polyprotein must be effectively processed and transported to the cell membrane. Cofactors such as the phospholipid phosphatidylinositol (4,5) biphosphate, the ADP ribosylation factor binding proteins or tumour susceptibility gene 101, are required for the intracellular transport and budding of HIV particles. While these are just a few examples of virus-host cell interactions, each one represents a potential new target under rigorous research with

The functional structure of HIV-1 PT is a homodimer containing an active site created in the cleft between the monomers as part of a four-stranded β turn. The active site region is capped by two identical β-hairpin loops (the flaps, residues 45-55 in each monomer), which undergo significant conformational changes upon substrate binding. All PIs currently licensed for the treatment of HIV infections mimic the substrate and block the active site. Another strategy is to develop compounds that bind to the subunit interface and thus block dimerization. As a result, drug discovery efforts continue to focus on the identification of new inhibitors against this validated target that are active against HIV-1 variants which are resistant to the currently available HIV-1 PIs. In line with these efforts, the assays described here may be conducted with wild-type proteins or variants that contain mutations

FRET assays are more commonly used for HIV-1 PT. Synthetic peptide substrates typically consist of a cleavage sequence flanked with fluorescent donor and acceptor labels. The fluorescence signal is low in the intact peptide because the donor is quenched by the nearby acceptor. Once the substrate is cleaved by HIV-1 PT, the FRET interaction is removed, and the fluorescence increases. Hamilton et al. (2003) described a biochemical detection method for peptide products of enzymatic reactions, based on the formation of PSD95/Disc-large/ZO-1 (PDZ) domain\* peptide ligand complexes. The product sensor involves using masked or cryptic PDZ domain peptide ligands as enzyme substrates. The practical applicability of this PDZ-based detection method is determined by the affinity of the PDZ\* peptide ligand interaction, and the efficiency of the enzyme to process the masked peptide ligand. These results showed that the Na+/H+ exchanger regulatory factor, which binds to the consensus sequence Thr/Ser-X-Leu-COOH, can be used to extend the flexibility in the recognition of the carboxy-terminal amino acid of the ligand, and monitor the enzymatic activity of HIV PT. In addition to enzyme assays, a number of cell-based assays have been reported for HIV-1 PT. A green fluorescent protein (GFP)-PT chimera was developed that can be expressed in mammalian cells, causing minimal toxicity until autocatalytic cleavage occurs (Lindsten et al., 2001). The precursor is activated *in vivo* by autocatalytic cleavage, resulting in rapid elimination of PT-expressing cells. Treatment with therapeutic doses of HIV-1 PIs results in a dose-dependent accumulation of the fluorescent precursor that can be easily detected and quantified by flow cytometric and fluorimetric assays. More recently, Majerova-Uhlikova et al. (2006) described a new assay that might serve as a non-infectious, rapid, cheap and reliable alternative to the currently used phenotypic assays. These investigations showed that in the GFP-PT reporter, the HIV wild-type PT can be replaced by a drug-resistant HIV PT mutant, yielding a simple and biologically relevant tool for the quantitative analysis of

Although biochemical high-throughput screening and structure-based drug design approaches are currently preferred over holistic approaches, HIV-1 replication screens have

their validation being actively pursued.

conferring resistance to current HIV-1 PIs.

drug-resistant HIV PT mutant susceptibility to HIV PTs.

**5. HIV-1 replication screens** 

historically been used to identify antiviral compounds. HIV-1 replication assays offer the advantage of screening for multiple targets in the context of a natural infection.

As the methodology used in the determination of the antiviral activity and the interpretation of the results have been virtually specific to each laboratory and are thus not comparable to one another, simple procedures and guidelines for evaluating antiviral and/or virucidal activities of compounds are needed. Various cell culture-based assays are currently available and can be successfully applied for the antiviral or virucidal determination of substances. Antiviral agents interfere with one or more dynamic processes during virus biosynthesis, making them candidates for clinically useful antiviral drugs; whereas virucidal substances inactivate virus infectivity extracellularly and are therefore better candidates for antiseptics, exhibiting a broad spectrum of germicidal activities.

Cost, simplicity, accuracy and reproducibility are the key factors determining the selection of the assay system, but selectivity, specificity and sensitivity also need to be taken into account. The methods commonly used for evaluation of *in vitro* antiviral activities are based on the different abilities of viruses to replicate in cultured cells. Some viruses can cause cytopathic effect (CPE) or form plaques. Others are capable of producing specialized functions or cell transformation. Virus replications in cell culture may also be monitored by the detection of viral products such as viral DNA, RNA or polypeptides. Thus, the antiviral test selected may be based on inhibition of CPE, reduction or inhibition of plaque formation. Several different HIV-1 replication assays have been described that could be adapted for medium-to-high-throughput screening. Such assays can generally be subdivided into one of three categories: reporter virus assays, reporter cell assays or cell protection assays.

In reporter virus assays, a reporter gene is introduced into the virus genome, usually in place of a viral gene not required for replication, in the target cells of interest. The concept of using HIV-1 reporter viruses to monitor HIV-1 replication was first introduced using a replication competent HIV-1 reporter virus containing the chloramphenicol acetyltransferase gene in place of HIV-1 Nef sequences. Cells are then infected with the recombinant reporter virus and virus replication is quantified by measuring the expression of the virally encoded reporter gene (Adelson et al., 2003; Dey & Berger, 2003).

For reporter cell assays, the target cells of interest are engineered to contain a reporter gene, which is activated upon viral infection. Virus replication is measured by monitoring induction of the reporter gene in the infected target cells. These assays have been used for some time to monitor HIV-1 infection and measure the activity of HIV-1 inhibitors. Kremb et al. (2010) presented a full HIV-replication system for the identification and analysis of HIV inhibitors. This technology is based on adherently growing HIV-susceptible cells, with a stable fluorescent reporter gene activated by HIV Tat and Rev. A fluorescence-based assay was designed to measure HIV infection through two parameters relating to the early and the late phases of HIV replication respectively. These results concluded that this technology is a versatile tool for the discovery and characterization of HIV inhibitors. Reporter cell assays have also been adapted to allow analysis of CCR5 as well as CXCR4 tropic HIV strains (Miyake et al., 2003).

In cell protection assays, CPE resulting from virus replication are measured by determining cell viability using a dye reduction method. These assays represent a more conventional approach to antiviral screening and have been used successfully to execute antiviral screens and identify new HIV-1 inhibitors. Although cell protection assay formats have been available for some time, they continue to be the cornerstone of many HIV-1 drug discovery programs.

HIV-Screening Strategies for the Discovery of Novel HIV-Inhibitors 467

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activities. *J. Biomol. Screen.*, 10, 5, (August 2005), 456-462, ISSN 1087-0571 Wang, T., Duan, Y. (2009). HIV co-receptor CCR5: structure and interactions with inhibitors. *Infect. Disord. Drug Targets*, 9, 3, (March 2009), 279-288, ISSN 1871-5265 Warnke, D., Barreto, J. (2007). Antiretroviral drugs. *J. Clin. Pharmacol.*, 47, 12, (December

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update and perspectives. *Curr. Top. Med. Chem.*, 9, 11, (November 2009), 1016-1037,

CCR5- and CXCR4-using human immunodeficiency virus type 1 and its application to drug susceptibility tests. *J. Clin. Microbiol.*, 41, 6, (June 2003), 2515-

assay for screening inhibitors against the ribonuclease H activity of HIV-1 reverse

HIV drug screening using EGFP as a quantitative marker of HIV-1 replication. *Bio* 

based high throughput screening assay for inhibitors of human immunodeficiency virus-1 reverse transcriptase-associated ribonuclease H activity. *Anal. Biochem.*, 322,

and molecular interactions. *J. Gen. Virol.*, 90, 8, (August 2009), 1795-1805, ISSN 0022-

human immunodeficiency virus type 1. *J. Gen. Virol.*, 91, 1, (January 2010), 1-12,

E. (2009). Structure and function of HIV-reverse transcriptase: molecular mechanism of polymerization and inhibition. *J. Mol. Biol.*, 385, 3, (January 2009),

underpinning the development of novel techniques for the treatment of HIV infection. *Drug Discover. Today*, 15, 5/6, (March 2010), 186-197, ISSN 1359-6446 Wahba, A.S., Esmaeili, A., Damha, M.J., Hudson, R.M. (2010). A single-label

phenylpyrrolocytidine provides a molecular beacon-like response reporting HIV-1 RT RNase H activity. *Nucleic Acid Res.*, 38, 3, (January 2010), 1048-1056, ISSN 0305-

Hale, J., Lesley, S., Kuhen, K., Caldwell, J., Brinker, A. (2005). Homogeneous highthroughput screening assays for HIV-1 integrase 3β-processing and strand transfer

#### **6. Acknowledgements**

The technical assistance of Ms. Brooke-Turner is gratefully acknowledged.

#### **7. References**


Adelson, M.E., Pacchia, A.L., Kaul, M., Rando, R.F., Ron, Y., Peltz, S.W., Dougherty, J.P.

Buonaguro, L., Tornesello, M.L., Buonaguro, F.M. (2007). Human immunodeficiency virus

Chilba-Mizutani, T., Mivra, H., Matsuda, M., Matsuda, Z., Yokomaku, Y., Miyauchi, K.,

Dey, B., Berger, E.A. (2003). Current Protocols in Immunology, In: *Detection and Analysis of HIV*, Straber, W., 10, John Wiley & Sons, Inc., ISBN1934-368X, Sussex, England Grande, F., Garofalo, A., Neamati, N. (2008). Small molecules anti-HIV therapeutics targeting CXCR4. *Curr. Pharm. Des.*, 14, 4, (April 2008), 385-404, ISSN 1381-6128 Greene, W.C., Debyser, Z., Ikeda, Y., Freed, E.O., Stephens, E., Yonemoto, W., Buckheit,

Hamilton, A.C., Inglese, J., Ferrer, M. (2003). A PDZ domain-based assay for measuring HIV

Herschhorn, A., Hiz, A. (2010). Retroviral reverse transcriptases. *Cell Mol. Life Sci.*, 67, 16,

John, S., Fletcher, T.M., Jonsson, C.B. (2005). Development and application of a high-

Kremb, S., Helfer, M., Heller, W., Hoffmann, D., Wolff, H., Kleinschmidt, A., Cepok, S.,

Lindsten, K., Uhlikova, T., Konvalinka, J., Masucci, M.G., Dantuna, N.P. (2001). Cell-based

*Agents Chemother.*, 54, 12, (December 2010), 5257-5268, ISSN 1098:6596 Liang, X. (2008). CXCR4 inhibitors and mechanism of action. *Chem. Biol. Drug Des.*, 72, 2,

mutants. *J. Clin. Virol.*, 36, 1, (May 2006), 50-59, ISSN 1386-6532

*Agents Chemother.*, 47, 2, (February 2003), 501-508, ISSN 1098:6596

(2003). Toward the development of a virus-cell-based assay for the discovery of novel compounds against human immunodeficiency virus type 1. *Antimicrob.* 

type 1 subtype distribution in the Worlwide epidemic: pathogenetic and therapeutic implications. *J. Virol.*, 81, 19, (October 2007), 10209-10219, ISSN 0022-

Nishizawa, M., Yamamoto, N., Sigiura, W. (2007). Use of new T-cell-based cell lines expressing two luciferase reporters for accurately evaluating susceptibility to antihuman immunodeficiency virus type 1 drugs. *J. Clin. Microbiol.*, 45, 2, (February

R.W., Este, J.A., Cihlar, T. (2008). Novel targets for HIV therapy. *Antiviral Res.*, 80, 3,

protease activity: assay design considerarions. *Protein Sci.*, 12, 3, (March 2003), 458-

throughput screening assay for HIV-1 integrase enzyme activities. *J. Biomol. Screen.*,

Hemmer, B., Durner, J., Brack-Werner, R. (2010). EASY-HIT: HIV full-replication technology for broad discovery of multiple classes of HIV inhibitors. *Antimicrob.* 

fluorescence assay for human immunodeficiency virus type 1 protease activity. *Antimicrob. Agents Chemother.*, 45, 9, (September 2001), 2616-2622, ISSN 1098:6596 Majerova-Uhlikova, T., Dantuma, N.P., Lindstein, K., Masucci, M.G., Konvalinka, J. (2006).

Non-infectious fluorimetric assay for phenotyping of drug-resistant HIV proteinase

The technical assistance of Ms. Brooke-Turner is gratefully acknowledged.

**6. Acknowledgements** 

**7. References** 

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(December 2008), 251-265, ISSN 0166-3542

(August 2010), 2717-2747, ISSN 1420-682X

(August 2008), 97-110, ISSN 1747-0285

10, 6, (September 2005), 606-614, ISSN 1087-0571


**Part 8** 

**Vaccine Development** 


## **Part 8**

**Vaccine Development** 

468 Recent Translational Research in HIV/AIDS

Westby, M., Nakayama, G.R., Butler, S.L., Blair, W.S. (2005). Cell-based and biochemical

Xuei, X., David, C.A., Middleton, T.R., Lim, B., Pithawalla, R., Chen, C.M., Tripathi, R.L.,

Zhan, P., Lia, X., Li, Z. (2009). Recent advances in the discovery and development of novel

Zhao, Q., Lu, H., Schols, D., De Clercq, E., Jiang, S. (2003). Development of a cell-based

assays. *J. Biomol. Screen.*, 8, 3, (June 2003), 273-282, ISSN 1087-0571

3, (September 2005), 121-140, ISSN 0166-3542

11, (November 2003), 947-955, ISSN 0889-2229

2876-2889, ISSN 0929-8673

screening approaches for the discovery of novel HIV-1 inhibitors. *Antiviral Res.*, 67,

Burns, D.J., Warrior, V. (2003). Use of SAM2® biotin capture membrane in microarrayed compound screening (μARCS) format for nucleic acid polymerization

HIV-1 NNRTI platforms: 2006-2008 update. *Curr. Med. Chem.*, 16, 22, (July 2009),

enzyme-linked immunosorbent assay for high-throughput screening of HIV type entry inhibitors targeting the coreceptor CXCR4. *AIDS Res. Hum. Retroviruses*, 19,

**23** 

*Brazil* 

**HIV Vaccine** 

*University of São Paulo* 

Alexandre de Almeida, Telma Miyuki Oshiro,

Alessandra Pontillo and Alberto José da Silva Duarte

More than three decades after the discovery of Human Immunodeficiency Virus (HIV) as the causative agent of Acquired Immunodeficiency Syndrome (AIDS), a vaccine is still considered as the best hope for controlling the epidemic. In fact the history of medicine

Remarkable success in the AIDS treatment has been achieved with the development of antiretroviral drugs that, by interfering with various aspects of the HIV life cycle, allowed an impressive control of infection (Fischl et al., 1987; Egger et al., 1997). The use of these drugs, however, was accompanied by new challenges related to side effects, high cost and resistance development (Carr, 2000; Hawkins, 2010; Menéndez-Arias, 2010;

Antiretroviral treatment cannot prevent early infection events, such as transmission to sexual partners during the post-infection peak of viremia (Wawer et al., 1999) and the massive destruction of intestinal CD4+T cells during the first weeks of infection (Brenchley et al., 2004). Furthermore drugs delivery to poor and endemic areas is often hard due to practical limitations. In resource-limited countries only 1 out 4 HIV-positive individuals has access to antiretroviral medications, and for each person who begins the therapy, there are about 6 new infections (www.who.int/entity/hiv/mediacentre/universal\_access\_progress\_report\_en.pdf).

Others approaches could be taken in account to reduce HIV-1 infection in subjects at risk of exposure, including public health involvement (i.e.: screening of donor blood products), educational effort (i.e.: risk reduction counselling), or social imprinting (i.e.: male circumcision and behaviour modifications such as condom usage). In high seropositive communities, pre-exposure or post-exposure antiretroviral prophylaxis may reduce susceptibility to HIV infection, as well as the vertical HIV transmission from mother to

The creation of an HIV-1 vaccine represents an unprecedented scientific challenge and it's an absolute priority in field of HIV prevention. We must remember that vaccines are one of the most effective public health interventions ever known, but unfortunately, in HIV infection, the current perspective is that we will not have a product, even moderately

The truth is that often in the history of vaccinology it takes a long time since the discovery of infectious agents to the licensing of an effective vaccine (Heyward et al., 1998). This is due in

These factors made difficult to control the pandemic through antiretroviral therapy.

shows that no viral disease have ever been controlled without a vaccine.

www.hivresourcetracking.org/treatments/vaccines).

**1. Introduction** 

child.

effective, in the coming years.

## **HIV Vaccine**

Alexandre de Almeida, Telma Miyuki Oshiro, Alessandra Pontillo and Alberto José da Silva Duarte *University of São Paulo Brazil* 

#### **1. Introduction**

More than three decades after the discovery of Human Immunodeficiency Virus (HIV) as the causative agent of Acquired Immunodeficiency Syndrome (AIDS), a vaccine is still considered as the best hope for controlling the epidemic. In fact the history of medicine shows that no viral disease have ever been controlled without a vaccine.

Remarkable success in the AIDS treatment has been achieved with the development of antiretroviral drugs that, by interfering with various aspects of the HIV life cycle, allowed an impressive control of infection (Fischl et al., 1987; Egger et al., 1997). The use of these drugs, however, was accompanied by new challenges related to side effects, high cost and resistance development (Carr, 2000; Hawkins, 2010; Menéndez-Arias, 2010; www.hivresourcetracking.org/treatments/vaccines).

Antiretroviral treatment cannot prevent early infection events, such as transmission to sexual partners during the post-infection peak of viremia (Wawer et al., 1999) and the massive destruction of intestinal CD4+T cells during the first weeks of infection (Brenchley et al., 2004). Furthermore drugs delivery to poor and endemic areas is often hard due to practical limitations. In resource-limited countries only 1 out 4 HIV-positive individuals has access to antiretroviral medications, and for each person who begins the therapy, there are about 6 new infections (www.who.int/entity/hiv/mediacentre/universal\_access\_progress\_report\_en.pdf). These factors made difficult to control the pandemic through antiretroviral therapy.

Others approaches could be taken in account to reduce HIV-1 infection in subjects at risk of exposure, including public health involvement (i.e.: screening of donor blood products), educational effort (i.e.: risk reduction counselling), or social imprinting (i.e.: male circumcision and behaviour modifications such as condom usage). In high seropositive communities, pre-exposure or post-exposure antiretroviral prophylaxis may reduce susceptibility to HIV infection, as well as the vertical HIV transmission from mother to child.

The creation of an HIV-1 vaccine represents an unprecedented scientific challenge and it's an absolute priority in field of HIV prevention. We must remember that vaccines are one of the most effective public health interventions ever known, but unfortunately, in HIV infection, the current perspective is that we will not have a product, even moderately effective, in the coming years.

The truth is that often in the history of vaccinology it takes a long time since the discovery of infectious agents to the licensing of an effective vaccine (Heyward et al., 1998). This is due in

HIV Vaccine 473

HIV presents a genome of about 10,000 base pairs, composed of three structural genes (*gag*, *pol* and *env*) beyond the six accessory genes (*vif*,*vpr*, *rev*, *tat*,*vpu* and *nef*). The *gag* gene encodes the viral core protein as the capsid, matrix and nucleocapsid, *pol* encodes the viral enzymes (reverse transcriptase, protease, ribonuclease and integrase) and *env* gene encodes the envelope glycoproteins. Some products of these genes are targets of choice for the study

The great genetic diversity of HIV represents a major obstacle to developing an effective vaccine. Such diversity is the result of a highly HIV replicative rate (new 1010 viral particles/day) and of its prone to errors retrotranscriptase (1 new nucleotide

The highest degree of HIV diversity is found in the envelope glycoproteins. The amino acid sequences of Env may differ by about 15% between isolates of the same clade and in more

As a consequence of this high degree of mutational rate, HIV can counteract the selective pressure imposed by the host immune response, and it soon become able to evade an effective response. This aspect makes it difficult to identify potential HIV targets against

Another important point is that an effective vaccine may protect against various HIV subtypes and clades prevalent in every region of the world. HIV is classified into two types: HIV-1 and HIV-2 that have a genetic homology around 40-50%. While HIV-2 is less pathogenic and its incidence is confined to Africa, HIV-1 is the causative agent of a worldwide pandemic. HIV-1 is divided into three groups M, O and N. The groups O and N

Within group M, HIV-1 isolates are divided into six subtypes and clades (A, B, C, D, E and G) and have distinct geographic distributions. While subtype B is prevalent in the Americas and Europe, subtype C, which accounts for more than 50% of AIDS cases worldwide, is prevalent in Southeast Asia and Africa. The difference between the amino acid sequences among viral clades differs by 20% and the variation within clades can reach over 10% in amino acid sequence. Furthermore, different subtypes may be associated with generating circulating recombinant forms (CRFs), further increasing the

Another major challenge that hinders the design of an effective vaccine is the HIV tropism for immune cells. HIV uses the CD4 molecule as a receptor for cell entry. This molecule, in turn, is expressed mainly by T helper lymphocytes and to a lesser degree by dendritic cells, macrophages and monocytes. Since these are strategic cells within the immune system, the

Belonging to retroviruses, HIV integrates its genetic material into host cell genome. Days after infection the virus begins its haematogenous spreading from mucosal to lymphoid sites, particularly gut-associated lymphoid tissue (GALT) where a lot of CD4+ CCR5+ T

The massive loss of CD4+ T cells compromises the host immune response during the infection. Moreover, since the HIV genome is integrated latently until cells become activated, establishing viral reservoirs that hinder the complete elimination of

immune response in HIV-infected individuals is compromised (Figure1).

memory lymphocytes are destructed (Matapallil et al., 2005).

are restricted to Central Africa, while group M is responsible for the AIDS pandemic.

substitution/replication for a genome of approximately 10 000 bp).

than 35% between envelopes of different clades (Gaschen et al 2002).

**2.1 HIV heterogeneity and cell targets** 

which the immune system could be directed.

of vaccines.

viral diversity.

infection.

part to the fact that even today no one knows for sure how the immune system protects us against infections and, consequently, how to handle it for this to occur. In HIV/AIDS, the stimulation of a specific immune response is unlikely to immunize against HIV: there are no established immune correlates of protection (i.e.: humoral or cellular response), no documented cases of spontaneous recovery from AIDS or HIV infection, and no animal model that faithfully predicts HIV disease or vaccine responses in humans beyond the variability of the virus. Moreover HIV entries predominantly through mucosal surfaces, targets preferentially CD4+T cells, and rapidly establishes a persistent reservoir of latently infected cells, making difficult the study of the host/virus interaction as well as the development of an interventional strategy.

Novel approaches for an HIV vaccination need a rational vaccine design, including a better integration of emerging scientific concepts and knowledge derived from vaccinology research fields.

Models of natural resistance to HIV infection, including individuals able to control the infection (elite controllers) and some species of non-human primates, show that some level of control can be achieved (Dunham et al., 2006; Sumpter et al., 2007; Walker, 2007; Lederman et al., 2010; Poropatich & Sullivan, 2011).

The creation of an effective HIV vaccine will require continued scientific research and cooperation between academic community and biotechnology industry with the contributions of brightest scientists, long-term commitments of stable and flexible funding, trials and vaccines accessibility for developing countries. In coming years, the prospect is that several area of scientific community will be involved seeking to combine the knowledge necessary to develop new strategies, new candidates and evaluating these products.

This chapter will describe some aspects of the development of HIV vaccines, with emphasis on scientific efforts and challenges made to producing a safe and effective vaccine, strategies and methods used in the development of anti-HIV vaccines, current outlook and perspectives in this area.

### **2. Major challenges to get an HIV vaccine**

Primary in prevention and control of infectious diseases, vaccines are a highly effective way to stimulate the immune system to fight pathogens. In the case of HIV infection, it has not yet been possible to obtain a vaccine to control infection, despite the efforts of the scientific community, the large financial investment and scientific and technological progress achieved.

Considering the natural history of infection, an HIV vaccine has the principal aim to prevent the integration of HIV genetic material into the genome of the host cell in order to prevent systemic infection and the establishment of viral reservoirs. This occurs within a few days after exposure, when HIV rapidly replicates in the lymphoid tissues, so the window of opportunity to prevent the establishment of a persistent infection is very brief. Therefore an effective HIV vaccine should be able to activate the immune system against the virus very early after the infection.

The complexity and diversity of HIV, its high capacity to evade the immune system and the missing gap in effective host immune response against the virus represent some major challenges to design an optimal vaccine. Moreover the absence of an experimental model able to mimic human infection represents another limit for pre-clinical studies.

part to the fact that even today no one knows for sure how the immune system protects us against infections and, consequently, how to handle it for this to occur. In HIV/AIDS, the stimulation of a specific immune response is unlikely to immunize against HIV: there are no established immune correlates of protection (i.e.: humoral or cellular response), no documented cases of spontaneous recovery from AIDS or HIV infection, and no animal model that faithfully predicts HIV disease or vaccine responses in humans beyond the variability of the virus. Moreover HIV entries predominantly through mucosal surfaces, targets preferentially CD4+T cells, and rapidly establishes a persistent reservoir of latently infected cells, making difficult the study of the host/virus interaction as well as the

Novel approaches for an HIV vaccination need a rational vaccine design, including a better integration of emerging scientific concepts and knowledge derived from vaccinology

Models of natural resistance to HIV infection, including individuals able to control the infection (elite controllers) and some species of non-human primates, show that some level of control can be achieved (Dunham et al., 2006; Sumpter et al., 2007; Walker, 2007;

The creation of an effective HIV vaccine will require continued scientific research and cooperation between academic community and biotechnology industry with the contributions of brightest scientists, long-term commitments of stable and flexible funding, trials and vaccines accessibility for developing countries. In coming years, the prospect is that several area of scientific community will be involved seeking to combine the knowledge necessary to develop new strategies, new candidates and evaluating these

This chapter will describe some aspects of the development of HIV vaccines, with emphasis on scientific efforts and challenges made to producing a safe and effective vaccine, strategies and methods used in the development of anti-HIV vaccines, current outlook and

Primary in prevention and control of infectious diseases, vaccines are a highly effective way to stimulate the immune system to fight pathogens. In the case of HIV infection, it has not yet been possible to obtain a vaccine to control infection, despite the efforts of the scientific community, the large financial investment and scientific and technological progress

Considering the natural history of infection, an HIV vaccine has the principal aim to prevent the integration of HIV genetic material into the genome of the host cell in order to prevent systemic infection and the establishment of viral reservoirs. This occurs within a few days after exposure, when HIV rapidly replicates in the lymphoid tissues, so the window of opportunity to prevent the establishment of a persistent infection is very brief. Therefore an effective HIV vaccine should be able to activate the immune system against the virus very

The complexity and diversity of HIV, its high capacity to evade the immune system and the missing gap in effective host immune response against the virus represent some major challenges to design an optimal vaccine. Moreover the absence of an experimental model

able to mimic human infection represents another limit for pre-clinical studies.

development of an interventional strategy.

Lederman et al., 2010; Poropatich & Sullivan, 2011).

**2. Major challenges to get an HIV vaccine** 

research fields.

products.

achieved.

perspectives in this area.

early after the infection.

#### **2.1 HIV heterogeneity and cell targets**

HIV presents a genome of about 10,000 base pairs, composed of three structural genes (*gag*, *pol* and *env*) beyond the six accessory genes (*vif*,*vpr*, *rev*, *tat*,*vpu* and *nef*). The *gag* gene encodes the viral core protein as the capsid, matrix and nucleocapsid, *pol* encodes the viral enzymes (reverse transcriptase, protease, ribonuclease and integrase) and *env* gene encodes the envelope glycoproteins. Some products of these genes are targets of choice for the study of vaccines.

The great genetic diversity of HIV represents a major obstacle to developing an effective vaccine. Such diversity is the result of a highly HIV replicative rate (new 1010 viral particles/day) and of its prone to errors retrotranscriptase (1 new nucleotide substitution/replication for a genome of approximately 10 000 bp).

The highest degree of HIV diversity is found in the envelope glycoproteins. The amino acid sequences of Env may differ by about 15% between isolates of the same clade and in more than 35% between envelopes of different clades (Gaschen et al 2002).

As a consequence of this high degree of mutational rate, HIV can counteract the selective pressure imposed by the host immune response, and it soon become able to evade an effective response. This aspect makes it difficult to identify potential HIV targets against which the immune system could be directed.

Another important point is that an effective vaccine may protect against various HIV subtypes and clades prevalent in every region of the world. HIV is classified into two types: HIV-1 and HIV-2 that have a genetic homology around 40-50%. While HIV-2 is less pathogenic and its incidence is confined to Africa, HIV-1 is the causative agent of a worldwide pandemic. HIV-1 is divided into three groups M, O and N. The groups O and N are restricted to Central Africa, while group M is responsible for the AIDS pandemic.

Within group M, HIV-1 isolates are divided into six subtypes and clades (A, B, C, D, E and G) and have distinct geographic distributions. While subtype B is prevalent in the Americas and Europe, subtype C, which accounts for more than 50% of AIDS cases worldwide, is prevalent in Southeast Asia and Africa. The difference between the amino acid sequences among viral clades differs by 20% and the variation within clades can reach over 10% in amino acid sequence. Furthermore, different subtypes may be associated with generating circulating recombinant forms (CRFs), further increasing the viral diversity.

Another major challenge that hinders the design of an effective vaccine is the HIV tropism for immune cells. HIV uses the CD4 molecule as a receptor for cell entry. This molecule, in turn, is expressed mainly by T helper lymphocytes and to a lesser degree by dendritic cells, macrophages and monocytes. Since these are strategic cells within the immune system, the immune response in HIV-infected individuals is compromised (Figure1).

Belonging to retroviruses, HIV integrates its genetic material into host cell genome. Days after infection the virus begins its haematogenous spreading from mucosal to lymphoid sites, particularly gut-associated lymphoid tissue (GALT) where a lot of CD4+ CCR5+ T memory lymphocytes are destructed (Matapallil et al., 2005).

The massive loss of CD4+ T cells compromises the host immune response during the infection. Moreover, since the HIV genome is integrated latently until cells become activated, establishing viral reservoirs that hinder the complete elimination of infection.

HIV Vaccine 475

d. The existence of hidden CD4/receptor binding sites which difficult the access of the

Fig. 2. Sites of antibody binding to gp120. Neutralizing and non-neutralizing sites of gp120.

Considering that HIV infects CD4+ T cells, the stimulation of a cellular response is important for the destruction of infected cells by cytotoxic T lymphocytes (CTLs) before the release of new viral particles. Thus, in a context of an anti-HIV vaccine CD4+ T cells should be rapidly expanded to stimulate the right cytotoxic response against infected cells and drive memory

Although many studies provide strong evidence that a cellular response can effectively suppress HIV (Rowland-Jones et al., 1998; Hladik et al., 2003), it remains unclear how this viral suppression occurs and how a vaccine could stimulate it. Peculiar characteristics of the virus and the nature of infection hinder the development of an appropriate cellular

a. The reduction of the expression of MHC class I molecules mediated by the HIV Nef

c. The massive destruction of T cells specific or not for HIV, caused mainly by activation-

In this context, even though there are not clear evidences regarding the type of immune response to be induced by an HIV vaccine, it is reasonable to assume that elements of the immune response important to ensure a real effectiveness of an HIV vaccine may depend on both neutralizing antibody and specific cellular immunity, and besides, also on innate immunity. In a simplified view, neutralizing antibodies prevent the entry of virus into the

cells to sites potentially susceptible to infection such as mucosal and lymph nodes.

b. The establishment and maintenance of latent viral reservoirs in cells

induced apoptosis. (Cadogan & Dalgleish, 2008).

b. The large number of subtypes of HIV that exhibit little cross-reactivity;

c. The high rate of glycosylation on the viral envelope

The sites of glycosylation of the molecule are also shown.

antibodies. (Figure 2)

response, for example,

protein;

Fig. 1. HIV cell target. HIV predominantly infects CD4+ lymphocytes, which play a fundamental role in the induction of specific immune response. APC: Antigen-presenting cell CTL: Cytotoxic T lymphocyte

#### **2.2 Host/virus interaction**

A key feature of the immune system is to "remember" and respond to antigens with which they've previously met. This property, called immunological memory, is the basis of the vaccination process. To play its role, a vaccine must therefore deliver the antigen to the immune system in order to stimulate it and to enable the development of memory.

In this sense, a basic problem in developing an HIV vaccine is the lack of definition of the correlates of immune protection in HIV infection, so that is not completely clear what types of immune response should ideally be stimulated by vaccination and consequently what measure and criteria should be used to evaluate the effectiveness of the vaccine.

Initially the first strategies implied to obtain an HIV vaccine were focused on inducing neutralizing antibodies against the viral envelope proteins (Dolin, 1995) and from mid-1990, studies began to focus on the activation of a cellular immune response.

Stimulation of neutralizing antibodies with broad specificity for all HIV variants would be definitely interesting for a vaccine strategy. Evidence in nonhuman primates suggest that a protection could be afforded if neutralizing antibodies could be present in high concentration both in blood and mucosa at the time of first infection (Parren et al., 2001). However, the induction of neutralizing antibodies against HIV is hampered by some specific characteristics of the virus, such as

a. The high epitopes mutation rate, which causes loss of recognition capacity by antibodies;

Fig. 1. HIV cell target. HIV predominantly infects CD4+ lymphocytes, which play a

immune system in order to stimulate it and to enable the development of memory.

measure and criteria should be used to evaluate the effectiveness of the vaccine.

studies began to focus on the activation of a cellular immune response.

A key feature of the immune system is to "remember" and respond to antigens with which they've previously met. This property, called immunological memory, is the basis of the vaccination process. To play its role, a vaccine must therefore deliver the antigen to the

In this sense, a basic problem in developing an HIV vaccine is the lack of definition of the correlates of immune protection in HIV infection, so that is not completely clear what types of immune response should ideally be stimulated by vaccination and consequently what

Initially the first strategies implied to obtain an HIV vaccine were focused on inducing neutralizing antibodies against the viral envelope proteins (Dolin, 1995) and from mid-1990,

Stimulation of neutralizing antibodies with broad specificity for all HIV variants would be definitely interesting for a vaccine strategy. Evidence in nonhuman primates suggest that a protection could be afforded if neutralizing antibodies could be present in high concentration both in blood and mucosa at the time of first infection (Parren et al., 2001). However, the induction of neutralizing antibodies against HIV is hampered by some

a. The high epitopes mutation rate, which causes loss of recognition capacity by

fundamental role in the induction of specific immune response.

APC: Antigen-presenting cell CTL: Cytotoxic T lymphocyte

**2.2 Host/virus interaction** 

specific characteristics of the virus, such as

antibodies;


Fig. 2. Sites of antibody binding to gp120. Neutralizing and non-neutralizing sites of gp120. The sites of glycosylation of the molecule are also shown.

Considering that HIV infects CD4+ T cells, the stimulation of a cellular response is important for the destruction of infected cells by cytotoxic T lymphocytes (CTLs) before the release of new viral particles. Thus, in a context of an anti-HIV vaccine CD4+ T cells should be rapidly expanded to stimulate the right cytotoxic response against infected cells and drive memory cells to sites potentially susceptible to infection such as mucosal and lymph nodes.

Although many studies provide strong evidence that a cellular response can effectively suppress HIV (Rowland-Jones et al., 1998; Hladik et al., 2003), it remains unclear how this viral suppression occurs and how a vaccine could stimulate it. Peculiar characteristics of the virus and the nature of infection hinder the development of an appropriate cellular response, for example,


In this context, even though there are not clear evidences regarding the type of immune response to be induced by an HIV vaccine, it is reasonable to assume that elements of the immune response important to ensure a real effectiveness of an HIV vaccine may depend on both neutralizing antibody and specific cellular immunity, and besides, also on innate immunity. In a simplified view, neutralizing antibodies prevent the entry of virus into the

HIV Vaccine 477

viral proteins or peptides artificially synthesized (mimetopos) or produced by the

 proteins expressed in the form of virus like particles (VLP), consisting of structurally preserved viral epitopes (i.e.: parts of the virus surface proteins), without the viral

 HIV genetic material to insert directly into cells that will express their products. Usually this material is inserted in the form of plasmids, which are molecules of extrachromosomal circular DNA, with independent replication. The insertion of genetic material in the body can be made directly (eg. electroporation or gene guns using

Live attenuated virus vaccines have not been investigated in anti-HIV vaccines due to the risk of development of virulence, as evidenced in a model of NHP (Whatmore et al., 1995).

Fig. 3. Immunogens used in the composition of HIV vaccines. There are three types of immunogens: whole vital particle, peptides or recombinant proteins and genetic material of the virus, which may or may not be inserted within vectors. Sometimes the combination of

different immunogens can be used (prime-boost strategy).

chemical (eg. alcohols) or heat inactivated virus particles;

genetic material, thus preventing their replication.

compressed gas) or through biological vectors.

insertion of relevant genes in biological vectors (recombinants);

cell by blocking the transmission and infection, while the cellular response would act destroying HIV-infected cells before the release of new viral particles in order to control an yet established infection.

Another interesting tips is that being mucosal the primary site of natural HIV infection (Kozlowski & Neutra, 2003) an effective vaccine must induce anti-HIV-1 neutralizing antibodies at mucosal surfaces to prevent the infection and cytotoxic T lymphocytes (CTLs) in sub-mucosal areas to kill virus-infected cells, or a combination of both. Unfortunately, the immune response within the mucosa may be associated with a high viral replication and dissemination: HIV activates and recruits a lot of target cells and the virus uptake by dendritic cells allow its dissemination to draining lymph nodes avoiding antibody recognition. The challenge to an effective vaccine is to activate mucosal immunity at the right time. Moreover, all mucosal vaccines have to overcome tolerance, which is related with regulatory cells and depend on the nature of the antigen, the dosage, the method of delivery and on whether or not adjuvant is used.

#### **2.3 Lack of animal models**

Actually there is no animal model capable of to mimic the human HIV infection and AIDS development. The use of animal models could help the investigation of disease pathogenesis and provide information about toxicity and efficacy of drugs and vaccines to reduce risk, duration and cost of a clinical trial.

Despite its relatively low cost and ease of maintenance in animal houses, the use of small rodents as experimental models for HIV infection is not appropriate since HIV is unable to sustain infection in murine cells. More recently it has been demonstrated the use of humanized mice models (Van Duyne et al., 2009).

Studies in non-human primates (NHP; i.e.: *Macaca rhesus*), when allowed, even if expensive, gave some good results and have the advantage of sharing a high genetic background with humans. NHP are the natural host of a retrovirus of the same HIV family, SIV (Simian Immunodeficiency Virus) which has a very low mutational rate compared to HIV. In some studies the SHIV, a hybrid virus composed of parts of the genome of HIV and SIV, has been implied to create the infection model (Stapransan et al, 2010).

Although providing crucial information about viral immunobiology and vaccine design, it must be taken in account that important differences in the viral infection exist between humans and NHP. Data from NHP models should be critically evaluated for their predictive value in human trials (Shedlock et al., 2009).

#### **3. Strategies and methods used in the development of anti-HIV vaccines**

Like most vaccines, candidates for HIV vaccine contained weakened or killed forms of the virus or viral components which resembling original HIV and could be able to stimulate the immune system to develop an appropriate response. Taking in account all these considerations, in the past decades several aspects related to the vaccine composition, route of immunization and vaccine strategy have been tested in the effort to develop an effective HIV vaccine.

#### **3.1 Vaccine composition**

#### **3.1.1 Immunogen production**

Many techniques have been employed in order to produce relevant immunogenic HIV antigens, such as:

cell by blocking the transmission and infection, while the cellular response would act destroying HIV-infected cells before the release of new viral particles in order to control an

Another interesting tips is that being mucosal the primary site of natural HIV infection (Kozlowski & Neutra, 2003) an effective vaccine must induce anti-HIV-1 neutralizing antibodies at mucosal surfaces to prevent the infection and cytotoxic T lymphocytes (CTLs) in sub-mucosal areas to kill virus-infected cells, or a combination of both. Unfortunately, the immune response within the mucosa may be associated with a high viral replication and dissemination: HIV activates and recruits a lot of target cells and the virus uptake by dendritic cells allow its dissemination to draining lymph nodes avoiding antibody recognition. The challenge to an effective vaccine is to activate mucosal immunity at the right time. Moreover, all mucosal vaccines have to overcome tolerance, which is related with regulatory cells and depend on the nature of the antigen, the dosage, the method of delivery

Actually there is no animal model capable of to mimic the human HIV infection and AIDS development. The use of animal models could help the investigation of disease pathogenesis and provide information about toxicity and efficacy of drugs and vaccines to reduce risk,

Despite its relatively low cost and ease of maintenance in animal houses, the use of small rodents as experimental models for HIV infection is not appropriate since HIV is unable to sustain infection in murine cells. More recently it has been demonstrated the use of

Studies in non-human primates (NHP; i.e.: *Macaca rhesus*), when allowed, even if expensive, gave some good results and have the advantage of sharing a high genetic background with humans. NHP are the natural host of a retrovirus of the same HIV family, SIV (Simian Immunodeficiency Virus) which has a very low mutational rate compared to HIV. In some studies the SHIV, a hybrid virus composed of parts of the genome of HIV and SIV, has been

Although providing crucial information about viral immunobiology and vaccine design, it must be taken in account that important differences in the viral infection exist between humans and NHP. Data from NHP models should be critically evaluated for their predictive

Like most vaccines, candidates for HIV vaccine contained weakened or killed forms of the virus or viral components which resembling original HIV and could be able to stimulate the immune system to develop an appropriate response. Taking in account all these considerations, in the past decades several aspects related to the vaccine composition, route of immunization and vaccine strategy have been tested in the effort to develop an effective

Many techniques have been employed in order to produce relevant immunogenic HIV

**3. Strategies and methods used in the development of anti-HIV vaccines** 

yet established infection.

and on whether or not adjuvant is used.

**2.3 Lack of animal models** 

duration and cost of a clinical trial.

humanized mice models (Van Duyne et al., 2009).

value in human trials (Shedlock et al., 2009).

HIV vaccine.

antigens, such as:

**3.1 Vaccine composition 3.1.1 Immunogen production** 

implied to create the infection model (Stapransan et al, 2010).


Live attenuated virus vaccines have not been investigated in anti-HIV vaccines due to the risk of development of virulence, as evidenced in a model of NHP (Whatmore et al., 1995).

Fig. 3. Immunogens used in the composition of HIV vaccines. There are three types of immunogens: whole vital particle, peptides or recombinant proteins and genetic material of the virus, which may or may not be inserted within vectors. Sometimes the combination of different immunogens can be used (prime-boost strategy).

HIV Vaccine 479

receptors - TLRs) and result in an improvement in the quantity or quality of the ensuing

**CLASS EXAMPLE** 

**NKT cell ligand** CD1d-binding NKT

Table 2. Molecular adjuvants for HIV vaccine strategy.

**CD40 agonists** CD40L or its derivates, Agonistic anti-CD40

and TLR9 (pDCs)

**NLRs agonists** MDP act on NOD2 and NALP3, extracellular

**Chemokines** MIP-1a, CCL19/EBI1-ligand and CCL21/SLC

Many adjuvants have been developed in the past, but were never accepted for routine vaccination because of safety concerns (e.g. acute toxicity and the possibility of delayed side

Vaccine delivery systems are the sum of pharmacologic technologies (including drug preparation, route of administration, site targeting, metabolism and toxicity) and have the principal aim to make the vaccine preparation faster and easier available to immune system. In its broadest sense, the concept of vaccine delivery systems can be expanded to include a diverse range of devices and physical delivery systems that are designed to improve the potency of vaccines or to allow immunization using novel, non-invasive routes (eg. genegun approach, devices designed to fire powdered vaccines into the skin through the use of helium gas and vaccine patches). Delivery systems may function to improve antigen access

a. increase antigens presenting cells (APCs), generally dendritic cells (DC), infiltration into

c. deliver antigen from the injection site to the local lymph node trough into the lymphatic

Various routes of HIV vaccine administration have been used starting from the most common (eg. subcutaneous, intramuscular) to more specific such as mucosal with different outcomes. HIV transmission occurs through mucosal (specially of the genital tract) and many efforts have been done to better characterize the mucosal associated lymphoid tissue (MALT) and its involvement in early HIV infection, with the final purpose to use the mucosal route of immunization. For vaccine design, the choice of mucosal inductive site is critical in determining the distal effector site to which induced memory cells will home. In

b. promote the uptake of antigen in APCs through activating phagocytosis;

antibodies, Heat shock protein (Hsp70)

TLR3, TLR4, and TLR7 (mDCs)

poly(I:C), LPS, and imidazoquinolines act on

imidazoquinolines and CpG ODP act on TLR7

flagellin act on TLR5 (Vassilieva et al., 2011)

ATP and pathogen RNA act on NALP3

immune response (Kornbluth & Stone, 2006).

**TLRs agonists** 

**3.2 Route of administration** 

to lymph nodes in a number of ways:

the injection site;

system

effects).


The advantages and disadvantages of some types of immunogens used in the development of HIV vaccines are summarized in Table 1.

Table 1. Advantages and disadvantages of some types of immunogens used in the development of HIV vaccines

#### **3.1.2 Adjuvants**

A vaccine adjuvant is a component that potentiates the immune responses to an antigen and/or modulates it towards the desired immune responses (i.e.: with respect to immunoglobulin classes and induction of cytotoxic or helper T lymphocyte responses). In addition, certain adjuvants can be used to promote antibody responses at mucosal surfaces.

Activation of innate immune system and in particular of dendritic cells (DCs) is a crucial mechanism by which adjuvants stimulate protective adaptive immunity against the vaccine antigen.

As immune response is typically initiated by activation of antigen presenting cells (APCs), notably dendritic cells (DCs). There has been significant interest in improving APCstimulating adjuvants as a key step in constructing better vaccines

Adjuvants have several forms, ranging from mineral salts such as alum to oil-based emulsions. Moreover molecular adjuvants such as proteins, lipids, nucleic acids, carbohydrates, or chemical compounds have a known receptor in DCs (i.e.: Toll Like

The advantages and disadvantages of some types of immunogens used in the development

PRODUCT ADVANTAGE DISADVANTAGE









simple and inexpensive


Table 1. Advantages and disadvantages of some types of immunogens used in the

A vaccine adjuvant is a component that potentiates the immune responses to an antigen and/or modulates it towards the desired immune responses (i.e.: with respect to immunoglobulin classes and induction of cytotoxic or helper T lymphocyte responses). In addition, certain adjuvants can be used to promote antibody responses at mucosal surfaces. Activation of innate immune system and in particular of dendritic cells (DCs) is a crucial mechanism by which adjuvants stimulate protective adaptive immunity against the

As immune response is typically initiated by activation of antigen presenting cells (APCs), notably dendritic cells (DCs). There has been significant interest in improving APC-

Adjuvants have several forms, ranging from mineral salts such as alum to oil-based emulsions. Moreover molecular adjuvants such as proteins, lipids, nucleic acids, carbohydrates, or chemical compounds have a known receptor in DCs (i.e.: Toll Like

stimulating adjuvants as a key step in constructing better vaccines

**DNA** -production relatively

of HIV vaccines are summarized in Table 1.

**Inactivated virus** 

**Peptide** 

**Recombinant proteins** 

**Vaccines based on biological vectors** 

development of HIV vaccines

**3.1.2 Adjuvants** 

vaccine antigen.

receptors - TLRs) and result in an improvement in the quantity or quality of the ensuing immune response (Kornbluth & Stone, 2006).


Table 2. Molecular adjuvants for HIV vaccine strategy.

Many adjuvants have been developed in the past, but were never accepted for routine vaccination because of safety concerns (e.g. acute toxicity and the possibility of delayed side effects).

#### **3.2 Route of administration**

Vaccine delivery systems are the sum of pharmacologic technologies (including drug preparation, route of administration, site targeting, metabolism and toxicity) and have the principal aim to make the vaccine preparation faster and easier available to immune system. In its broadest sense, the concept of vaccine delivery systems can be expanded to include a diverse range of devices and physical delivery systems that are designed to improve the potency of vaccines or to allow immunization using novel, non-invasive routes (eg. genegun approach, devices designed to fire powdered vaccines into the skin through the use of helium gas and vaccine patches). Delivery systems may function to improve antigen access to lymph nodes in a number of ways:


Various routes of HIV vaccine administration have been used starting from the most common (eg. subcutaneous, intramuscular) to more specific such as mucosal with different outcomes. HIV transmission occurs through mucosal (specially of the genital tract) and many efforts have been done to better characterize the mucosal associated lymphoid tissue (MALT) and its involvement in early HIV infection, with the final purpose to use the mucosal route of immunization. For vaccine design, the choice of mucosal inductive site is critical in determining the distal effector site to which induced memory cells will home. In

HIV Vaccine 481

Once proven its potential as a vaccine candidate, the clinical phase of the study will start to evaluate safety, immunogenicity and efficacy of the product. Phases I-III are required for licensing the product. In the process of vaccine development, clinical trials may last for many years and the number of volunteers is increasing at every step. The goals set for each stage involves pharmacological and clinical issues, evaluated in a progressive manner

The clinical trial itself begins in Phase I: the candidate vaccine is first evaluated in a small group of human volunteers in order to evaluate its safety, tolerability, pharmacokinetics and

In Phase I trial it is also possible to evaluate efficacy markers (e.g.: the generation of antibodies and/or cytotoxic T response), allowing a preliminary assessment of the ability of the vaccine to generate an immune response. Once the safety of the candidate product has

The objective of Phase II is to test the candidate vaccine in a larger number of volunteers with two principal purposes: identify side effects related to the product use (within the perspective of future safety analysis, i.e.: toxicity) and collect preliminary indications of

Sometimes these aims are studied in different moments and the Phase II trials are divided

The Phase III trials are the last stage before possible licensing of the vaccine for marketing. They are randomized controlled trials, often multi-centric, involving large numbers of patients. The primary objective of this step is to evaluate the effectiveness of the product. Achieving a high level of effectiveness at this stage, however, does not necessarily guarantee that the product is effective in the general population, which will be evaluated in phase IV. Phase IV trials, also called post-evaluation of efficacy, are pharmacovigilance studies performed after licensing the product and that aim to measure the effect of the product in a population. The importance of this phase is to assess the real impact of a vaccine in the

The conduct of clinical trials involving prophylactic and therapeutic vaccines for HIV remains a challenge. In terms of design and implementing Phase I and early Phase II are relatively easy to do, although studies involving analysis of effectiveness show a higher degree of complexity. For prophylactic vaccines, the statistical requirements to demonstrate a real prevention of infection require a very large number of patients and they are sometimes prohibitive. Clinical trials for HIV vaccines require the appropriate preclinical

The role of society is essential for the success of all the program to develop HIV vaccines and the establishment of a genuine dialogue with the community facilitates clinical research

Obtaining an HIV vaccine has been one of the biggest challenges of this century. To date numerous clinical trials have been conducted to test candidate products as prophylactic

a. Phase IIa: designed to determine the optimal dose of vaccine (dose-response studies).

throughout the process (www.ich.org - General Considerations for Clinical Trials).

pharmacodynamics and identify possible side effects.

b. Phase IIb: designed to study the vaccine efficacy.

product potential effectiveness (efficacy).

into:

epidemic.

with HIV vaccines.

vaccine.

**4. Current outlook** 

been checked, the research could proceed to the next step (Phase II).

studies and the development of better laboratory markers of efficacy.

HIV vaccine research, many mucosal sites were chosen to administer the immunogen: nasal mucosal (Vajdy & Singh, 2006), intratracheal/aerosol vaccination (Corbett et al., 2008), oral vaccination (Stahl-Hennig et al., 2007, N. Cuburu et al., 2007), rectal/colonic vaccination (Belyakov et al., 2006), intravaginal vaccination- (Pialoux et al., 2008).

#### **3.3 Vaccination strategies**

The vaccination strategy comprehends the combination of diverse types of vaccine (immunogen, adjuvant, route of immunization) with different schedules to increasing the vaccine potential immunogenic effect. In HIV vaccine several combinations of immunogens (inactivated virus, viral proteins, recombinant viral DNA), routes and schedules have been tested to augment the delivery of HIV antigens to the immune system.

For example, viral vectors are efficient to place HIV relevant epitopes within the target cells and, due to their composition, to stimulate the innate response, promoting an adjuvant effect, although it has been observed that the immunogenicity of these vectors could be affected by the competition between HIV and vector epitopes in the context of antigen presentation. For these reason it was developed the strategy to combined this type of vaccine with a vaccine based on HIV proteins or peptides in a such called prime-boost regimen.

Prime-boost strategy was first time used in 1992 in NHP studies (Hu et al., 1992). Briefly, it consists in the administration of one type of vaccine, such as a live-vector vaccine, followed by or together with a second type of vaccine, such as a recombinant subunit vaccine. The intent of this combination regimen is to induce different types of immune responses and enhance the overall immune response, a result that may not occur if only one type of vaccine were to be given for all doses (Ranasinghe et al., 2009).

#### **3.4 Steps of vaccine development**

The development of a vaccine is a process that requires several steps aiming to answer specific questions and to test concepts through experimental practice. Scientific knowledge generated from the execution of each stage or phase will give useful data for planning the next step.

The development of an effective vaccine against HIV infection, due to its unique aspects, will be an unprecedented challenge, and scientific rigor and discipline, statistical principles and bioethics should be required to achieve success.

The preliminary step is to generate more ideas. Established scientists in universities, research institutes and industry use the existing scientific knowledge and technology to develop ideas of how a vaccine might work.

From there, preclinical studies should be performed before human trials to assess whether a novel product has scientific merit to be a candidate vaccine. Such pre-clinical studies involve *in vitro* experiments and *in vivo* tests in available animal models to obtain information regarding the efficacy, toxicity and pharmacokinetics, using varying doses of the product being tested.

The transformation process of a candidate product in a vaccine logarithmic increases the cost and complexity of the research, as it moves from laboratory to clinical application. It is also important to emphasize that only a small percentage of the candidate products being studied in preclinical development is considered safe and promising enough to be evaluated in humans (clinical phase).

HIV vaccine research, many mucosal sites were chosen to administer the immunogen: nasal mucosal (Vajdy & Singh, 2006), intratracheal/aerosol vaccination (Corbett et al., 2008), oral vaccination (Stahl-Hennig et al., 2007, N. Cuburu et al., 2007), rectal/colonic vaccination

The vaccination strategy comprehends the combination of diverse types of vaccine (immunogen, adjuvant, route of immunization) with different schedules to increasing the vaccine potential immunogenic effect. In HIV vaccine several combinations of immunogens (inactivated virus, viral proteins, recombinant viral DNA), routes and schedules have been

For example, viral vectors are efficient to place HIV relevant epitopes within the target cells and, due to their composition, to stimulate the innate response, promoting an adjuvant effect, although it has been observed that the immunogenicity of these vectors could be affected by the competition between HIV and vector epitopes in the context of antigen presentation. For these reason it was developed the strategy to combined this type of vaccine with a vaccine based on HIV proteins or peptides in a such called prime-boost

Prime-boost strategy was first time used in 1992 in NHP studies (Hu et al., 1992). Briefly, it consists in the administration of one type of vaccine, such as a live-vector vaccine, followed by or together with a second type of vaccine, such as a recombinant subunit vaccine. The intent of this combination regimen is to induce different types of immune responses and enhance the overall immune response, a result that may not occur if only one type of vaccine

The development of a vaccine is a process that requires several steps aiming to answer specific questions and to test concepts through experimental practice. Scientific knowledge generated from the execution of each stage or phase will give useful data for planning the

The development of an effective vaccine against HIV infection, due to its unique aspects, will be an unprecedented challenge, and scientific rigor and discipline, statistical principles

The preliminary step is to generate more ideas. Established scientists in universities, research institutes and industry use the existing scientific knowledge and technology to

From there, preclinical studies should be performed before human trials to assess whether a novel product has scientific merit to be a candidate vaccine. Such pre-clinical studies involve *in vitro* experiments and *in vivo* tests in available animal models to obtain information regarding the efficacy, toxicity and pharmacokinetics, using varying doses of the product

The transformation process of a candidate product in a vaccine logarithmic increases the cost and complexity of the research, as it moves from laboratory to clinical application. It is also important to emphasize that only a small percentage of the candidate products being studied in preclinical development is considered safe and promising enough to be evaluated

(Belyakov et al., 2006), intravaginal vaccination- (Pialoux et al., 2008).

tested to augment the delivery of HIV antigens to the immune system.

were to be given for all doses (Ranasinghe et al., 2009).

and bioethics should be required to achieve success.

develop ideas of how a vaccine might work.

**3.4 Steps of vaccine development** 

**3.3 Vaccination strategies** 

regimen.

next step.

being tested.

in humans (clinical phase).

Once proven its potential as a vaccine candidate, the clinical phase of the study will start to evaluate safety, immunogenicity and efficacy of the product. Phases I-III are required for licensing the product. In the process of vaccine development, clinical trials may last for many years and the number of volunteers is increasing at every step. The goals set for each stage involves pharmacological and clinical issues, evaluated in a progressive manner throughout the process (www.ich.org - General Considerations for Clinical Trials).

The clinical trial itself begins in Phase I: the candidate vaccine is first evaluated in a small group of human volunteers in order to evaluate its safety, tolerability, pharmacokinetics and pharmacodynamics and identify possible side effects.

In Phase I trial it is also possible to evaluate efficacy markers (e.g.: the generation of antibodies and/or cytotoxic T response), allowing a preliminary assessment of the ability of the vaccine to generate an immune response. Once the safety of the candidate product has been checked, the research could proceed to the next step (Phase II).

The objective of Phase II is to test the candidate vaccine in a larger number of volunteers with two principal purposes: identify side effects related to the product use (within the perspective of future safety analysis, i.e.: toxicity) and collect preliminary indications of product potential effectiveness (efficacy).

Sometimes these aims are studied in different moments and the Phase II trials are divided into:

a. Phase IIa: designed to determine the optimal dose of vaccine (dose-response studies).

b. Phase IIb: designed to study the vaccine efficacy.

The Phase III trials are the last stage before possible licensing of the vaccine for marketing. They are randomized controlled trials, often multi-centric, involving large numbers of patients. The primary objective of this step is to evaluate the effectiveness of the product. Achieving a high level of effectiveness at this stage, however, does not necessarily guarantee that the product is effective in the general population, which will be evaluated in phase IV.

Phase IV trials, also called post-evaluation of efficacy, are pharmacovigilance studies performed after licensing the product and that aim to measure the effect of the product in a population. The importance of this phase is to assess the real impact of a vaccine in the epidemic.

The conduct of clinical trials involving prophylactic and therapeutic vaccines for HIV remains a challenge. In terms of design and implementing Phase I and early Phase II are relatively easy to do, although studies involving analysis of effectiveness show a higher degree of complexity. For prophylactic vaccines, the statistical requirements to demonstrate a real prevention of infection require a very large number of patients and they are sometimes prohibitive. Clinical trials for HIV vaccines require the appropriate preclinical studies and the development of better laboratory markers of efficacy.

The role of society is essential for the success of all the program to develop HIV vaccines and the establishment of a genuine dialogue with the community facilitates clinical research with HIV vaccines.

#### **4. Current outlook**

Obtaining an HIV vaccine has been one of the biggest challenges of this century. To date numerous clinical trials have been conducted to test candidate products as prophylactic vaccine.

HIV Vaccine 483

Multiclade Recombinant HIV-1 Adenoviral Vector Vaccine, VRCHIVADV014-00-VP

Recombinant adenovirus serotype

A candidate prophylactic DNA prime-rFPV boost HIV vaccination strategy (rFPV-HIV-AE;pHIS-HIV-

HIV-1 adenovirus vector vaccine VRC-HIVADV027-00VP: dose escalation and prime-boost with an

Recombinant Adenoviral Subtype 35 (rAd35) and Subtype 5 (rAd5) HIV-1 Vaccines When Given as a Heterologous Prime-Boost Regimen or as Boosts to a Recombinant DNA Vaccine in Healthy, Ad5-Naïve and Ad5-Exposed (VRC-HIVDNA044- 00-VP;VRC-HIVADV027-00- VP;VRC-HIVADV038-00-VP)

HIV-1 adenovirus vector vaccine, VRC-HIVADV038-00-VP

Canarypox viral vector with

VICHREPOL with polyoxidonium

Sanofi Pasteur Live Recombinant ALVAC-HIV (vCP205, HIV-1 Env/Gag/Pol) subcutaneously, intradermally, or intramuscularly

Recombinant HIV-1 multi-envelope

VRC-HIVADV014-00-VP alone or as a boost to VRCHIVDNA009-00-

VP

Table 3. Ongoing clinical trials (www.avac.org/ht/a/GetDocumentAction/i/3436). The results of Phase II and Phase III major prophylactic trials are summarized above.

*env*and*gag-pol* <sup>50</sup>Ugand

adjuvant 15 Russia I

DNA plasmid vaccine with *env* 6 USA I

26 (rAd26) vaccine 48 USA I

SITE fase

40 USA I

<sup>8</sup>Thaila

35 USA I

192 USA I

<sup>a</sup><sup>I</sup>

36 USA I

<sup>30</sup>Ugand

<sup>a</sup><sup>I</sup>

nd I/II

PROTOCOL SPONSOR PRODUCT N TRIAL

AE)

Crossroads, Chris Hani BaragwanathHospita

Center

Center, Crucell

NIAID, VRC, NIH Clinical

NIAID, IPCAVD, Brigham and Women's Hospital, Beth Israel Deaconess Medical

NCHECR, University of New South Wales, Thai Red Cross AIDS Research Centre

NIAID, HVTN, Alabama Vaccine, San Francisco Vaccine and Prevention, Hope Clinic of the Emory Vaccine Center, NY Blood Ctr./Union Square, NY Blood Ctr./Bronx, University of Rochester

HVTN, International Maternal Pediatric Adolescent AIDS Clinical Trials Group,Makerere University, Johns Hopkins University, Mulago Hospital,

Sanofi-Pasteur

**RV 138; B011** Walter Reed Army Institute

Research Hospital

MHRP, Makerere U.

*EnvDNA* St. Jude's Children's

**RV 156A** NIAID, HVTN, VRC,

Moscow Institute of Immunology, FederalMedical and Biological Agency, Russian Federation Ministry of Education and Science

of Research, US Military HIV

**VRC 015 (08-1-**

**Ad26.ENVA.01** 

**NCHECR-AE1** 

**HVTN 077** 

**HPTN 027** 

**HVRF-380- 131004** 

**VRC 012** NIAID, VRC

HVTN

**0171)** 

Most initial approaches have focused on the gp120 HIV envelope protein. At least thirteen different gp120 candidates have been evaluated in Phase I trials in the USA predominantly through the AIDS Vaccine Evaluation Group, showing to be safe and immunogenic in diverse populations. They have induced neutralizing antibody, but rarely induced CD8+ cytotoxic T lymphocytes (CTL). Moreover it was very difficult to induce and maintain the high anti-gp120 antibody titers necessary to have any hope of neutralizing an HIV exposure. The availability of several recombinant vectors (adenovirus, canarypox) carrying HIV gens (gag, pol, nef or env) has provided interesting results characterized principally by a polyfunctional CTL responses.


Currently, about 20 clinical trials are underway, most protocols for Phase I.


Most initial approaches have focused on the gp120 HIV envelope protein. At least thirteen different gp120 candidates have been evaluated in Phase I trials in the USA predominantly through the AIDS Vaccine Evaluation Group, showing to be safe and immunogenic in diverse populations. They have induced neutralizing antibody, but rarely induced CD8+ cytotoxic T lymphocytes (CTL). Moreover it was very difficult to induce and maintain the high anti-gp120 antibody titers necessary to have any hope of neutralizing an HIV exposure. The availability of several recombinant vectors (adenovirus, canarypox) carrying HIV gens (gag, pol, nef or env) has provided interesting results characterized principally by a poly-

PROTOCOL SPONSOR PRODUCT N TRIAL

**PedVacc002** Medical Research Council MVA.HIVA 48 Kenya I

gp140 *env*

tat)

SITE fase

zland I/II

1,350 US II

42 USA I

32 India I

<sup>225</sup>USA,

48

Peru II

I

USA, South Africa

<sup>n</sup><sup>I</sup>

HIVADV014-00-VP USA I

VP 80 Switer

VRC-HIVDNA016-00-VP; VRC-

Prime: VRC-HIVDNA016-00-VP

Adenovirus serotype 35 vector. Ad35-GRIN/ENV consists of two vectors: Ad35-GRIN vector with *gag*, reverse transcriptase, integrase, and *nef*Ad35-ENV vector with

Prime: ADVAX (DNA vaccine containing env, gag, pol, nef and

Boost: TBC-M4 (MVA vector with env, gag, RT, rev, tat and nef)

HIV-1 CN54 gag, pol and env genes

Recombinant Adenovirus HIV-1

Prime: DNA vaccine containing gag, pol, env, rat,rev, vpu Boost: MVA vaccine containing

gag, pol, env

*nef, env*

Prime: SAAVI DNA-C2 Boost: SAAVI MVA-C; DNA plasmid vaccine with *gag, RT, tat,* 

with DNA and rTV vectors 80 China I

Vaccine, Ad5HVR48.ENVA.01 48 USA I

HIVADV014-00-VP

Infectious disease Control MVA-CMDR 24 Swede

Currently, about 20 clinical trials are underway, most protocols for Phase I.

**HVTN 082** NIAID, HVTN VRC-HIVDNA016-00-VP; VRC-

**HVTN 078** NIAID, EuroVacc, HVTN NYVAC-B; VRC-HIVADV038-00-

functional CTL responses.

**HVTN 505** NIAID, HVTN

**B001** IAVI, University of

**HIVIS 05** Swedish Institute for

Pune

**HVTN205** GeoVax, HVTN

Rochester Medical Center

IAVI, Indian Council of Medical Research, Tuberculosis Research Centre, Chennai; National AIDS Research Institute,

Chinese Center for Disease Control and Prevention, National Vaccine and SerumInstitute, Peking Union Medical College

HVTN, SAAVI, Brigham and Women's Hospital CRS, Fenway Community Health, Clinical Research Boston,

NIAID, Brigham and Women's Hospital

**PedVacc001 &** 

**P001** 

**HIV Vaccine** 

**Tiantianvaccinia** 

**Ad5HVR48.EN VA.01** 

**HVTN 073** 


Table 3. Ongoing clinical trials (www.avac.org/ht/a/GetDocumentAction/i/3436).

The results of Phase II and Phase III major prophylactic trials are summarized above.

HIV Vaccine 485

The rate of HIV infection among volunteers who received the experimental vaccine being tested in the trial was 31% lower than the rate of HIV infection among volunteers who

Although showing only a modest benefit, this work has renewed optimism in this field of research. However, criticisms related primarily to the study design and statistical method employed to analyse data generated debate about the results (Cohen, 2009; Letvin, 2009).

In addition to trials aimed at obtaining prophylactic HIV vaccine, has been also developed protocols for therapeutic vaccination using dendritic cells (DC) for the treatment of

DCs are potent antigen presenting cells that act as controllers and regulators of the immune system and are the only cells capable of fully activate naive CD4 lymphocytes and thus initiate a specific response (Banchereau & Steinman, 1998). In the context of an HIV vaccine

Fig. 4. Treatment of HIV infected patients with monocyte-derived DCs. Peripheral blood mononuclear cells (PBMC) are obtained by leukapheresis and monocytes are separated and cultured in the presence of IL-4 and GM-CSF to obtain immature DCs (iDCs). iDCs are loaded with the antigen of interest and are activated by different stimuli for maturation. Mature DCs (MDCs), potentially able to migrate and to present antigens, are reinoculate

received placebo (Rerks-Ngarm et al., 2009).

**4.4 Dendritic cell based immunotreatment** 

individuals already infected with HIV.

into the patient.

#### **4.1 VAX 004 trial (Phase III, USA 1998-2002)**

The phase III VAX 004 trial enrolled 5,403 USA participants between 1998 and 1999. Volunteers received 7 injections of either vaccine or placebo (ratio, 2:1) over 30 months.

The study vaccine contained 2 rgp120 HIV-1 envelope antigens (300 mg each of two recombinant proteins rgp120/HIV-1 MN and GNE8) (AIDSVAX B/B; VaxGen) that had been derived from 2 different subtype B strains and that were adsorbed onto 600 mg of alum. GNE8 gp120 was cloned directly from peripheral-blood mononuclear cells and had the CCR5 phenotype; the GNE8 gp120 DNA sequence was deposited in GenBank.

The vaccine did not prevent HIV-1 acquisition and there was no overall protective effect (Flynn et al., 2005).

#### **4.2 STEP trial (Phase II, USA, 2004-2007)**

On December 13, 2004, the HIV Vaccine Trials Network (HVTN) began recruiting for the STEP study, a 3,000-participant phase II clinical trial of a novel HIV vaccine, at sites in North America, South America, the Caribbean and Australia.

The trial was co-funded by the National Institute of Allergy and Infectious Diseases (NIAID/NIH, USA), and the pharmaceutical company Merck & Co. Merck developed the experimental vaccine called V520 which contains a adenoviral vector rAd5 carrying three subtype B HIV genes (gag/pol/nef). The vaccine was administered in prime-boost regimen at 0, 1 and 6 months. The follow up of vaccinated subjects showed the lack of efficacy of this vaccine, as well as an increment in HIV-1 infection in individuals with prior immunity to adenovirus. Adenovirus vectors and many other viral vectors currently used in HIV vaccines, will induce a rapid memory immune response against the vector. This results in an impediment to the development of a T cell response against the inserted antigen.

For this reason the phase II trial was closed in September 2007 and other vaccine protocols in progress including the same vector vaccine such as the HVTN503 (Phambili) were cancelled or modificated (Barouch & Korber, 2010).

While the final results of STEP have been disappointing, this study has raised its contribution to redefine the priorities in HIV vaccines research field, demonstrating the need to focus on basic research, preclinical and clinical studies.

#### **4.3 RV144 trial (Phase III, Thailand, 2003-2009)**

The phase III HIV vaccine RV144 involved more than 16,000 young Thailandese adults at variable risk for infection between October 2003 and September 2009. Ever six months, volunteers received a prime-boost vaccination including six injections of a vaccine called ALVAC-HIV (vCP1521, Sanofi Pasteur) with the last two of the six injections being a combination of that vaccine and another one called AIDSVAX B/E (gp120, Genentech).

ALVACHIV consists of a viral vector containing genetically engineered versions of three HIV genes (env, gag and pro).The ALVAC vector is an inert form of canarypox, a bird virus which cannot cause disease or replicate in humans. AIDSVAX B/E is composed of genetically engineered gp120. The RV 144 protocol was sponsored by the Surgeon General of the United States Army and conducted by the Thailand Ministry of Public Health with support from the United States Army Medical Research and Materiel Command and the NIAD/NIH.

The phase III VAX 004 trial enrolled 5,403 USA participants between 1998 and 1999. Volunteers received 7 injections of either vaccine or placebo (ratio, 2:1) over 30 months. The study vaccine contained 2 rgp120 HIV-1 envelope antigens (300 mg each of two recombinant proteins rgp120/HIV-1 MN and GNE8) (AIDSVAX B/B; VaxGen) that had been derived from 2 different subtype B strains and that were adsorbed onto 600 mg of alum. GNE8 gp120 was cloned directly from peripheral-blood mononuclear cells and had

The vaccine did not prevent HIV-1 acquisition and there was no overall protective effect

On December 13, 2004, the HIV Vaccine Trials Network (HVTN) began recruiting for the STEP study, a 3,000-participant phase II clinical trial of a novel HIV vaccine, at sites in North

The trial was co-funded by the National Institute of Allergy and Infectious Diseases (NIAID/NIH, USA), and the pharmaceutical company Merck & Co. Merck developed the experimental vaccine called V520 which contains a adenoviral vector rAd5 carrying three subtype B HIV genes (gag/pol/nef). The vaccine was administered in prime-boost regimen at 0, 1 and 6 months. The follow up of vaccinated subjects showed the lack of efficacy of this vaccine, as well as an increment in HIV-1 infection in individuals with prior immunity to adenovirus. Adenovirus vectors and many other viral vectors currently used in HIV vaccines, will induce a rapid memory immune response against the vector. This results in an

For this reason the phase II trial was closed in September 2007 and other vaccine protocols in progress including the same vector vaccine such as the HVTN503 (Phambili) were

While the final results of STEP have been disappointing, this study has raised its contribution to redefine the priorities in HIV vaccines research field, demonstrating the need

The phase III HIV vaccine RV144 involved more than 16,000 young Thailandese adults at variable risk for infection between October 2003 and September 2009. Ever six months, volunteers received a prime-boost vaccination including six injections of a vaccine called ALVAC-HIV (vCP1521, Sanofi Pasteur) with the last two of the six injections being a combination of that vaccine and another one called AIDSVAX B/E (gp120, Genentech). ALVACHIV consists of a viral vector containing genetically engineered versions of three HIV genes (env, gag and pro).The ALVAC vector is an inert form of canarypox, a bird virus which cannot cause disease or replicate in humans. AIDSVAX B/E is composed of genetically engineered gp120. The RV 144 protocol was sponsored by the Surgeon General of the United States Army and conducted by the Thailand Ministry of Public Health with support from the United States Army Medical Research and Materiel Command and the

the CCR5 phenotype; the GNE8 gp120 DNA sequence was deposited in GenBank.

impediment to the development of a T cell response against the inserted antigen.

**4.1 VAX 004 trial (Phase III, USA 1998-2002)** 

**4.2 STEP trial (Phase II, USA, 2004-2007)** 

America, South America, the Caribbean and Australia.

cancelled or modificated (Barouch & Korber, 2010).

**4.3 RV144 trial (Phase III, Thailand, 2003-2009)** 

to focus on basic research, preclinical and clinical studies.

(Flynn et al., 2005).

NIAD/NIH.

The rate of HIV infection among volunteers who received the experimental vaccine being tested in the trial was 31% lower than the rate of HIV infection among volunteers who received placebo (Rerks-Ngarm et al., 2009).

Although showing only a modest benefit, this work has renewed optimism in this field of research. However, criticisms related primarily to the study design and statistical method employed to analyse data generated debate about the results (Cohen, 2009; Letvin, 2009).

#### **4.4 Dendritic cell based immunotreatment**

In addition to trials aimed at obtaining prophylactic HIV vaccine, has been also developed protocols for therapeutic vaccination using dendritic cells (DC) for the treatment of individuals already infected with HIV.

DCs are potent antigen presenting cells that act as controllers and regulators of the immune system and are the only cells capable of fully activate naive CD4 lymphocytes and thus initiate a specific response (Banchereau & Steinman, 1998). In the context of an HIV vaccine

Fig. 4. Treatment of HIV infected patients with monocyte-derived DCs. Peripheral blood mononuclear cells (PBMC) are obtained by leukapheresis and monocytes are separated and cultured in the presence of IL-4 and GM-CSF to obtain immature DCs (iDCs). iDCs are loaded with the antigen of interest and are activated by different stimuli for maturation. Mature DCs (MDCs), potentially able to migrate and to present antigens, are reinoculate into the patient.

HIV Vaccine 487

becomes desirable to induce a specific and effective activation of the immune system against

Protocols of immunotherapy with DCs began in the late 1990 and since then a growing number of studies evaluating this strategy. Because it is an individualized protocol, the number of individuals in the tests is always limited, never exceeding a few tens of

It is a strategy that involves the collection of mononuclear cells from HIV-infected individual, separation of monocytes and stimulation of these cells with cytokines to differentiate into immature dendritic cells. Dendritic cells are then sensitized (pulsed or loaded) with the antigen of interest, activated and reinoculated into the individuals (Figure 4). The objective of this strategy is to stimulate the immune response by enhancing antigen

An overview of the works conducted so far (Table 4) shows although that the products are always safe and the results are quite heterogeneous (Kundu et al., 1998; García et al., 2005,

Considering the difficulty to obtain an HIV prophylactic vaccine, the immunotherapy offers a unique opportunity to study the mechanisms of immune response against the virus and contribute to the definition of correlates of protection in HIV infection. Knowledge generated from studies of DC-based immunotherapy may contribute also to the

Despite numerous difficulties and great scientific challenges that must be overcome to obtain an HIV vaccine, the extraordinary advance in biomedical research and the

Knowledge has been accumulated on the biology and diversity of HIV; new methods have been used for the production of immunologically relevant antigens; the study of immune response in exposed not-infected individuals and in elite controllers has generated important information regarding the type of effective immune response against HIV. Furthermore, immunotherapy protocols in infected individuals provide a unique

Lessons from the failure of the previous protocols can effectively guide the design and refinement of the next generation of candidate vaccines. In this scenario, the perspective is that knowledge of the various interdisciplinary areas of science can provide an environment

Banchereau, J., Steinman, R.M. (1998). Dendritic cells and the control of immunity. *Nature* ,

Barouch, D.H., Korber B. (2010). HIV-1 vaccine development after STEP. Annu Rev Med,

Belyakov, I.M., Kuznetsov, V.A., Kelsall, B., Klinman, D., Moniuszko, M., Lemon, M.,

Markham, P.D., Pal, R., Clements, J.D., Lewis, M.G., Strober, W., Franchini, G. and Berzofsky J.A. (2006). Impact of vaccine-induced mucosal high-avidity CD8+ CTLs

Vol 392, No. 6673, (Mar 1998), pp. 245-252, ISSN 0028-0836.

2011; Ide at al., 2006; Connolly et al., 2008, Lu et al., 2004, Ghandhi, 2009)

remarkable progress achieved show clear reasons for optimism.

opportunity to studying immune mechanisms against the virus.

Vol. 61, (Feb 2010), pp.153-167, ISSN 0066-4219.

leading to overcome these scientific challenges.

the viral chronic infection.

presentation mediated by dendritic cells.

development of prophylactic vaccines.

**5. Conclusions** 

**6. References** 

individuals.


Table 4. Parameters used in the post-vaccine immune response assessment HLA= **Human Leukocyte Antigen.** IL-2= **Interleukin**-**2** . PBMCs=Peripheral Blood Mononuclear Cells.

IMMUNOGENICITY ASSESSMENT

Envelope-specific CTL- and lymphocyte-proliferative responses, IFN-gamma and IL-2 production, peptide-specific lymphocyte-proliferative

neutralizingantibodytiters, HIV-1-specific interferon-γ (IFN-γ) expressing CD4+ T and CD8+ cells, HIV-1-specific IL-2- expressing CD4+ T cells, HIV-1 gag−specific CD8+ T cells, HIV-1 gag−specific CD8+ T cells expressing perforin

Lymphoproliferation, Th1 cell levels, cytotoxic T lymphocyte [CTL] levels,

neutralizingantibodytiters and changes in lymphoid tissue

IFN-g production in CD8

Gamma interferon (IFN-) producing cells (PBMC)

Gamma interferon (IFN-) producing cells (PBMC), lymphocyte-proliferative

Lymphoproliferation, serum neutralizing antibody titers,

responses

Serum

serum

lymphocytes

responses

ELISPOT

ANTIGEN

Recombinant HIV-1 MN gp160 or HLA-A2-restricted synthetic peptides of envelope, Gag,

and Pol

**2004** 18 Chemically inactivated

autologous HIV-1

Heat-inactivated autologous human immunodeficiency virus type 1 (HIV-1)

HIV-1-derived cytotoxic T

peptides

<sup>18</sup>Gag, Env, and Pol peptides

> Viral vector (canarypox) expressing HIV-1 envandgag and a

synthetic polypeptide encompassing epitopes from nefandpol

Heat-inactivated autologous human immunodeficiency virus type 1 (HIV-1)

Table 4. Parameters used in the post-vaccine immune response assessment

HLA= **Human Leukocyte Antigen.** IL-2= **Interleukin**-**2** . PBMCs=Peripheral Blood

lymphocytes (CTL)

AUTHORS n TYPE OF

**Kundu et al., 1998** <sup>6</sup>

**Lu W et al.,** 

**Garcia F et al.,2005** <sup>12</sup>

**Ide F et al., <sup>2006</sup>**<sup>4</sup>

**Connolly NC et al., 2008** 

**Ghandhi RT et al., 2009** 

**Garcia F et al, 2011** <sup>24</sup>

Mononuclear Cells.

29

becomes desirable to induce a specific and effective activation of the immune system against the viral chronic infection.

Protocols of immunotherapy with DCs began in the late 1990 and since then a growing number of studies evaluating this strategy. Because it is an individualized protocol, the number of individuals in the tests is always limited, never exceeding a few tens of individuals.

It is a strategy that involves the collection of mononuclear cells from HIV-infected individual, separation of monocytes and stimulation of these cells with cytokines to differentiate into immature dendritic cells. Dendritic cells are then sensitized (pulsed or loaded) with the antigen of interest, activated and reinoculated into the individuals (Figure 4). The objective of this strategy is to stimulate the immune response by enhancing antigen presentation mediated by dendritic cells.

An overview of the works conducted so far (Table 4) shows although that the products are always safe and the results are quite heterogeneous (Kundu et al., 1998; García et al., 2005, 2011; Ide at al., 2006; Connolly et al., 2008, Lu et al., 2004, Ghandhi, 2009)

Considering the difficulty to obtain an HIV prophylactic vaccine, the immunotherapy offers a unique opportunity to study the mechanisms of immune response against the virus and contribute to the definition of correlates of protection in HIV infection. Knowledge generated from studies of DC-based immunotherapy may contribute also to the development of prophylactic vaccines.

#### **5. Conclusions**

Despite numerous difficulties and great scientific challenges that must be overcome to obtain an HIV vaccine, the extraordinary advance in biomedical research and the remarkable progress achieved show clear reasons for optimism.

Knowledge has been accumulated on the biology and diversity of HIV; new methods have been used for the production of immunologically relevant antigens; the study of immune response in exposed not-infected individuals and in elite controllers has generated important information regarding the type of effective immune response against HIV. Furthermore, immunotherapy protocols in infected individuals provide a unique opportunity to studying immune mechanisms against the virus.

Lessons from the failure of the previous protocols can effectively guide the design and refinement of the next generation of candidate vaccines. In this scenario, the perspective is that knowledge of the various interdisciplinary areas of science can provide an environment leading to overcome these scientific challenges.

#### **6. References**


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and Burton, D.R. (2001). Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro. *J Virol*, Vol. 75, No. 17, pp. 8340-8347, ISSN

Mathieu, E., Gaillard, C., Petitprez, K., Launay, O. and Chene, G. (2008). Phase I study of a candidate vaccine based on recombinant HIV-1 gp160 (MN/LAI) administered by the mucosal route to HIV-seronegative volunteers: The ANRS

non-progressors: the viral, genetic and immunological basis for disease non-

strategies for improved systemic and mucosal immunity. *Expert Rev Vaccines* , Vol.

Premsri, N., Namwat, C., de Souza, M., Adams, E., Benenson, M., Gurunathan, S., Tartaglia, J., McNeil, J.G., Francis, D.P., Stablein, D., Birx, D.L., Chunsuttiwat, S., Khamboonruang, C., Thongcharoen, P., Robb, M.L., Michael, N.L., Kunasol, P., Kim, J.H. and MOPH-TAVEG Investigators. (2009). Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. *,* Vol. 361, No. 23, pp. 2209-2220, ISSN 1533-4406.


**24** 

*Norway* 

**Towards a Functional Cure for HIV Infection:** 

Human immunodeficiency virus (HIV-1) currently infects 33.3 million people globally. In 2009, 1.8 million people died from acquired immunodeficiency syndrome (AIDS) marking a decline in AIDS deaths by 19% since 1999, the estimated peak of the pandemic. This is largely due to the introduction of combination antiretroviral therapy (ART) in 1996 and its expanding access in recent years. However, despite continued efforts to improve ART availability worldwide, only 5 of the estimated 15 million people living with HIV-1 in lowand middle-income countries have access (UNAIDS, 2010). Furthermore, the number of new infections continues to outpace the number of people being put on ART each day. ART is costly, and places a formidable financial burden on healthcare services. This in turn

Combination ART has made a significant impact on HIV-1 morbidity and mortality (Vittinghoff et al., 1999, Palella et al., 2006) and represents the 'gold standard' for HIV-1 treatment. Despite early optimism that combination ART could potentially eradicate infection (Perelson et al., 1996, Ho, 1997), it has since become clear that virus invariably returns if ART is stopped. As a result, ART remains a daily lifelong treatment requiring a

Where ART is available, the diagnosis 'AIDS' becomes less frequent, and HIV-1 infection may no longer be considered a irrevocable terminal disease but rather a chronic manageable infection. However, recent studies have observed that ART does not restore life expectancy completely (Neuhaus et al., 2010a; The Antiretroviral Therapy Cohort, 2008). Furthermore, as those living with HIV-1 do become older, age-related toxicities emerge (Powderly, 2007, 2010) as well as other ART co-morbidities such as increased risk of cardiovascular disease, metabolic disorders, neurocognitive abnormalities, liver and renal disease, bone disorders,

Untreated HIV-1 infection is characterised by a substantial depletion of CD4+ T-cells in the mucosa as well as a gradual progressive decline of CD4+ T-cells in peripheral blood. When CD4+ T-cell levels in peripheral blood fall below 200 cells/mm3, immune competence is reduced leading to susceptibility to opportunistic infections and conditions that characterise AIDS as well as significant increases in viral load (Levy, 2007). It is primarily the level of CD4+ T-cells in peripheral blood that determines the requirement for ART (Panel on

high level of compliance to avoid the development of (multi) drug resistance.

**1. Introduction**

compromises efforts for universal access.

malignancy and frailty (Deeks & Phillips, 2009).

Antiretroviral Guidelines for Adults and Adolescents, 2011).

**The Potential Contribution of** 

**Therapeutic Vaccination** 

Maja A. Sommerfelt *Bionor Pharma ASA* 

Towards Universal Acess: Scaling up priority HIV/AIDS Interventions in the health sector. WHO, UNAIDS, UNICEF, 2007. www.who.int/entity/hiv/mediacentre/ universal\_access\_progress\_report\_en.pdf www.ich.org - General Considerations for Clinical Trials www.hivresourcetracking.org/treatments/vaccines

www.avac.org/ht/a/GetDocumentAction/i/3436

### **Towards a Functional Cure for HIV Infection: The Potential Contribution of Therapeutic Vaccination**

Maja A. Sommerfelt *Bionor Pharma ASA Norway* 

#### **1. Introduction**

492 Recent Translational Research in HIV/AIDS

Towards Universal Acess: Scaling up priority HIV/AIDS Interventions in the health sector.

universal\_access\_progress\_report\_en.pdf www.ich.org - General Considerations for Clinical Trials www.hivresourcetracking.org/treatments/vaccines www.avac.org/ht/a/GetDocumentAction/i/3436

WHO, UNAIDS, UNICEF, 2007. www.who.int/entity/hiv/mediacentre/

Human immunodeficiency virus (HIV-1) currently infects 33.3 million people globally. In 2009, 1.8 million people died from acquired immunodeficiency syndrome (AIDS) marking a decline in AIDS deaths by 19% since 1999, the estimated peak of the pandemic. This is largely due to the introduction of combination antiretroviral therapy (ART) in 1996 and its expanding access in recent years. However, despite continued efforts to improve ART availability worldwide, only 5 of the estimated 15 million people living with HIV-1 in lowand middle-income countries have access (UNAIDS, 2010). Furthermore, the number of new infections continues to outpace the number of people being put on ART each day. ART is costly, and places a formidable financial burden on healthcare services. This in turn compromises efforts for universal access.

Combination ART has made a significant impact on HIV-1 morbidity and mortality (Vittinghoff et al., 1999, Palella et al., 2006) and represents the 'gold standard' for HIV-1 treatment. Despite early optimism that combination ART could potentially eradicate infection (Perelson et al., 1996, Ho, 1997), it has since become clear that virus invariably returns if ART is stopped. As a result, ART remains a daily lifelong treatment requiring a high level of compliance to avoid the development of (multi) drug resistance.

Where ART is available, the diagnosis 'AIDS' becomes less frequent, and HIV-1 infection may no longer be considered a irrevocable terminal disease but rather a chronic manageable infection. However, recent studies have observed that ART does not restore life expectancy completely (Neuhaus et al., 2010a; The Antiretroviral Therapy Cohort, 2008). Furthermore, as those living with HIV-1 do become older, age-related toxicities emerge (Powderly, 2007, 2010) as well as other ART co-morbidities such as increased risk of cardiovascular disease, metabolic disorders, neurocognitive abnormalities, liver and renal disease, bone disorders, malignancy and frailty (Deeks & Phillips, 2009).

Untreated HIV-1 infection is characterised by a substantial depletion of CD4+ T-cells in the mucosa as well as a gradual progressive decline of CD4+ T-cells in peripheral blood. When CD4+ T-cell levels in peripheral blood fall below 200 cells/mm3, immune competence is reduced leading to susceptibility to opportunistic infections and conditions that characterise AIDS as well as significant increases in viral load (Levy, 2007). It is primarily the level of CD4+ T-cells in peripheral blood that determines the requirement for ART (Panel on Antiretroviral Guidelines for Adults and Adolescents, 2011).

Towards a Functional Cure for HIV Infection:

**2. HIV-1 persistence in reservoirs** 

individuals (Finzi et al., 1999, Siliciano, 2010).

**3. The concept of a functional cure** 

activation induced by HIV-1.

functional cure).

vaccination to achieve a functional cure for HIV-1 infection.

The Potential Contribution of Therapeutic Vaccination 495

Therapeutic vaccines have the advantage of being able to penetrate sanctuary sites less well accessed by ART such as lymphoid tissue (Pantaleo et al., 1991; Fox et al., 1991) and the central nervous system (Alexaki et al., 2008), that represent regions for viral persistence. This relates to therapeutic interventions targeting both the virus itself as well as HIV-associated immune activation. This chapter will discuss the potential contribution of therapeutic

The failure of ART to eradicate HIV-1 infection lies in the observation that HIV-1 remains quiescent in latent reservoirs. Latently infected resting CD4+ cells (either naive or long lived memory cells) carry transcriptionally silent HIV-1 and represent the predominant reservoir of HIV-1 infection. Other cells may also act as reservoirs (Reviewed in Alexaki et al., 2008) such as macrophages, dendritic cells and astrocytes (where HIV-1 infection occurs via a CD4-independent mechanism). It is these latent reservoirs that represent the major challenge to eradication of HIV-1 infection. More than 80% of individuals on suppressive ART have persistent viraemia below the level of detection (Maldarelli et al., 2007). This low level viraemia is not reduced further despite ART intensification (Dinoso et al., 2009) supporting the concept that HIV-1 rebounds on ART cessation from the rapid reactivation of virus from latently infected cells rather than from continuous ongoing low level replication (Joos et al., 2008). Long lived memory cells comprise approximately 1 cell per million with an extremely low decay rate explaining why 73 years is required to eliminate HIV-1 from infected

It is clear that to achieve a functional cure, therapeutic vaccination will need to induce not only effective antigen-specific immune responses but also combat the generalised immune

The ultimate aim of a functional cure for HIV-1 infection is to induce long-term remission by depleting virus reservoirs to such an extent that a 'controller' status is achieved. In this way virus is maintained at low levels for long periods of time in the absence of ART, equivalent to that observed in known HIV-1 controllers (Lambotte et al., 2005) natural virus suppressors (Sajadi et al., 2007) and elite controllers (Deeks & Walker, 2007). This concept can be compared to achieving a sustained virological response for hepatitis C virus (HCV) infection following interferon/ribavirin treatment. If a sustained virological response is observed for HCV (undetectable virus for at least 6 months), the patient is considered cured. The potential for curing HCV infection is theoretically greater than for HIV-1 since HCV, a separate genus *Hepacivirus* within the virus family Flaviviridae, replicates solely in the cytoplasm of infected cells. As such, on cell division, the virus may remain in only one of the daughter cells. In contrast, HIV-1 is a retrovirus that integrates into the host genome and as

a. Indefinite virus control below the limits of detection (<50 copies HIV-1 RNA/ml)

b. Long-term low level virus replication, as for a natural virus suppressor or long-term non progressor, with concomitant low levels of immune activation (equivalent to a

such, on cell division will be automatically present in both daughter cells. A sustained virological response for HIV-1 could be envisaged as either:

(equivalent to a sterilising cure/eradication).

In recent years it has become apparent that disease progression in HIV-1 infection is not simply due to a loss of CD4+ T-cells as a result of chronic cytopathic viral infection. Instead, HIV-1 infection is accompanied by a progressive generalised immune activation (Neuhaus et al., 2010b; Kuller et al., 2008). Indeed, expression of the activation marker CD38 particularly on CD8+ T-cells has been found to be more predictive of disease progression than viral load (Giorgi et al., 1993; Hazenberg et al., 2003). Although immune activation may be reduced on effective ART, it is not completely absent but remains higher than in uninfected individuals. This may in part explain the loss and/or lack of optimal gain in CD4+ T-cell counts despite effective viral suppression below the level of detection (Hunt et al., 2003). It is intriguing that a similar immune activation is also observed in rhesus macaques infected with simian immunodeficiency virus (SIVsmm or SIVagm) but not in the natural host for these viruses, the sooty mangabey and African green monkey respectively, despite high viral loads (Silvestri et al., 2003). Furthermore, HIV-2 in contrast to HIV-1, is associated with slower disease progression and lower levels of immune activation (Sousa et al., 2002).

The underlying causes of the generalised immune activation associated with HIV-1 infection are presently not fully understood, but are probably associated with multiple mechanisms. These may include reactivation of latent viruses during HIV-1 infection, such as cytomegalovirus (CMV) or Epstein-Barr virus (EBV). The most widely considered mechanism is based on the significant depletion of CD4+ T-cells in the mucosa leading to a disruption of the gut lining and translocation of microbial flora to the systemic immune system (Brenchley et al., 2006). HIV-1 is known to incorporate host human leukocyte antigens (HLA) into its envelope during budding, that may play a role in immune activation. Furthermore, the conserved C5 region of gp120 may also be involved in immune activation (Cadogan & Dalgleish, 2008) by virtue of similarity with the peptide binding domains of HLA molecules. This region of gp120 has been shown to bind peptide and promote activation of antigen-specific T-cell clones (Sheikh et al., 2000).

A small percent (<5%) of individuals have been found to control HIV-1 infection for long periods in the absence of ART. Virus levels, although very low – are never eliminated in these individuals (Hunt et al., 2011). These elite and viraemic controllers (that have a low viral load) have been shown to have narrow cell-mediated immune responses preferentially targeting Gag, and lower immune activation (Rosenberg et al., 1997; Zuniga et al., 2006; Walker, 2007; Saez-Cirion et al., 2007; Binley et al., 1997; Kiepiela et al., 2007). The fact that some individuals can control HIV-1 viraemia suggests that long-term immunological control of HIV-1 infection is possible. This therefore provides credence to the concept of therapeutic vaccination as a means to confer relevant immune stimulation that can ultimately lead to a sustained virological response, emulating a long-term nonprogressor status where the risk of virus transmission is reduced. As a result, more focus will need to be directed to understanding the mechanism(s) behind the control of HIV-1 in elite and viraemic controllers (Autran et al., 2011).

Long-term control of HIV-1 infection in the absence of ART forms the basis for the term 'functional cure' where virus and immune activation levels become equivalent to that found in elite controllers or natural virus suppressors (Jeffries, 2010). In contrast, a 'sterilising cure' relates to HIV-1 eradication, that is, the permanent removal of the HIV-1 by the complete elimination of viral reservoirs. The eradication concept has been inspired by 'The Berlin Patient' who received a bone marrow transplant from a donor that had the CCR5 32 mutation rendering the cells resistant to virus strains using this co-receptor for infection (Hütter et al., 2009). The Berlin patient has remained virus-free for four years to date (Allers et al., 2011).

In recent years it has become apparent that disease progression in HIV-1 infection is not simply due to a loss of CD4+ T-cells as a result of chronic cytopathic viral infection. Instead, HIV-1 infection is accompanied by a progressive generalised immune activation (Neuhaus et al., 2010b; Kuller et al., 2008). Indeed, expression of the activation marker CD38 particularly on CD8+ T-cells has been found to be more predictive of disease progression than viral load (Giorgi et al., 1993; Hazenberg et al., 2003). Although immune activation may be reduced on effective ART, it is not completely absent but remains higher than in uninfected individuals. This may in part explain the loss and/or lack of optimal gain in CD4+ T-cell counts despite effective viral suppression below the level of detection (Hunt et al., 2003). It is intriguing that a similar immune activation is also observed in rhesus macaques infected with simian immunodeficiency virus (SIVsmm or SIVagm) but not in the natural host for these viruses, the sooty mangabey and African green monkey respectively, despite high viral loads (Silvestri et al., 2003). Furthermore, HIV-2 in contrast to HIV-1, is associated with slower disease

The underlying causes of the generalised immune activation associated with HIV-1 infection are presently not fully understood, but are probably associated with multiple mechanisms. These may include reactivation of latent viruses during HIV-1 infection, such as cytomegalovirus (CMV) or Epstein-Barr virus (EBV). The most widely considered mechanism is based on the significant depletion of CD4+ T-cells in the mucosa leading to a disruption of the gut lining and translocation of microbial flora to the systemic immune system (Brenchley et al., 2006). HIV-1 is known to incorporate host human leukocyte antigens (HLA) into its envelope during budding, that may play a role in immune activation. Furthermore, the conserved C5 region of gp120 may also be involved in immune activation (Cadogan & Dalgleish, 2008) by virtue of similarity with the peptide binding domains of HLA molecules. This region of gp120 has been shown to bind peptide and

A small percent (<5%) of individuals have been found to control HIV-1 infection for long periods in the absence of ART. Virus levels, although very low – are never eliminated in these individuals (Hunt et al., 2011). These elite and viraemic controllers (that have a low viral load) have been shown to have narrow cell-mediated immune responses preferentially targeting Gag, and lower immune activation (Rosenberg et al., 1997; Zuniga et al., 2006; Walker, 2007; Saez-Cirion et al., 2007; Binley et al., 1997; Kiepiela et al., 2007). The fact that some individuals can control HIV-1 viraemia suggests that long-term immunological control of HIV-1 infection is possible. This therefore provides credence to the concept of therapeutic vaccination as a means to confer relevant immune stimulation that can ultimately lead to a sustained virological response, emulating a long-term nonprogressor status where the risk of virus transmission is reduced. As a result, more focus will need to be directed to understanding the mechanism(s) behind the control of HIV-1 in elite and viraemic

Long-term control of HIV-1 infection in the absence of ART forms the basis for the term 'functional cure' where virus and immune activation levels become equivalent to that found in elite controllers or natural virus suppressors (Jeffries, 2010). In contrast, a 'sterilising cure' relates to HIV-1 eradication, that is, the permanent removal of the HIV-1 by the complete elimination of viral reservoirs. The eradication concept has been inspired by 'The Berlin Patient' who received a bone marrow transplant from a donor that had the CCR5 32 mutation rendering the cells resistant to virus strains using this co-receptor for infection (Hütter et al., 2009). The Berlin patient

progression and lower levels of immune activation (Sousa et al., 2002).

promote activation of antigen-specific T-cell clones (Sheikh et al., 2000).

has remained virus-free for four years to date (Allers et al., 2011).

controllers (Autran et al., 2011).

Therapeutic vaccines have the advantage of being able to penetrate sanctuary sites less well accessed by ART such as lymphoid tissue (Pantaleo et al., 1991; Fox et al., 1991) and the central nervous system (Alexaki et al., 2008), that represent regions for viral persistence. This relates to therapeutic interventions targeting both the virus itself as well as HIV-associated immune activation. This chapter will discuss the potential contribution of therapeutic vaccination to achieve a functional cure for HIV-1 infection.

#### **2. HIV-1 persistence in reservoirs**

The failure of ART to eradicate HIV-1 infection lies in the observation that HIV-1 remains quiescent in latent reservoirs. Latently infected resting CD4+ cells (either naive or long lived memory cells) carry transcriptionally silent HIV-1 and represent the predominant reservoir of HIV-1 infection. Other cells may also act as reservoirs (Reviewed in Alexaki et al., 2008) such as macrophages, dendritic cells and astrocytes (where HIV-1 infection occurs via a CD4-independent mechanism). It is these latent reservoirs that represent the major challenge to eradication of HIV-1 infection. More than 80% of individuals on suppressive ART have persistent viraemia below the level of detection (Maldarelli et al., 2007). This low level viraemia is not reduced further despite ART intensification (Dinoso et al., 2009) supporting the concept that HIV-1 rebounds on ART cessation from the rapid reactivation of virus from latently infected cells rather than from continuous ongoing low level replication (Joos et al., 2008). Long lived memory cells comprise approximately 1 cell per million with an extremely low decay rate explaining why 73 years is required to eliminate HIV-1 from infected individuals (Finzi et al., 1999, Siliciano, 2010).

It is clear that to achieve a functional cure, therapeutic vaccination will need to induce not only effective antigen-specific immune responses but also combat the generalised immune activation induced by HIV-1.

### **3. The concept of a functional cure**

The ultimate aim of a functional cure for HIV-1 infection is to induce long-term remission by depleting virus reservoirs to such an extent that a 'controller' status is achieved. In this way virus is maintained at low levels for long periods of time in the absence of ART, equivalent to that observed in known HIV-1 controllers (Lambotte et al., 2005) natural virus suppressors (Sajadi et al., 2007) and elite controllers (Deeks & Walker, 2007). This concept can be compared to achieving a sustained virological response for hepatitis C virus (HCV) infection following interferon/ribavirin treatment. If a sustained virological response is observed for HCV (undetectable virus for at least 6 months), the patient is considered cured. The potential for curing HCV infection is theoretically greater than for HIV-1 since HCV, a separate genus *Hepacivirus* within the virus family Flaviviridae, replicates solely in the cytoplasm of infected cells. As such, on cell division, the virus may remain in only one of the daughter cells. In contrast, HIV-1 is a retrovirus that integrates into the host genome and as such, on cell division will be automatically present in both daughter cells.

A sustained virological response for HIV-1 could be envisaged as either:


Towards a Functional Cure for HIV Infection:

ultimately eradication.

established viral reservoirs.

expressing these antigens.

infected subjects that have robust CD4 T-cell counts.

The Potential Contribution of Therapeutic Vaccination 497

CD4+ T-cell counts represent the major parameter that determines the need for ART initiation. For this reason, earlier efforts within therapeutic vaccination aimed to improve CD4+ T-cell counts in order to slow disease progression. However, in light of the SILCAAT and ESPRIT studies that focused on improving CD4+ T-cell counts using IL-2 (which provides nonspecific immune stimulation unlike a therapeutic vaccine that is antigen-specific), the conclusion was that improving CD4 counts *per se* was not associated with clinical benefit (INSIGHT-ESPRIT and SILCAAT Study groups, 2009). Consequently, reducing viral load now represents the unequivocal major endpoint for any therapeutic vaccine or intervention aimed at effecting a functional cure or

The current scepticism regarding treatment interruption means that inclusion criteria for patients in such studies will take in to consideration both preART and nadir (lowest ever) CD4+ T-cell counts since this has been shown to be a critical parameter in determining the outcome of treatment interruption (Willberg & Nixon, 2007). In subjects with low CD4+ Tcells nadir (200-250 cells/mm3), CD4+ T-cell levels fall rapidly on treatment interruption requiring earlier re-initiation of ART (Toulson et al., 2005). Patients selected may therefore be relatively newly infected and have robust preART CD4+ T-cell levels and less well

**3.1.1 Functional cure scenario 1: Long lasting remission on ART interruption** 

One approach towards a functional cure could involve therapeutic vaccination in combination with ART followed by treatment interruption with the aim of providing long lasting sustained virological suppression. The advantage of immunising individuals in the presence of ART is that patients have usually regained CD4+ T-cell counts, including naive CD4+ T-cells that can be stimulated to target HIV-1. Furthermore, virus replication is controlled allowing for immunisation in the absence of circulating virus. The immunisation itself will provide some immune activation as CD4+ T-cells harbouring virus become activated leading to a virus burst which would nevertheless be contained by ART. It would therefore be important to allow for vaccine-induced immune activation to subside before stopping ART. Antigen-specific therapeutic vaccines inducing cell-mediated immune responses against gene products from multiply spliced RNA such as Tat may function in the presence of ART and remove infected cells. This is because these early gene products are not targeted by current antiretroviral therapy. Furthermore, Tat expression is not dependent on the activation state of the infected cell and is therefore also synthesized in quiescent T-cells in the absence of virus replication (Wu & Marsh, 2001). In contrast, for therapeutic vaccines targeting products requiring the expression of structural genes such as Gag and Env, ART would need to be stopped in order for the immune system to identify HIV-1 infected cells

Therapeutic vaccination using antigen-specific immune stimulation could be combined with other interventions to provide a long-lasting reduction of HIV-1-associated generalised immune activation and consequently reduce the level of viral rebound even further. The aim would be that when patients are removed from ART, CD4+ T-cell counts would remain sustained and a virus set point would be established at a level compatible with a long-term non-progressor, or elite controller for a significant period of time (Figure 1). The therapeutic vaccine may also attenuate the height of the initial peak rebound so that it does not necessarily overshoot the preART value. This scenario may be most beneficial for newly

Approaches towards eradication include attempts to purge reservoirs by selective activation of latently infected cells (such as memory cells) in the presence of ART such that released virus may not infect and replicate in neighbouring cells (Richman et al., 2009). Agents include histone deacetylase inhibitors, cytokines, such as IL-2 and IL-7, as well as bryostatin, the protein kinase C activator (Kovochich et al., 2011). However, such interventions may also be associated with side effects, resistance and high cost.

Maintaining HIV-1-infected cells in a continuously latent (transcriptionally silent) state, akin to true latency characteristic of herpesviruses, represents the opposite extreme that has received less attention. HIV-1 is produced from activated CD4+ T-cells. At present it is not clear how HIV-1 can be maintained transcriptionally silent whilst still allowing for the CD4+ T-cell activation required to mount an immune response.

#### **3.1 Functional cure and treatment interruption**

In order to demonstrate a sustained virological response (functional cure) for patients that are well controlled on ART, treatment will ultimately need to be stopped in order to show that virus levels remain controlled (low/undetectable).

Treatment interruption has been intensely investigated in the past as a means to overcome the limitations of lifelong ART which include side effects, drug resistance and high cost. Today, treatment interruption *per se*, is viewed with scepticism due to safety concerns arising from the SMART study, the largest treatment interruption study to date (El-Sadr et al., 2006). In the SMART study and numerous previous smaller studies, ART was interrupted without any additional immunological support. Treatment interruption in the SMART study was CD4-guided, where ART was discontinued when CD4 levels rose above 350 cells/mm3 and resumed if CD4 counts fell below 250 cells/mm3. However, the study was prematurely halted since patients in the treatment conservation group (treatment interruption) experienced greater side effects and adverse events than those in the continuous ART arm. The SMART study therefore concluded that treatment interruption was not safe and that ART should remain a continuous life-long treatment. These safety concerns have affected the design of all treatment interruption trials including those for therapeutic vaccines. Interestingly, a more recent large study of the Swiss Cohort, has suggested that treatment interruption of up to six months can be safely tolerated particularly if patients are well monitored (Kauffman et al., 2011).

Earlier clinical studies have shown that upon cessation of ART, and in the absence of therapeutic immunisation, CD4+ T-cell counts and virus load rebound to preART levels (i.e. the preART set point) (Oxenius et al., 2002a; Wit et al., 2005; Oxenius et al., 2002b; Mata et al., 2005). However, not all patients have available preART viral load information and therefore efforts have been made to identify alternate markers that may predict where the viral load may settle on treatment interruption in the absence of any other intervention. This is necessary in order to determine whether an intervention has lowered the viral load set point in a subject. Proviral DNA levels at baseline have been shown to correlate with the preART viral load, (Yerly et al., 2005), however, this approach will require further validation before it can be taken in to routine use. Until alternative markers are available, preART RNA values will remain the best predictor of the viral load set point that may be obtained on treatment interruption in the absence of therapeutic immunisation. Consequently, the effect of different therapeutic interventions on the viral load will therefore be compared to the preART values.

Approaches towards eradication include attempts to purge reservoirs by selective activation of latently infected cells (such as memory cells) in the presence of ART such that released virus may not infect and replicate in neighbouring cells (Richman et al., 2009). Agents include histone deacetylase inhibitors, cytokines, such as IL-2 and IL-7, as well as bryostatin, the protein kinase C activator (Kovochich et al., 2011). However, such interventions may

Maintaining HIV-1-infected cells in a continuously latent (transcriptionally silent) state, akin to true latency characteristic of herpesviruses, represents the opposite extreme that has received less attention. HIV-1 is produced from activated CD4+ T-cells. At present it is not clear how HIV-1 can be maintained transcriptionally silent whilst still allowing for the CD4+

In order to demonstrate a sustained virological response (functional cure) for patients that are well controlled on ART, treatment will ultimately need to be stopped in order to show

Treatment interruption has been intensely investigated in the past as a means to overcome the limitations of lifelong ART which include side effects, drug resistance and high cost. Today, treatment interruption *per se*, is viewed with scepticism due to safety concerns arising from the SMART study, the largest treatment interruption study to date (El-Sadr et al., 2006). In the SMART study and numerous previous smaller studies, ART was interrupted without any additional immunological support. Treatment interruption in the SMART study was CD4-guided, where ART was discontinued when CD4 levels rose above 350 cells/mm3 and resumed if CD4 counts fell below 250 cells/mm3. However, the study was prematurely halted since patients in the treatment conservation group (treatment interruption) experienced greater side effects and adverse events than those in the continuous ART arm. The SMART study therefore concluded that treatment interruption was not safe and that ART should remain a continuous life-long treatment. These safety concerns have affected the design of all treatment interruption trials including those for therapeutic vaccines. Interestingly, a more recent large study of the Swiss Cohort, has suggested that treatment interruption of up to six months can be safely tolerated

Earlier clinical studies have shown that upon cessation of ART, and in the absence of therapeutic immunisation, CD4+ T-cell counts and virus load rebound to preART levels (i.e. the preART set point) (Oxenius et al., 2002a; Wit et al., 2005; Oxenius et al., 2002b; Mata et al., 2005). However, not all patients have available preART viral load information and therefore efforts have been made to identify alternate markers that may predict where the viral load may settle on treatment interruption in the absence of any other intervention. This is necessary in order to determine whether an intervention has lowered the viral load set point in a subject. Proviral DNA levels at baseline have been shown to correlate with the preART viral load, (Yerly et al., 2005), however, this approach will require further validation before it can be taken in to routine use. Until alternative markers are available, preART RNA values will remain the best predictor of the viral load set point that may be obtained on treatment interruption in the absence of therapeutic immunisation. Consequently, the effect of different therapeutic interventions on the viral load will therefore be compared to the

also be associated with side effects, resistance and high cost.

T-cell activation required to mount an immune response.

that virus levels remain controlled (low/undetectable).

particularly if patients are well monitored (Kauffman et al., 2011).

preART values.

**3.1 Functional cure and treatment interruption** 

CD4+ T-cell counts represent the major parameter that determines the need for ART initiation. For this reason, earlier efforts within therapeutic vaccination aimed to improve CD4+ T-cell counts in order to slow disease progression. However, in light of the SILCAAT and ESPRIT studies that focused on improving CD4+ T-cell counts using IL-2 (which provides nonspecific immune stimulation unlike a therapeutic vaccine that is antigen-specific), the conclusion was that improving CD4 counts *per se* was not associated with clinical benefit (INSIGHT-ESPRIT and SILCAAT Study groups, 2009). Consequently, reducing viral load now represents the unequivocal major endpoint for any therapeutic vaccine or intervention aimed at effecting a functional cure or ultimately eradication.

The current scepticism regarding treatment interruption means that inclusion criteria for patients in such studies will take in to consideration both preART and nadir (lowest ever) CD4+ T-cell counts since this has been shown to be a critical parameter in determining the outcome of treatment interruption (Willberg & Nixon, 2007). In subjects with low CD4+ Tcells nadir (200-250 cells/mm3), CD4+ T-cell levels fall rapidly on treatment interruption requiring earlier re-initiation of ART (Toulson et al., 2005). Patients selected may therefore be relatively newly infected and have robust preART CD4+ T-cell levels and less well established viral reservoirs.

#### **3.1.1 Functional cure scenario 1: Long lasting remission on ART interruption**

One approach towards a functional cure could involve therapeutic vaccination in combination with ART followed by treatment interruption with the aim of providing long lasting sustained virological suppression. The advantage of immunising individuals in the presence of ART is that patients have usually regained CD4+ T-cell counts, including naive CD4+ T-cells that can be stimulated to target HIV-1. Furthermore, virus replication is controlled allowing for immunisation in the absence of circulating virus. The immunisation itself will provide some immune activation as CD4+ T-cells harbouring virus become activated leading to a virus burst which would nevertheless be contained by ART. It would therefore be important to allow for vaccine-induced immune activation to subside before stopping ART. Antigen-specific therapeutic vaccines inducing cell-mediated immune responses against gene products from multiply spliced RNA such as Tat may function in the presence of ART and remove infected cells. This is because these early gene products are not targeted by current antiretroviral therapy. Furthermore, Tat expression is not dependent on the activation state of the infected cell and is therefore also synthesized in quiescent T-cells in the absence of virus replication (Wu & Marsh, 2001). In contrast, for therapeutic vaccines targeting products requiring the expression of structural genes such as Gag and Env, ART would need to be stopped in order for the immune system to identify HIV-1 infected cells expressing these antigens.

Therapeutic vaccination using antigen-specific immune stimulation could be combined with other interventions to provide a long-lasting reduction of HIV-1-associated generalised immune activation and consequently reduce the level of viral rebound even further. The aim would be that when patients are removed from ART, CD4+ T-cell counts would remain sustained and a virus set point would be established at a level compatible with a long-term non-progressor, or elite controller for a significant period of time (Figure 1). The therapeutic vaccine may also attenuate the height of the initial peak rebound so that it does not necessarily overshoot the preART value. This scenario may be most beneficial for newly infected subjects that have robust CD4 T-cell counts.

Towards a Functional Cure for HIV Infection:

The Potential Contribution of Therapeutic Vaccination 499

Stippled line at 350 indicates CD4 count below which ART should be initiated. Thick solid line: CD4

Fig. 2. Scenario 2: Functional cure over time: intermittent ART supported by therapeutic vaccination, where viral rebound achieves a lower set point for each successive treatment

Any therapeutic vaccination approach involving treatment interruption involves concerns that viral reservoirs would become repopulated. It is interesting to note that viral reservoirs are also repopulated in elite controller patients since they never manage to eliminate their virus despite maintaining a viral set point below the level of detection

Although potentially applicable to all patient categories, this third scenario for achieving a functional cure on continuous ART may be particularly suited for subjects where treatment interruption is not considered a viable option due to poor CD4+ T-cell reconstitution on ART, low CD4+ T-cell nadir or a very high preART viral load set point. This approach could involve combining continuous ART with therapeutic vaccination and reservoir purging

In this scenario, subjects would be maintained on continuous ART. Therapeutic vaccination would be carried out in the presence of ART as in scenarios 1 and 2 with the aim of generating more effective responses to HIV-1. However, instead of removing patients from ART as in scenarios 1 and 2, reservoir purging agents would be used to reverse latency and allow for the expression of viral genes. Viral replication and spread would be hindered due to the presence of ART. Expression of viral genes would render infected cells 'visible' to the immune system allowing for their removal as a consequence of the improved immune responses resulting from therapeutic immunisation. However, to show ultimately that viral reservoirs have been reduced significantly or even fully depleted, subjects will need to be

count. Thin line: viral load (VL). Dashed line: PreART viral load

**3.1.3 Functional cure scenario 3: On continuous ART** 

(Hunt et al., 2011).

agents (Figure 3).

removed from ART.

interruption with a concomitant slower CD4+ T-cell decline over time

Stippled line at 350 indicates CD4 count below which ART should be initiated. Thick solid line: CD4 count. Thin line: viral load (VL). Dashed line: PreART viral load

Fig. 1. Scenario 1: Therapeutic vaccination in combination with ART leading to sustained virological response (long-lasting remission). Viral rebound may not necessarily overshoot the preART viral load. CD4+ T-cell levels would remain above the level of 350 cells/mm3 that necessitates a return to ART according to current guidelines

#### **3.1.2 Functional cure scenario 2: Remission following intermittent ART**

It is possible that on treatment interruption as in scenario 1, viral load levels may stabilize at a lower set point, but not sufficiently low to be compatible with an HIV controller. This may be the case for individuals that started ART later on in disease course, where the number of viral reservoirs is greater, and the CD4+ T-cell nadir lower. To address this, therapeutic vaccination may be used to allow ART to become safely intermittent and where the viral set point may be sequentially reduced following multiple cycles of ART and booster immunisations with the therapeutic vaccine (Figure 2). In such a scenario, due to the safety concerns, the duration of the ART-free period should not exceed the 6 month time period shown to be safe in the Swiss cohort study (Kauffman et al., 2011). This approach of intermittent ART in combination with therapeutic immunisation and booster immunisations has not been investigated to date and may be viewed with scepticism due to the safety concerns arising from the SMART study. However, the underlying basis for the SMART study, i.e. a need to combat ART side effects, drug resistance and high cost remain relevant issues that need to be resolved.

Similarly to scenario 1, therapeutic vaccination may also attenuate the size of the initial peak of rebound during the first treatment interruption allowing the set point to establish below the preART level. Following subsequent booster immunisations on ART in this scenario, as the viral load set point is lowered, CD4+ T-cell decline would also become less marked and would ultimately stabilise above the level necessitating ART (350 cells/mm3).

Stippled line at 350 indicates CD4 count below which ART should be initiated. Thick solid line: CD4

Fig. 1. Scenario 1: Therapeutic vaccination in combination with ART leading to sustained virological response (long-lasting remission). Viral rebound may not necessarily overshoot the preART viral load. CD4+ T-cell levels would remain above the level of 350 cells/mm3

It is possible that on treatment interruption as in scenario 1, viral load levels may stabilize at a lower set point, but not sufficiently low to be compatible with an HIV controller. This may be the case for individuals that started ART later on in disease course, where the number of viral reservoirs is greater, and the CD4+ T-cell nadir lower. To address this, therapeutic vaccination may be used to allow ART to become safely intermittent and where the viral set point may be sequentially reduced following multiple cycles of ART and booster immunisations with the therapeutic vaccine (Figure 2). In such a scenario, due to the safety concerns, the duration of the ART-free period should not exceed the 6 month time period shown to be safe in the Swiss cohort study (Kauffman et al., 2011). This approach of intermittent ART in combination with therapeutic immunisation and booster immunisations has not been investigated to date and may be viewed with scepticism due to the safety concerns arising from the SMART study. However, the underlying basis for the SMART study, i.e. a need to combat ART side effects, drug resistance and high cost remain relevant

Similarly to scenario 1, therapeutic vaccination may also attenuate the size of the initial peak of rebound during the first treatment interruption allowing the set point to establish below the preART level. Following subsequent booster immunisations on ART in this scenario, as the viral load set point is lowered, CD4+ T-cell decline would also become less marked and

would ultimately stabilise above the level necessitating ART (350 cells/mm3).

count. Thin line: viral load (VL). Dashed line: PreART viral load

issues that need to be resolved.

that necessitates a return to ART according to current guidelines

**3.1.2 Functional cure scenario 2: Remission following intermittent ART** 

Stippled line at 350 indicates CD4 count below which ART should be initiated. Thick solid line: CD4 count. Thin line: viral load (VL). Dashed line: PreART viral load

Fig. 2. Scenario 2: Functional cure over time: intermittent ART supported by therapeutic vaccination, where viral rebound achieves a lower set point for each successive treatment interruption with a concomitant slower CD4+ T-cell decline over time

Any therapeutic vaccination approach involving treatment interruption involves concerns that viral reservoirs would become repopulated. It is interesting to note that viral reservoirs are also repopulated in elite controller patients since they never manage to eliminate their virus despite maintaining a viral set point below the level of detection (Hunt et al., 2011).

#### **3.1.3 Functional cure scenario 3: On continuous ART**

Although potentially applicable to all patient categories, this third scenario for achieving a functional cure on continuous ART may be particularly suited for subjects where treatment interruption is not considered a viable option due to poor CD4+ T-cell reconstitution on ART, low CD4+ T-cell nadir or a very high preART viral load set point. This approach could involve combining continuous ART with therapeutic vaccination and reservoir purging agents (Figure 3).

In this scenario, subjects would be maintained on continuous ART. Therapeutic vaccination would be carried out in the presence of ART as in scenarios 1 and 2 with the aim of generating more effective responses to HIV-1. However, instead of removing patients from ART as in scenarios 1 and 2, reservoir purging agents would be used to reverse latency and allow for the expression of viral genes. Viral replication and spread would be hindered due to the presence of ART. Expression of viral genes would render infected cells 'visible' to the immune system allowing for their removal as a consequence of the improved immune responses resulting from therapeutic immunisation. However, to show ultimately that viral reservoirs have been reduced significantly or even fully depleted, subjects will need to be removed from ART.

Towards a Functional Cure for HIV Infection:

interruption.

Argos Therapeutics

Baylor University/ ANRS

Bionor Pharma

Genetic Immunity

NIAID/ Profectus Biosciences

topical patches/plasters.

The Potential Contribution of Therapeutic Vaccination 501

functional cure (Tables 1-3). The majority of products aim to induce T-cell immunity

The viral antigens used as therapeutic vaccine candidates include peptides, polypeptides, fusion proteins, recombinant proteins, DNA, RNA either alone or with viral vectors such as poxviruses or adenoviruses, as well as inactivated autologous virus. These antigens can be injected directly or via *ex vivo* bombardment of autologous dendritic cells that are re-infused into the patient. The overall objective of therapeutic vaccine candidates is to reduce viral load, although some also aim to concurrently sustain CD4+ T-cell counts upon ART

The potency of *ex vivo* stimulation of dendritic cells with inactivated autologous virus was first appreciated following the original studies by Lu et al., (2004) and Garcia et al., (2005) where subjects experienced a significant although transient reduction of viral load. Such approaches require access to autologous virus prior to ART initiation either for purification and inactivation or use as the basis for amplification of viral genes. This approach requires access to advanced technology and may require intermittent boosting to maintain the effect. Therapeutic vaccines are also being developed that aim to target dendritic cells *in situ*. This usually involves intradermal administration. Since intradermal injection requires trained personnel, alternative approaches are being developed to target dendritic cells such as

> Autologous DCs co-electroporated with amplified *in vitro* transcribed RNA encoding CD40L and autologous HIV-1 antigens derived from the patient's own plasma taken immediately prior to the

DALIA study: *Ex vivo* administration of Lipopeptides to Nef, Gag and Env to DCs

Peptides to conserved domains of HIV p24 injected intradermally with GM-CSF.

followed by reinfusion to patient

Clade B DNA in nanoparticles and delivered to DCs in a patch (Dermavir).

Replication defective adenovirus vector

initiation of ART.

carrying HIV-1 gag.

whereas a minority aim to induce antibody responses to specific viral antigens.

**Company Product Clinical phase Technology** 

I n=19

n=137

n=16

the clinical trial identifier for trials listed on www.clinicaltrials.gov

II n=120

n=34

NCT00672191

NCT00796770

NCT00659789

NCT00918840

NCT00080106

Table 1. Therapeutic vaccine candidates immunising subjects on ART with a treatment interruption phase in the study. DC:dendritic cell, TI: treatment interruption. NCT provides

Viral vectors derived from adenoviruses or poxviruses have also been extensively used to deliver DNA-based vaccines most often in a prime boost strategy. For such approaches it

AGS-004 II

Vacc-4x IIb

LC002 II

MRK Ad5 HIV-1 gag

DC Vaccine

Stippled line at 350 indicates CD4 count below which ART should be initiated. Thick solid line: CD4 count. Thin line: viral reservoirs

Fig. 3. Scenario 3: Subjects remain on continuous ART. Therapeutic vaccination takes place in the presence of ART and the immune responses generated can remove infected cells that release virus following the use of reservoir purging agents. This procedure may need to be repeated to gradually remove virus reservoirs over time

#### **3.2 Functional cure and treatment naive patients**

Therapeutic vaccination of individuals that are treatment naive would be an attractive proposition in regions where ART availability is incomplete and where the financial burden to sustain life long treatment is greatest. In this case, subjects would be immunised in the presence of circulating virus to improve and direct immune responses to important epitopes such that viral load is decreased, CD4+ T-cell numbers have the potential to increase and the initiation of treatment delayed. However, therapeutic vaccination itself may result in a transient immune activation that could result in the seeding of further reservoirs with functional and 'fit' (replication competent) virus.

Treatment naive individuals currently represent a study population where the effects of therapeutic vaccination on viral load and CD4+ T-cell counts can be readily observed. However, clinical trials involving treatment naive subjects will likely involve enrolment of patients that are early in disease course and where ART is not yet indicated. Such patients would likely have robust CD4+ T-cell counts and viral loads below 100 000 copies/ml. It is likely that viral reservoirs in these patients would be less well established. The more robust the CD4+ T-cell count, the more likely that the patient may provide an immunological response to the therapeutic vaccine.

#### **4. Approaches to therapeutic vaccination in clinical development**

A number of different approaches to HIV-1 therapeutic vaccination are currently in clinical development, although not necessarily at this point in time directly aiming to achieve a

Stippled line at 350 indicates CD4 count below which ART should be initiated. Thick solid line: CD4

Fig. 3. Scenario 3: Subjects remain on continuous ART. Therapeutic vaccination takes place in the presence of ART and the immune responses generated can remove infected cells that release virus following the use of reservoir purging agents. This procedure may need to be

Therapeutic vaccination of individuals that are treatment naive would be an attractive proposition in regions where ART availability is incomplete and where the financial burden to sustain life long treatment is greatest. In this case, subjects would be immunised in the presence of circulating virus to improve and direct immune responses to important epitopes such that viral load is decreased, CD4+ T-cell numbers have the potential to increase and the initiation of treatment delayed. However, therapeutic vaccination itself may result in a transient immune activation that could result in the seeding of further reservoirs with

Treatment naive individuals currently represent a study population where the effects of therapeutic vaccination on viral load and CD4+ T-cell counts can be readily observed. However, clinical trials involving treatment naive subjects will likely involve enrolment of patients that are early in disease course and where ART is not yet indicated. Such patients would likely have robust CD4+ T-cell counts and viral loads below 100 000 copies/ml. It is likely that viral reservoirs in these patients would be less well established. The more robust the CD4+ T-cell count, the more likely that the patient may provide an immunological

A number of different approaches to HIV-1 therapeutic vaccination are currently in clinical development, although not necessarily at this point in time directly aiming to achieve a

**4. Approaches to therapeutic vaccination in clinical development** 

count. Thin line: viral reservoirs

repeated to gradually remove virus reservoirs over time

**3.2 Functional cure and treatment naive patients** 

functional and 'fit' (replication competent) virus.

response to the therapeutic vaccine.

functional cure (Tables 1-3). The majority of products aim to induce T-cell immunity whereas a minority aim to induce antibody responses to specific viral antigens.

The viral antigens used as therapeutic vaccine candidates include peptides, polypeptides, fusion proteins, recombinant proteins, DNA, RNA either alone or with viral vectors such as poxviruses or adenoviruses, as well as inactivated autologous virus. These antigens can be injected directly or via *ex vivo* bombardment of autologous dendritic cells that are re-infused into the patient. The overall objective of therapeutic vaccine candidates is to reduce viral load, although some also aim to concurrently sustain CD4+ T-cell counts upon ART interruption.

The potency of *ex vivo* stimulation of dendritic cells with inactivated autologous virus was first appreciated following the original studies by Lu et al., (2004) and Garcia et al., (2005) where subjects experienced a significant although transient reduction of viral load. Such approaches require access to autologous virus prior to ART initiation either for purification and inactivation or use as the basis for amplification of viral genes. This approach requires access to advanced technology and may require intermittent boosting to maintain the effect.

Therapeutic vaccines are also being developed that aim to target dendritic cells *in situ*. This usually involves intradermal administration. Since intradermal injection requires trained personnel, alternative approaches are being developed to target dendritic cells such as topical patches/plasters.


Table 1. Therapeutic vaccine candidates immunising subjects on ART with a treatment interruption phase in the study. DC:dendritic cell, TI: treatment interruption. NCT provides the clinical trial identifier for trials listed on www.clinicaltrials.gov

Viral vectors derived from adenoviruses or poxviruses have also been extensively used to deliver DNA-based vaccines most often in a prime boost strategy. For such approaches it

Towards a Functional Cure for HIV Infection:

Genetic Immunity

SEEK (previously PepTcell)

Statens

seruminstitutt, DK, EU clinical trials partnership

The Potential Contribution of Therapeutic Vaccination 503

(International HIV Controllers Study Study, 2010). However these HLA alleles are not present in a large proportion of individuals. It has been suggested that patients in clinical studies should be HLA tested to help explain and understand the results (Li et al., 2011). One salient difference between the preventative and therapeutic vaccines lies in their objectives. At present it is considered remote that a vaccine can be developed that will yield sterilising immunity and complete protection from HIV-1 infection. For this reason, the objective of a preventative vaccine is now to prevent infection as far as is possible, and should infection occur the immune system will be sufficiently primed to ensure that the disease course is milder (Johnston & Fauci, 2007). This was the aim of the STEP trial, which used an adenovirus vector. However, unexpectedly, prior exposure to adenovirus infection resulted in

greater susceptibility to HIV-1 infection in study participants (Buchbinder et al., 2008).

n=36

n=55

n=20

n=24

n=60

n= 160 NCT01029548

II n=60

NCT00711230

NCT01071031

NCT01141205

NCT00848211

NCT00402142

Table 3. Therapeutic vaccine candidates in clinical development immunising subjects that

The complexity of HIV-1 infection represents a challenge to achieving a functional cure or ultimately eradication of infection. A number of scenarios have been suggested in this chapter where therapeutic vaccination is combined with ART and also potentially with virus

Clade B DNA incorporated into nanoparticles and delivered to DCs

Mixture of polypeptide T-cell epitope sequences to conserved domains of HIV (internal proteins). Single subcutaneous injection

Peptides representing 3 CD4 and 17 CD8 minimal HIV epitopes.

Subcutaneous injection, acts as

Autologous dendritic cell pulsed *ex vivo* with patient's own virus.

Inactivated Tat protein injected intradermally (i.d.) to induce antibodies to Tat. This study is an

observational cohort.

DNA plasmid using GTU® Technology patented by FIT Biotech (Gene Transport Unit). Gag, Rev, Nef, Tat. Clade B.

in a patch (Dermavir).

Adjuvant CAF01.

Tat Lipopeptide.

own adjuvant.

**Company Product Clinical phase Technology** 

LC002 II

HIV-v I

AFO-18 I

DCV2 I/II

ISS T003 II

Thymon TUTI-16 I/II

(GTU-MultiHIV-B)

FIT Biotech FIT06

Hospital Clinic of

Istituto Superiore

are treatment naive.

**6. Conclusion** 

Barcelona

di Sanita

will likely be necessary to determine the serological status of vaccine recipients to the viruses that have been used as a basis for these vectors since prior immunity may negatively affect vaccine efficacy. Similarly, maintenance of vaccine effect may require boosting using a heterologous virus vector, to avoid inhibitory effects of prior vaccine-induced immunity to the original vector.

Although the induction of neutralising antibodies remains the major goal for an effective preventative vaccine, therapeutic vaccines aim to induce antibody responses to other viral antigens such as the HIV-1 Tat protein. Earlier studies have shown that loss of antibody responses to Tat correlated with disease progression (van Baalen et al., 1997; Rezza et al., 2005). Such a vaccine may also address pathogenic effects of Tat released from infected cells (Ensoli et al., 1993).


Table 2. Therapeutic vaccine candidates in clinical development where therapeutic vaccination occurs in the presence of continuous ART. DC: dendritic cell. NCT provides the clinical trial identifier for trials listed on www.clinicaltrials.gov

#### **5. The challenges facing therapeutic vaccination**

No preventative vaccine has yet been developed for HIV-1 infection. This is despite intense efforts since the virus was first isolated in 1983 (Barre-Sinoussi et al., 1983). The challenges faced by preventative and therapeutic vaccines are similar in that HIV-1 shows extensive genetic variation and a propensity for immune escape. Furthermore, human populations are also varied and this is characterised by a variety of human leukocyte antigens (HLA). HLA function to present HIV-1 epitopes at the surface of infected cells to allow for recognition and removal by cytotoxic T-lymphocytes. The association of certain HLA with virus control (e.g. HLA-B57) and disease progression (e.g. B35) has recently been highlighted

will likely be necessary to determine the serological status of vaccine recipients to the viruses that have been used as a basis for these vectors since prior immunity may negatively affect vaccine efficacy. Similarly, maintenance of vaccine effect may require boosting using a heterologous virus vector, to avoid inhibitory effects of prior vaccine-induced immunity to

Although the induction of neutralising antibodies remains the major goal for an effective preventative vaccine, therapeutic vaccines aim to induce antibody responses to other viral antigens such as the HIV-1 Tat protein. Earlier studies have shown that loss of antibody responses to Tat correlated with disease progression (van Baalen et al., 1997; Rezza et al., 2005). Such a vaccine may also address pathogenic effects of Tat released from infected cells

**Technology** 

Pol, Env.

IL-15)

and Nef mRNA.

the response.

DNA plasmid. Intradermal injections in combination with GM-CSF and IL-2 as

well as a growth hormone.

MVA vector encoding a DNA that carries conserved domains in Gag, Vif,

PENNVAX-B is a DNA vaccine encodes synthetic HIV-1 envelope protein, Gag and Pol. GENEVAX and pIL15 are DNA adjuvants (IL-12 and

Dendritic cells transfected with vectors encoding consensus (clade B) HIV Gag

Plasmid DNA with IL-12 to enhance

**phase** 

NCT01130376

NCT01024842

NCT00775424

NCT00833781

NCT01266616

Table 2. Therapeutic vaccine candidates in clinical development where therapeutic

vaccination occurs in the presence of continuous ART. DC: dendritic cell. NCT provides the

No preventative vaccine has yet been developed for HIV-1 infection. This is despite intense efforts since the virus was first isolated in 1983 (Barre-Sinoussi et al., 1983). The challenges faced by preventative and therapeutic vaccines are similar in that HIV-1 shows extensive genetic variation and a propensity for immune escape. Furthermore, human populations are also varied and this is characterised by a variety of human leukocyte antigens (HLA). HLA function to present HIV-1 epitopes at the surface of infected cells to allow for recognition and removal by cytotoxic T-lymphocytes. The association of certain HLA with virus control (e.g. HLA-B57) and disease progression (e.g. B35) has recently been highlighted

I n=30

I n=20

I n=38

n=21

I n=60

clinical trial identifier for trials listed on www.clinicaltrials.gov

**5. The challenges facing therapeutic vaccination** 

the original vector.

(Ensoli et al., 1993).

Imperial College

Univ. Oxford Med Research Council.

University Pennsylvania

Massachusett s General Hospital

NIAID HIV

/ Drexel University

**Company Product Clinical** 

GTU-MultiHIV-B (FIT06)

nsv

MVA.HIVco

PENNVAX B,GENEVA X IL-12- 4532, pIL15EAM

DNA I

Antigens & IL-12

(International HIV Controllers Study Study, 2010). However these HLA alleles are not present in a large proportion of individuals. It has been suggested that patients in clinical studies should be HLA tested to help explain and understand the results (Li et al., 2011). One salient difference between the preventative and therapeutic vaccines lies in their objectives. At present it is considered remote that a vaccine can be developed that will yield sterilising immunity and complete protection from HIV-1 infection. For this reason, the objective of a preventative vaccine is now to prevent infection as far as is possible, and should infection occur the immune system will be sufficiently primed to ensure that the disease course is milder (Johnston & Fauci, 2007). This was the aim of the STEP trial, which used an adenovirus vector. However, unexpectedly, prior exposure to adenovirus infection resulted in greater susceptibility to HIV-1 infection in study participants (Buchbinder et al., 2008).


Table 3. Therapeutic vaccine candidates in clinical development immunising subjects that are treatment naive.

#### **6. Conclusion**

The complexity of HIV-1 infection represents a challenge to achieving a functional cure or ultimately eradication of infection. A number of scenarios have been suggested in this chapter where therapeutic vaccination is combined with ART and also potentially with virus

Towards a Functional Cure for HIV Infection:

Team (2008). *Lancet* 372:1881–93

2006; 355:2283-96

*J. Virol* 67:277–87.

AIDS-related morbidity. *BMJ* 338:a3172.

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Barré-Sinoussi, F., Chermann, J.C., Rey, F., Nugeyre, M.T., Chamaret, S., Gruest, J., Dauguet,

Binley, J.M.; Klasse, P.J., Cao, Y.; Jones, I.; Markowitz, M.; Ho, D.D. & Moore, J.P.(1997).

Brenchley, J.M. Price, D.A.; Schacker, T.W.; Asher, T.E.; Silvestri, G.; Rao, S.; Kazzaz, Z.;

Buchbinder, S.P.; Mehrotra, D.V.; Duerr, A.; Fitzgerald, D.A.; Mogg, R.; Li, D. Gilbert, P.B.;

Cadogan, M. & Dalgleish, A.G. (2008). Pathogenesis of HIV: non-specific immune hyperactivity and its implications for vaccines. *Clinical Medicine* 8:267-71. Deeks, S.G. & Phillips, A.N. (2009). HIV infection, antiretroviral treatment, ageing and non-

Deeks, S.G. & Walker, B.D. (2007). Human immunodeficiency virus controllers: mechanisms of durable virus control in the absence of therapy. *Immunity* 27:406-416. Dinoso, J.B.; Kim, S.Y.; Wiegand, A.M.; Palmer, S.E.; Gange, S.J.; Cranmer, L.; O'Shea, A.;

highly active antiretroviral therapy. *Proc. Nat. Acad. Sci. USA* 106:9403-08. El-Sadr, W.M., Lundgren, J.D.; Neaton, J.D.; Gordin, F.; Abrams, D.; Arduino, R.C.; Babiker,

Ensoli, B.; Buonaguro, L.; Barillari, G.; Fiorelli, V.; Gendelman, R.; Morgan, R.A.; Wingfield,

Finzi, D.; Blankson, J.; Siliciano, J.D.; Margolick, J.B.; Chadwick, K.; Pierson, T.; Smith, K.;

effective combination therapy. *Nat. Med.* 5:512–517.

immune activation in chronic HIV infection. *Nat. Med.* 12:1365-71

immunodeficiency syndrome AIDS. *Science* 220:868-71.

immunodeficiency virus type 1. *J. Virol.* 71:2799-2809

C., Axler-Blin, C., Vézinet-Brun, F., Rouziousx, C., Rosenbaum, W., Montagnier, L. (1983). Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired

Differential regulation of the antibody responses to Gag and Env proteins of human

Bornstein, E.; Lambotte, O.; Altmann, D.; Blazar, B.R.; Rodriguez, B.; Teixeira-Johnson, L.; Landay, A.; Martin, J.N.; Hecht, F.M.; Picker, L.J.; Lederman, M.M.; Deeks, S.G. & Douek, D.C. (2006). Microbial translocation is a cause of systemic

Lama, J.R.; Marmor, M.; del Rio, C.; McElrath, J.; Casimiro, D.R.; Gottesdiener, K.M.; Chodakewitz, J.A.; Corey, L. & Robertson, M.N. The Step Study Protocol

Callender, M.; Spivak, A., Brennan, T.; Kearney, M.F.; Proschan, M.A.; Mican, J.M.; Rehm, C.A.; Coffin, J.M.; Mellors, J.W.; Siliciano, R.F. & Maldarelli, F. (2009). Treatment intensification does not reduce residual HIV-1 viraemia in patients in

A.; Burman, W.; Clumeck, N.; Cohen, C.J., Cohn, D.; Cooper, D.; Darbyshire, J.; Emery, S.; Fätkenheuer, G.; Gazzard, B.; Grund, B.; Hoy, J.; Klingman, K.; Losso, M.; Markowitz, N.; Neuhaus, J.; Phillips, A.; Rappoport, C. & the SMART Study Group. CD4+ count-guided interruption of antiretroviral treatment. *N. Engl. J. Med.*

P. & Gallo, R.C. (1993) Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation.

Lisziewicz, J.; Lori, F.; Flexner, C.; Quinn, T.C.; Chaisson, R.E.; Rosenberg, E.; Walker, B.; Gange, S.; Gallant, J. & Siliciano, R.F. (1999). Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on

purging agents. At present it is unlikely that any one scenario will suit all purposes, indeed, the choice of approach will likely depend upon the availability of ART, how far advanced the infection is on diagnosis and when during the disease course ART was initiated since these considerations will influence the size of viral reservoir.

It is unlikely that there will ever be a single product that will either prevent HIV-1 infection completely or eradicate HIV-1 infection. Therefore, combinations may be more appropriate. Harnessing the immune system is a rational approach to combine with ART bearing in mind that the immune system may penetrate regions of the body not reached by current therapy. Combination ART has been more successful than monotherapy. Similarly combining ART with therapeutic vaccination and/or virus purging agents will likely be more effective than any of these interventions on their own. The recent Thai study provides is an example where two preventative vaccine candidates that had not shown effect earlier, provided an improved response leading to a marginally significant effect when combined (Reks-Ngarm et al., 2009).

Ultimately a therapeutic vaccine will need to confer effective immune responses in all individuals regardless whether they possess HLA compatible with virus control or not. It is therefore important that therapeutic vaccine candidates take into consideration genetic variation in both human and viral populations in order to be able to elicit the most effective responses leading to control of infection. Strictly, the term 'functional cure' can be considered misleading since virus is not completely removed from the body, but rather the patient experiences remission from symptoms. The term 'functional control' would therefore be more appropriate.

Eradication approaches will require much research and development, where both novel and known compounds will be tested in new ways to determine a potential effect on eradication without incurring too many side effects. It may therefore take significant time before such products are available on the market. In contrast, a functional cure may be achievable in the shorter term and represent a more realistic goal since virus reduction has been shown for a number of therapeutic vaccine candidates. Approaches that aim to successfully combat HIV-1 infection will need to address both the virus (virus-specific approaches including ART and therapeutic vaccines) as well as the generalized immune activation that drives the infection. It is likely that to achieve a functional cure, a combination of different interventions may ultimately be required.

#### **7. Acknowledgments**

This work has been partly supported by a grant from the Research Council of Norway GLOBVAC programme. Many thanks to Birger Sørensen, Ingebjørg Baksaas, Vidar Wendel-Hansen and Giuseppe Pantaleo for reading and commenting the manuscript.

#### **8. References**


purging agents. At present it is unlikely that any one scenario will suit all purposes, indeed, the choice of approach will likely depend upon the availability of ART, how far advanced the infection is on diagnosis and when during the disease course ART was initiated since

It is unlikely that there will ever be a single product that will either prevent HIV-1 infection completely or eradicate HIV-1 infection. Therefore, combinations may be more appropriate. Harnessing the immune system is a rational approach to combine with ART bearing in mind that the immune system may penetrate regions of the body not reached by current therapy. Combination ART has been more successful than monotherapy. Similarly combining ART with therapeutic vaccination and/or virus purging agents will likely be more effective than any of these interventions on their own. The recent Thai study provides is an example where two preventative vaccine candidates that had not shown effect earlier, provided an improved response leading to a marginally significant effect when combined (Reks-Ngarm et al., 2009). Ultimately a therapeutic vaccine will need to confer effective immune responses in all individuals regardless whether they possess HLA compatible with virus control or not. It is therefore important that therapeutic vaccine candidates take into consideration genetic variation in both human and viral populations in order to be able to elicit the most effective responses leading to control of infection. Strictly, the term 'functional cure' can be considered misleading since virus is not completely removed from the body, but rather the patient experiences remission from symptoms. The term 'functional control' would

Eradication approaches will require much research and development, where both novel and known compounds will be tested in new ways to determine a potential effect on eradication without incurring too many side effects. It may therefore take significant time before such products are available on the market. In contrast, a functional cure may be achievable in the shorter term and represent a more realistic goal since virus reduction has been shown for a number of therapeutic vaccine candidates. Approaches that aim to successfully combat HIV-1 infection will need to address both the virus (virus-specific approaches including ART and therapeutic vaccines) as well as the generalized immune activation that drives the infection. It is likely that to achieve a functional cure, a combination of different interventions may

This work has been partly supported by a grant from the Research Council of Norway GLOBVAC programme. Many thanks to Birger Sørensen, Ingebjørg Baksaas, Vidar Wendel-

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these considerations will influence the size of viral reservoir.

therefore be more appropriate.

ultimately be required.

**7. Acknowledgments** 

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**Part 9** 

**Beyond Conventional** 


## **Part 9**

**Beyond Conventional** 

510 Recent Translational Research in HIV/AIDS

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**25** 

*USA* 

**Micronutrient Synergy in** 

*Dr. Rath Research Institute, Santa Clara, CA* 

**the Control of HIV Infection and AIDS** 

Raxit J. Jariwalla, Aleksandra Niedzwiecki and Matthias Rath

Acquired immune deficiency syndrome (AIDS) has become a global health pandemic and the most common cause of death among young adults aged 20-24 years (Patton et al., 2009). According to the UN/AIDS Global Report published in November 2010 (UNAIDS 2010), about 1.8 million persons died from AIDS-related causes in the year 2009 alone. At the end of that year, the epidemic had left behind totally 16.6 million orphans, defined as those under 18 who had lost one or both parents to AIDS. Since the beginning of the epidemic,

At the end of 2009, an estimated 30.8 million adults and 2.8 million children were living with HIV, the human immunodeficiency virus linked to AIDS; with women accounting for just over one-half of all adults living with HIV worldwide. During the same year, about 2.6 million persons became newly infected with HIV, including 370,000 children. Of all people

Despite these gruesome statistics, there is no cure in sight. Current treatment is based on the use of antiretroviral (ARV) drugs targeted against HIV at various steps in viral replication (Sleaseman and Goodenow 2003). Although ARV drugs can reduce viral load in the bloodstream, they neither cure HIV infection nor restore the immune system to combat AIDS (Roederer 1998, Pakker et al., 1998). Virus is known to persist indefinitely in reservoirs of latently-infected cells and emergence of drug-resistant strains is common. Furthermore, the effectiveness of ARVs in having any clinical benefits at all depends upon a number of factors, particularly the CD4 count and the nutritional status of patients at the point at which ARV treatment is commenced (Hong et al., 2001, Paton et al., 2006). Additionally, drugs are higly toxic and are often associated with adverse side effects to various organs of the body, including the bone marrow and liver, (Fischl et al., 1987, Richman et al., 1987, Costello et al., 1988, Abrescia et al., 2008), cellular mitochondria (Carr et al., 2001), and with

Consequently, there is need for safe and effective, nontoxic therapy that can not only restore the immune system and keep virus multiplication/spread in check but also block AIDS progression without harming cells of the host. This review will focus on the relationship of nutrition to infection and immunity and evidence from experimental and clinical studies on the potential value of micronutrients and their combinations in controlling HIV infection

nearly 30 million people have died from AIDS-related causes.

lipodystrophy and dyslipidemia (Carr et al., 1998).

and reducing symptoms associated with AIDS.

living with HIV, about 68% reside in Sub-Saharan Africa (UNAIDS 2010).

**1. Introduction** 

### **Micronutrient Synergy in the Control of HIV Infection and AIDS**

Raxit J. Jariwalla, Aleksandra Niedzwiecki and Matthias Rath *Dr. Rath Research Institute, Santa Clara, CA USA* 

#### **1. Introduction**

Acquired immune deficiency syndrome (AIDS) has become a global health pandemic and the most common cause of death among young adults aged 20-24 years (Patton et al., 2009). According to the UN/AIDS Global Report published in November 2010 (UNAIDS 2010), about 1.8 million persons died from AIDS-related causes in the year 2009 alone. At the end of that year, the epidemic had left behind totally 16.6 million orphans, defined as those under 18 who had lost one or both parents to AIDS. Since the beginning of the epidemic, nearly 30 million people have died from AIDS-related causes.

At the end of 2009, an estimated 30.8 million adults and 2.8 million children were living with HIV, the human immunodeficiency virus linked to AIDS; with women accounting for just over one-half of all adults living with HIV worldwide. During the same year, about 2.6 million persons became newly infected with HIV, including 370,000 children. Of all people living with HIV, about 68% reside in Sub-Saharan Africa (UNAIDS 2010).

Despite these gruesome statistics, there is no cure in sight. Current treatment is based on the use of antiretroviral (ARV) drugs targeted against HIV at various steps in viral replication (Sleaseman and Goodenow 2003). Although ARV drugs can reduce viral load in the bloodstream, they neither cure HIV infection nor restore the immune system to combat AIDS (Roederer 1998, Pakker et al., 1998). Virus is known to persist indefinitely in reservoirs of latently-infected cells and emergence of drug-resistant strains is common. Furthermore, the effectiveness of ARVs in having any clinical benefits at all depends upon a number of factors, particularly the CD4 count and the nutritional status of patients at the point at which ARV treatment is commenced (Hong et al., 2001, Paton et al., 2006). Additionally, drugs are higly toxic and are often associated with adverse side effects to various organs of the body, including the bone marrow and liver, (Fischl et al., 1987, Richman et al., 1987, Costello et al., 1988, Abrescia et al., 2008), cellular mitochondria (Carr et al., 2001), and with lipodystrophy and dyslipidemia (Carr et al., 1998).

Consequently, there is need for safe and effective, nontoxic therapy that can not only restore the immune system and keep virus multiplication/spread in check but also block AIDS progression without harming cells of the host. This review will focus on the relationship of nutrition to infection and immunity and evidence from experimental and clinical studies on the potential value of micronutrients and their combinations in controlling HIV infection and reducing symptoms associated with AIDS.

Micronutrient Synergy in the Control of HIV Infection and AIDS 515

Fig. 1. Impact of micronutrient deficiencies on infectious diseases

Fig. 2. Micronutrients are essential for sustaining all cellular functions

Micronutrients are also critical for optimum functioning of the immune system including cell-mediated immunity, antibody production (humoral immunity) and optimum thymus

The pathological basis of AIDS is a dysfunctional immune system clinically indicated by abnormally low levels of white blood cells. Micronutrients are essential for blood formation,

structures.

function (Fig. 3).

**4. Essential role of micronutrients in cell physiology and immunity** 

Micronutrients are essential for sustaining all cellular functions including metabolic reactions in the cytosol and biochemical functions within cellular organelles (Fig 2). Vitamins and minerals are needed in smaller amounts than proteins, fats and sugars but without them, cells cannot convert food into biological energy and build different body

#### **2. Nutritional deficiencies in HIV and AIDS**

The relationship between nutrition, infection and immunity is well established since the early 1940's (Scrimshaw 2003, Webb and Villamor 2007). It is for instance well recognized that nutritional deficiency can lower immunity and predispose individuals to microbial infection. Conversely, nutritional supplementation can improve immune function and prevent/confer resistance to infection.

As the latent period between HIV infection and AIDS manifestation has been estimated at 8- 10 years (Morgan et al 2002), nutritional cofactors, besides HIV, have been implicated in AIDS development (Beach et al., 1992, Baum et al., 1995, Jariwalla et al., 2008a, 2009). Furthermore, nutrient supplementation in asymptomatic HIV-infected individuals was shown to delay the onset of AIDS (Abrams et al., 1993, Tang et al., 1993), supporting involvement of nutritional status as a contributory factor in AIDS development.

It is universally known since the emergence of the AIDS epidemic in the early 1980's that nutritional deficiencies are prevalent in persons with HIV infection and AIDS (Gray 1984, Beach et al., 1992, Jariwalla 1995; see also Table 1). These deficits include: (i) specific micronutrient abnormalities such as reduced blood levels of the common ACE vitamins, minerals, trace elements including selenium, amino acids such as cysteine, and the tri-peptide glutathione, which displays a global systemic deficiency; (ii) macronutrient abnormalities such as protein calorie malnutrition, which has been linked to a wasting disease, characteristic of AIDS. Malnutrition has also been linked to the spread of AIDS and TB in developing countries and with reduced survival (Paton et al., 2006, Turchenko et al., 2008)


Table 1. Commonly occurring nutritional deficiencies in HIV infection and AIDS

#### **3. Impact of nutritional deficiencies**

Micronutrient deficiencies in particular vitamin and mineral deficiencies can promote and strengthen microbial growth by weakening the immune system of the host, making it prone to acquiring new infections (Scrimshaw 2003, Webb and Villamor 2007; see Fig 1).

The relationship between nutrition, infection and immunity is well established since the early 1940's (Scrimshaw 2003, Webb and Villamor 2007). It is for instance well recognized that nutritional deficiency can lower immunity and predispose individuals to microbial infection. Conversely, nutritional supplementation can improve immune function and

As the latent period between HIV infection and AIDS manifestation has been estimated at 8- 10 years (Morgan et al 2002), nutritional cofactors, besides HIV, have been implicated in AIDS development (Beach et al., 1992, Baum et al., 1995, Jariwalla et al., 2008a, 2009). Furthermore, nutrient supplementation in asymptomatic HIV-infected individuals was shown to delay the onset of AIDS (Abrams et al., 1993, Tang et al., 1993), supporting

It is universally known since the emergence of the AIDS epidemic in the early 1980's that nutritional deficiencies are prevalent in persons with HIV infection and AIDS (Gray 1984, Beach et al., 1992, Jariwalla 1995; see also Table 1). These deficits include: (i) specific micronutrient abnormalities such as reduced blood levels of the common ACE vitamins, minerals, trace elements including selenium, amino acids such as cysteine, and the tri-peptide glutathione, which displays a global systemic deficiency; (ii) macronutrient abnormalities such as protein calorie malnutrition, which has been linked to a wasting disease, characteristic of AIDS. Malnutrition has also been linked to the spread of AIDS and TB in developing countries

> MICRONUTRIENT ABNORMALITIES Vitamins Trace Elements

Vitamin A Selenium Vitamin B12 Zinc

Vitamin C Amino Acids Vitamin E Cysteine

> Peptides Glutathione

MACRONUTRIENT DEFECITS Protein Calorie Malnutrition Abnormal Lipids (Dyslipidemia)

Micronutrient deficiencies in particular vitamin and mineral deficiencies can promote and strengthen microbial growth by weakening the immune system of the host, making it prone

Table 1. Commonly occurring nutritional deficiencies in HIV infection and AIDS

to acquiring new infections (Scrimshaw 2003, Webb and Villamor 2007; see Fig 1).

involvement of nutritional status as a contributory factor in AIDS development.

and with reduced survival (Paton et al., 2006, Turchenko et al., 2008)

Vitamin B6

**3. Impact of nutritional deficiencies** 

**2. Nutritional deficiencies in HIV and AIDS** 

prevent/confer resistance to infection.

Fig. 1. Impact of micronutrient deficiencies on infectious diseases

#### **4. Essential role of micronutrients in cell physiology and immunity**

Micronutrients are essential for sustaining all cellular functions including metabolic reactions in the cytosol and biochemical functions within cellular organelles (Fig 2). Vitamins and minerals are needed in smaller amounts than proteins, fats and sugars but without them, cells cannot convert food into biological energy and build different body structures.

Fig. 2. Micronutrients are essential for sustaining all cellular functions

Micronutrients are also critical for optimum functioning of the immune system including cell-mediated immunity, antibody production (humoral immunity) and optimum thymus function (Fig. 3).

The pathological basis of AIDS is a dysfunctional immune system clinically indicated by abnormally low levels of white blood cells. Micronutrients are essential for blood formation,

Micronutrient Synergy in the Control of HIV Infection and AIDS 517

with antioxidant properties. They include: vitamins C and E, amino acid thiols such as cysteine or its derivative N-acetyl cysteine (NAC), disulfides such as alpha-lipoic acid, tripeptides such as glutathione and its derivative glutathione monoester, polyphenols such as epigallo-catecheine gallate (EGCG from green tea) and the trace element selenium. Among them, ascorbic acid (vitamin C or ascorbate) is the most versatile, capable of blocking HIV replication in all phases of HIV infection namely, acute, chronic and latent infection (Harakeh et al., 1990; Harakeh and Jariwalla, 1991, 1995). Cysteine and glutathione monoester inhibit chronic HIV expression (Mihm et al., 1991; Kalebic et al., 1991) whereas NAC and selenium are effective in inhibiting HIV activation in latently-infected cells (Roederer et al., 1990, Harakeh and Jariwalla 1991; Sappey et al., 1994). It has been reported that alpha-lipoic acid can block acute infection (Bauer et al., 1991) and flavonoids including the polyphenol EGCG inhibit HIV at an early stage, blocking interaction of the virus with

Fig. 4. Nutrients can directly suppress viral infections

including white blood cells. Of particular importance are: vitamin B-3, vitamin B-5, vitamin B-6, vitamin B-12, vitamin C, folic acid and iron. Any textbook of biology or biochemistry documents these scientific facts. Moreover, no less than nine Nobel Prizes in Medicine have been awarded to date on the discovery of the health benefits of vitamins, relevant to their role in cellular physiology and impact on the immune system (Nobel Prize Committee website, Nobelprize.org).

Fig. 3. Nutrients are critical for optimum immune defense of a host

#### **5. Role of micronutrients in suppression of virus infection**

Additionally, experimental studies have shown that specific micronutrients can suppress virus infection at various steps in the viral life cycle that include blocking (a) virus entry, (b) virus multiplication, (c) virus activation in latently infected cells and (d) virus spread (Fig 4). Prevent viral entry into cells (Vitamin C, EGCG)

Stop viral multiplication (Vitamin C, N-Acetylcysteine)

Prevent activation of "silent" viruses (Vitamin C)

Limit spread of infections (Lysine, Vitamin C)

In the case of HIV, micronutrients have been shown to block virus expression at all stages of virus-host interactions, which include acute infection, chronic expression and activation from latently infected cells (Fig 5). The specific micronutrients demonstrated to affect different phases of virus infection are listed in Table 2. Most of them are reducing agents

including white blood cells. Of particular importance are: vitamin B-3, vitamin B-5, vitamin B-6, vitamin B-12, vitamin C, folic acid and iron. Any textbook of biology or biochemistry documents these scientific facts. Moreover, no less than nine Nobel Prizes in Medicine have been awarded to date on the discovery of the health benefits of vitamins, relevant to their role in cellular physiology and impact on the immune system (Nobel Prize Committee

Fig. 3. Nutrients are critical for optimum immune defense of a host

**5. Role of micronutrients in suppression of virus infection** 

Prevent viral entry into cells (Vitamin C, EGCG) Stop viral multiplication (Vitamin C, N-Acetylcysteine) Prevent activation of "silent" viruses (Vitamin C) Limit spread of infections (Lysine, Vitamin C)

Additionally, experimental studies have shown that specific micronutrients can suppress virus infection at various steps in the viral life cycle that include blocking (a) virus entry, (b) virus multiplication, (c) virus activation in latently infected cells and (d) virus spread (Fig 4).

In the case of HIV, micronutrients have been shown to block virus expression at all stages of virus-host interactions, which include acute infection, chronic expression and activation from latently infected cells (Fig 5). The specific micronutrients demonstrated to affect different phases of virus infection are listed in Table 2. Most of them are reducing agents

website, Nobelprize.org).

with antioxidant properties. They include: vitamins C and E, amino acid thiols such as cysteine or its derivative N-acetyl cysteine (NAC), disulfides such as alpha-lipoic acid, tripeptides such as glutathione and its derivative glutathione monoester, polyphenols such as epigallo-catecheine gallate (EGCG from green tea) and the trace element selenium. Among them, ascorbic acid (vitamin C or ascorbate) is the most versatile, capable of blocking HIV replication in all phases of HIV infection namely, acute, chronic and latent infection (Harakeh et al., 1990; Harakeh and Jariwalla, 1991, 1995). Cysteine and glutathione monoester inhibit chronic HIV expression (Mihm et al., 1991; Kalebic et al., 1991) whereas NAC and selenium are effective in inhibiting HIV activation in latently-infected cells (Roederer et al., 1990, Harakeh and Jariwalla 1991; Sappey et al., 1994). It has been reported that alpha-lipoic acid can block acute infection (Bauer et al., 1991) and flavonoids including the polyphenol EGCG inhibit HIV at an early stage, blocking interaction of the virus with

Fig. 4. Nutrients can directly suppress viral infections

Micronutrient Synergy in the Control of HIV Infection and AIDS 519

acid Chronic and acute infection Mihm *et al* 1991, Baur *et al*<sup>1991</sup>

Harakeh and Jariwalla (1991, 1995)

Roederer *et al* 1990, Harakeh & Jariwalla 1991, Sappey *et al* 1994.

*al* 2002, Jariwalla et al 2010

**Nutrient Inhibitory Effect Targeted at Reference**  Vitamin E Latent infection Suzuki *et al* 1993

monoester Chronic infection Kalebic *et al*<sup>1991</sup>

Synergistic HIV suppression in

\* containing (vitamin C, green tea extract, lysine, proline, arginine, NAC, selenium)

**6. Our approach to controlling virus infection with nutrient synergy** 

micronutrient supplement in AIDS patients in a community wide setting.

green tea extract Acute infection Mahmood et al 1993, Fassina *et* 

Although specific, single nutrients have been shown to suppress HIV in previous studies, little attention has been directed at blocking virus expression with nutrient combinations. To investigate this, we have utilized the principle of nutrient synergy i.e. use of nutrients in combination at low to moderate (physiological) levels for prevention and control of disease (Rath and Niedzwiecki 1996, Rath et al., 2005, Jariwalla et al., 2008a, 2009). The principle underlying nutrient synergy is that nutrients work in the body in harmonious synergy, not isolation, and they allow for maximal benefits when used in combination at physiological doses. In nutrient synergy 1 + 1 is more than 2 (Fig 6). We have applied this principle to both experimental studies of HIV infection as well as the in vivo evaluation of a defined multi-

chronic and latent infection Jariwalla *et al* <sup>2010</sup>

infection

Vitamin C Acute, chronic and latent

NAC, Selenium Chronic and latent infection

Table 2. Action of micronutrients on phases of HIV infection

Cysteine, alpha-lipoic

Glutathione

Flavonoids, EGCG,

Nutrient mixture (NM)\*

Fig. 6. The benefits of nutrient synergy

host-cells receptor (Mahmood et al., 1993, Fassina et al., 2002). More recently, green tea extract enriched in such polyphenols (80% by weight) was shown to suppress HIV production in chronically and latently infected cells (Jariwalla et al., 2010).

Fig. 5. Micronutrients can target different stages in HIV-host cell interaction

host-cells receptor (Mahmood et al., 1993, Fassina et al., 2002). More recently, green tea extract enriched in such polyphenols (80% by weight) was shown to suppress HIV

production in chronically and latently infected cells (Jariwalla et al., 2010).

Fig. 5. Micronutrients can target different stages in HIV-host cell interaction


Table 2. Action of micronutrients on phases of HIV infection

\* containing (vitamin C, green tea extract, lysine, proline, arginine, NAC, selenium)

#### **6. Our approach to controlling virus infection with nutrient synergy**

Although specific, single nutrients have been shown to suppress HIV in previous studies, little attention has been directed at blocking virus expression with nutrient combinations. To investigate this, we have utilized the principle of nutrient synergy i.e. use of nutrients in combination at low to moderate (physiological) levels for prevention and control of disease (Rath and Niedzwiecki 1996, Rath et al., 2005, Jariwalla et al., 2008a, 2009). The principle underlying nutrient synergy is that nutrients work in the body in harmonious synergy, not isolation, and they allow for maximal benefits when used in combination at physiological doses. In nutrient synergy 1 + 1 is more than 2 (Fig 6). We have applied this principle to both experimental studies of HIV infection as well as the in vivo evaluation of a defined multimicronutrient supplement in AIDS patients in a community wide setting.

Fig. 6. The benefits of nutrient synergy

Micronutrient Synergy in the Control of HIV Infection and AIDS 521

changes in severity of symptoms seen after the first 3 visits (8-12) weeks from the beginning of micronutrient supplementation. Table 3 lists the AIDS-defining symptoms for Africa, other physical symptoms, pain symptoms and symptoms of well-being. Tables 4-6 show a summary of the impact on these symptoms from micronutrient supplementation. The results showed that within 10-12 weeks, the micronutrient supplement statistically significantly suppressed all AIDS-defining symptoms compared to baseline. The supplement also significantly suppressed other physical symptoms frequently seen in AIDS

**AIDS-Defining Symptoms Symptoms of Well Being**

Table 3. AIDS-Related Symptoms, Conditions and Diseases Monitored in Community Wide

The micronutrient supplement evaluated in Khayelitsha was also rolled out in KwaZulu Natal district (near Durban) where a very large group (522 patients) completed all 3 exams and questionnaires. Similar to Khayelitsha, the same trend in reduction of AIDS-defining symptoms, other physical AIDS-associated symptoms and pain symptoms was seen (Tables 4-6). The results were also confirmed in two other townships (Western Cape and Free State),

Depression

**% decrease in AIDS-defining symptoms from baseline after 3 visits \*** 

patients including state of well-being (Jariwalla et al., 2008a).

Fever Appetite Diarrhoea Energy

**Other Physical Symptoms** Insomnia Swollen glands Fatigue

Colds, flu Rashes

Headache Bloating, gas

Micronutrient Program

community wide program

Wounds, sores, ulcers

Other physical symptoms

for a total of 813 participants from all 4 townships (Tables 4-6).

**patients** 

Table 4. Impact of micronutrient supplementation on AIDS defining symptom in a

**Site Total no of** 

Khayelitsha 50 33-61% Kwazulu-Natal (KZN) 473 37-48% Western Cape 153 51-78% Free State 82 23-26%

\*8-12 weeks except Free State (= 40 weeks)

Cough Enjoyment of life Weight loss Fear of future TB Concentration Oppurtunistic infections Anxiety

All nutrients work in our bodies in harmonious synergy, not in isolation. Nutrient Synergy allows for achieving maximum health benefits and keeping cellular processes in balance using smaller quantities of nutrients. Use of single vitamins in very high-doses or a randomly selected nutrient combination is not recommended as an optimal approach to health.

#### **7. Experimental studies in HIV infection**

Studies conducted by us of micronutrient combinations in laboratory cultures of HIV infected cells have provided further support for nutritional efficacy in viral immunodeficiency disease (Jariwalla et al., 2010). In these studies, we compared the ability of micronutrient combinations to single nutrients in the suppression of HIV replication in both chronically and latently infected cells. H9-HTLV IIIB is a model, chronically-infected T lymphocytic cell line that constitutively produces HIV cytopathic virus in the cell culture supernatant (Popovic et al., 1984, Gallo et al., 1984, Harakeh et al., 1990, Harakeh and Jariwalla 1991). Exposure of these cells to low/moderate concentrations of single micronutrients such as ascorbic acid, green tea extract and the amino acids such lysine produced only small inhibitory effects on virus production. In contrast, exposure of cells to combinations of micronutrients conferred significantly greater HIV suppression compared to single nutrients, indicating a synergistic effect. A nutritional mixture (NM), consisting of vitamin C, green tea extract, amino acids (lysine, proline, arginine), NAC and selenium also gave enhanced suppression of HIV production in this cell line compared to single nutrients (Jariwalla et al., 2010; see also Table 2). A similar inhibitory effect on cytokine-stimulated virus expression was obtained in latently infected T cells, indicating that micronutrients cooperate to suppress virus expression in both chronically and latently-stimulated cells (Jariwalla et al., 2010; Table 2).

#### **8. Clinical nutrition studies in AIDS patients**

Based on the above scientific evidence of micronutrient effectiveness in laboratory cultures of virally-infected cells, we have incorporated the use of micronutrients in natural control of HIV infection. Our studies conducted in persons with AIDS symptoms have provided further support for micronutrient efficacy in viral immunodeficiency disease (Jariwalla et al., 2008a, 2009). This in vivo confirmation of micronutrient efficacy was demonstrated in AIDS patients in a community wide program conducted in South Africa between 2005 and 2008. In this community program, the Dr. Rath Foundation donated a micronutrient supplement to the South African National Civic Organization (SANCO) who distributed it among people affected by AIDS in various townships in South Africa.

The micronutrient supplement contained vitamins and trace elements (except iron) that are known to modulate the immune system (listed in Fig. 3) plus selenium, essential minerals and other important nutrients such as amino acids, green tea extract, bioflavonoids, N-acetyl cysteine, inositol and coenzyme Q10. This supplement was given to subjects to be taken 3 times a day with meals. The characteristics of participants, patient selection, informed consent, administration of questionnaire grading AIDS-defining symptoms and the evaluation methodology were reported previously (Jariwalla et al., 2008a, 2009).

The first township where a pilot nutritional program was evaluated was Khayelitsha, a township near Cape Town (Jariwalla et al., 2008a). In this pilot protocol, 56 AIDS patients completed all 3 examinations and their completed questionnaires were evaluated for

Nutrient Synergy allows for achieving maximum health benefits and keeping cellular processes in balance using smaller quantities of nutrients. Use of single vitamins in very high-doses or a randomly selected nutrient combination is not recommended as an optimal

Studies conducted by us of micronutrient combinations in laboratory cultures of HIV infected cells have provided further support for nutritional efficacy in viral immunodeficiency disease (Jariwalla et al., 2010). In these studies, we compared the ability of micronutrient combinations to single nutrients in the suppression of HIV replication in both chronically and latently infected cells. H9-HTLV IIIB is a model, chronically-infected T lymphocytic cell line that constitutively produces HIV cytopathic virus in the cell culture supernatant (Popovic et al., 1984, Gallo et al., 1984, Harakeh et al., 1990, Harakeh and Jariwalla 1991). Exposure of these cells to low/moderate concentrations of single micronutrients such as ascorbic acid, green tea extract and the amino acids such lysine produced only small inhibitory effects on virus production. In contrast, exposure of cells to combinations of micronutrients conferred significantly greater HIV suppression compared to single nutrients, indicating a synergistic effect. A nutritional mixture (NM), consisting of vitamin C, green tea extract, amino acids (lysine, proline, arginine), NAC and selenium also gave enhanced suppression of HIV production in this cell line compared to single nutrients (Jariwalla et al., 2010; see also Table 2). A similar inhibitory effect on cytokine-stimulated virus expression was obtained in latently infected T cells, indicating that micronutrients cooperate to suppress virus expression in both

Based on the above scientific evidence of micronutrient effectiveness in laboratory cultures of virally-infected cells, we have incorporated the use of micronutrients in natural control of HIV infection. Our studies conducted in persons with AIDS symptoms have provided further support for micronutrient efficacy in viral immunodeficiency disease (Jariwalla et al., 2008a, 2009). This in vivo confirmation of micronutrient efficacy was demonstrated in AIDS patients in a community wide program conducted in South Africa between 2005 and 2008. In this community program, the Dr. Rath Foundation donated a micronutrient supplement to the South African National Civic Organization (SANCO) who distributed it among

The micronutrient supplement contained vitamins and trace elements (except iron) that are known to modulate the immune system (listed in Fig. 3) plus selenium, essential minerals and other important nutrients such as amino acids, green tea extract, bioflavonoids, N-acetyl cysteine, inositol and coenzyme Q10. This supplement was given to subjects to be taken 3 times a day with meals. The characteristics of participants, patient selection, informed consent, administration of questionnaire grading AIDS-defining symptoms and the

The first township where a pilot nutritional program was evaluated was Khayelitsha, a township near Cape Town (Jariwalla et al., 2008a). In this pilot protocol, 56 AIDS patients completed all 3 examinations and their completed questionnaires were evaluated for

evaluation methodology were reported previously (Jariwalla et al., 2008a, 2009).

All nutrients work in our bodies in harmonious synergy, not in isolation.

chronically and latently-stimulated cells (Jariwalla et al., 2010; Table 2).

people affected by AIDS in various townships in South Africa.

**8. Clinical nutrition studies in AIDS patients** 

approach to health.

**7. Experimental studies in HIV infection** 

changes in severity of symptoms seen after the first 3 visits (8-12) weeks from the beginning of micronutrient supplementation. Table 3 lists the AIDS-defining symptoms for Africa, other physical symptoms, pain symptoms and symptoms of well-being. Tables 4-6 show a summary of the impact on these symptoms from micronutrient supplementation. The results showed that within 10-12 weeks, the micronutrient supplement statistically significantly suppressed all AIDS-defining symptoms compared to baseline. The supplement also significantly suppressed other physical symptoms frequently seen in AIDS patients including state of well-being (Jariwalla et al., 2008a).


Table 3. AIDS-Related Symptoms, Conditions and Diseases Monitored in Community Wide Micronutrient Program

The micronutrient supplement evaluated in Khayelitsha was also rolled out in KwaZulu Natal district (near Durban) where a very large group (522 patients) completed all 3 exams and questionnaires. Similar to Khayelitsha, the same trend in reduction of AIDS-defining symptoms, other physical AIDS-associated symptoms and pain symptoms was seen (Tables 4-6). The results were also confirmed in two other townships (Western Cape and Free State), for a total of 813 participants from all 4 townships (Tables 4-6).


Table 4. Impact of micronutrient supplementation on AIDS defining symptom in a community wide program

\*8-12 weeks except Free State (= 40 weeks)

Micronutrient Synergy in the Control of HIV Infection and AIDS 523

positive asymptomatic persons

1) Reduced oxidative stress, viral load 2) Prevention of AZT-induced

reduced viral load in advanced AIDS

Abrams *et al* 1993, Tang

Herzenberg *et al* 1997

Allard *et al* 1998, de la Asuncion *et al* 1998

Fawzi *et al* 1998, 2004

Namulemia *et al* 2007

Jariwalla *et al* 2008b

Jariwalla *et al* 2008a;

2009

Muller *et al* 2000

Jiamton *et al* 2003

*et al* 1993

**Micronutrient Supplement Clinical change Reference** 

miochondrial damage

infected Tanzanian women

Micronutrient Supplement Improved CD4 count Kaiser *et al* 2006

Increased survival among HIVinfected persons in Bangkok

improved lymphocyte function

Reduced AIDS related and pain symptoms; improved state of well

Table 7. Clinical improvements seen upon micronutrient supplementation in HIV and AIDS

We would like to thank Lisa Smith for help with formatting/presentation of the graphics

Abrams B, Duncan D & Hertz-Picciotto I. (1993*).* A prospective study of dietary intake and

Abrescia N, D'Abbraccio M, Figoni M, Busto A, Maddaloni A & De Marco M. (2005).

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is an effective inhibitor of human immuno-deficiency virus (HIV-1) replication. *Klin* 

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Vitamin C plus E

Multi-micronutrient Supplement

Micronutrient Supplement (see text, page 8)

**10. Acknowledgement** 

3697-3710.

(July 1), pp. 4-9.

**11. References** 

patients, in peer-reviewed published studies.

N-acetylcysteine (NAC) Increased survival compared to placebo

NAC plus vitamin C Enhanced immune responses and

Multivitamin Supplement Reduced fetal death among HIV-

Nutritional Supplements Delayed AIDS progression in HIV-

Alpha-lipoic acid Enhanced blood glutathione and

being

and Anupriya Pandit for tabulating data and organizing references.

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Table 5. Impact of micronutrient supplementation on other physical symptoms in AIDS patients in community wide program

\* 8-12 weeks except Free State (= 40 weeks)


Table 6. Impact of micronutrient supplementation on pain symptoms in AIDS patients in community wide program

\* 8-12 weeks except Free State (= 40 weeks)

#### **9. Conclusion**

The results we have seen are not in isolation. Beneficial effects of micronutrients and their combinations have been seen in clinical studies conducted by other researchers as summarized in Table 7. These studies have evaluated nutrients in combination and reported beneficial effects on various outcomes including improvement in viral and immune parameters, antioxidant protection from cellular damage, slowing of disease progression, reduction of AIDS-related symptoms and improvement of birth outcomes in pregnant women. The impact of nutritional support and vitamin and micronutrient supplementation in the treatment of HIV and AIDS is a seriously under-investigated area. Repeated calls have been made for more studies in this area by international health agencies. Although micronutrients are not a cure for AIDS, in the absence of an effective cure or vaccine and in the face of the toxicity and limited efficacy of ARVs, they are a safe, effective and affordable way to halt progression towards and even reduce the symptoms of the AIDS disease and to improve the quality of life of AIDS patients.

The implications of micronutrient supplementation results for public health and control of infectious and immunodeficiency disease are enormous. If properly evaluated, micronutrients have the potential of being incorporated into strategies for fighting viral pandemics on a global scale. Implementation of the above positive findings could save millions of lives.


Table 7. Clinical improvements seen upon micronutrient supplementation in HIV and AIDS patients, in peer-reviewed published studies.

#### **10. Acknowledgement**

We would like to thank Lisa Smith for help with formatting/presentation of the graphics and Anupriya Pandit for tabulating data and organizing references.

#### **11. References**

522 Recent Translational Research in HIV/AIDS

Table 5. Impact of micronutrient supplementation on other physical symptoms in AIDS

Table 6. Impact of micronutrient supplementation on pain symptoms in AIDS patients in

The results we have seen are not in isolation. Beneficial effects of micronutrients and their combinations have been seen in clinical studies conducted by other researchers as summarized in Table 7. These studies have evaluated nutrients in combination and reported beneficial effects on various outcomes including improvement in viral and immune parameters, antioxidant protection from cellular damage, slowing of disease progression, reduction of AIDS-related symptoms and improvement of birth outcomes in pregnant women. The impact of nutritional support and vitamin and micronutrient supplementation in the treatment of HIV and AIDS is a seriously under-investigated area. Repeated calls have been made for more studies in this area by international health agencies. Although micronutrients are not a cure for AIDS, in the absence of an effective cure or vaccine and in the face of the toxicity and limited efficacy of ARVs, they are a safe, effective and affordable way to halt progression towards and even reduce the symptoms of the AIDS disease and to

The implications of micronutrient supplementation results for public health and control of infectious and immunodeficiency disease are enormous. If properly evaluated, micronutrients have the potential of being incorporated into strategies for fighting viral pandemics on a global scale. Implementation of the above positive findings could save

**% decrease in other physical symptoms from baseline after 3 visits \*** 

> **% decrease in pain symptoms from baseline after 3 visits \***

**Site Total no of** 

patients in community wide program \* 8-12 weeks except Free State (= 40 weeks)

community wide program

**9. Conclusion** 

millions of lives.

\* 8-12 weeks except Free State (= 40 weeks)

improve the quality of life of AIDS patients.

Khayelitsha 45 37-60% Kwazulu-Natal (KZN) 522 17-54% Western Cape 153 44-83% Free State 78 17-47%

**Site Total no of** 

Khayelitsha 44 38-49% Kwazulu-Natal (KZN) 511 32-50% Western Cape 149 43-64% Free State 79 24-35%

**patients** 

**patients** 


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SA, Miedema F & Schellekens PT. (1998). Biphasic kinetics of peripheral blood T


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**26** 

*USA* 

**HIV Prevention** 

**Substance Abuse Treatment Utilizing** 

Thomas F Kresina, Robert Lubran and Laura W. Cheever

*Centre for Substance Abuse Treatment, Substance Abuse and Mental Health Services Administration and HIV/AIDS Bureau, Health Resources and Services Administration* 

International guidelines have been developed for the use of medications in the treatment of substance use disorders (WHO, 2008; WHO, 2009). Medications used in the detoxification from drug abuse and dependence provide symptomatic relief of drug and alcohol withdrawal. For long term treatment or medical maintenance treatment, medications eliminate the physiological effects of drug use by blocking drug-receptor binding in the brain and are an important part of the recovery process. The use of medication assistant treatment (MAT) is part of a comprehensive treatment plan for drug and alcohol dependence that addresses the medical, social, and psychological needs of the patient (SAMHSA, 2005; SAMHSA, 2009). An effective long term treatment paradigm for the successful treatment of alcohol or opioid dependence is the concomitant use of medications that block the effects of drug use in concert with behavior change counseling and psychotherapy. Medications which have demonstrated effectiveness in the long term treatment of opioid dependence are methadone, buprenorphine (subutex®, suboxone®), and naltrexone (Revia®, Depade®) or extended release injectable naltrexone (vivitrol®). Pharmacotherapies used in the treatment of alcohol dependence include acamprosate (Campral®), disulfiram (antabuse®, antabus®) and naltrexone (Revia®, Depade®) or extended

Time in treatment is a reliable indicator for successful treatment of drug dependence. Patients remain in treatment for longer periods of time when they perceive that their health care environment is supportive and non-stigmatizing, have a good patient-provider relationship, and feel that their needs are identified and met. Access to community-based substance abuse treatment that includes MAT is fundamental to achieving broad service coverage. Given that substance abuse treatment is Human Immunodeficiency Virus (HIV) prevention and the frequent co-morbidity of substance abuse and HIV infection, the provision of prevention, care and treatment for both need to be addressed in a coordinated manner for ideal patient outcomes. There are several models to achieve excellent patient outcomes for both HIV infection and the treatment of substance abuse (Proeschold-Bell et al 2010; Weiss et al, 2011). The highest level of coordinated care model has MAT and HIV services fully integrated with both the same medical record and health providers for both

**1. Introduction** 

release injectable naltrexone (vivitrol®).

**Medication Assisted Treatment as** 

cells after triple combination therapy in HIV-1 infection: a composite of redistribution and proliferation. *Nat Med.,* Vol. 4, No. 2, pp. 208-14.


### **Substance Abuse Treatment Utilizing Medication Assisted Treatment as HIV Prevention**

Thomas F Kresina, Robert Lubran and Laura W. Cheever *Centre for Substance Abuse Treatment, Substance Abuse and Mental Health Services Administration and HIV/AIDS Bureau, Health Resources and Services Administration USA* 

#### **1. Introduction**

526 Recent Translational Research in HIV/AIDS

Paton NI, Sangeetha S, Earnest A & Bellamy R. (2006). The impact of malnutrition on

Patton GC, Coffey C, Sawyer SM, Viner RM, Haller DM, Bose K, Vos T,Ferguson J & Mathers

population health data. *Lancet*, Vol. 374, No. 9693, (September 12), pp. 881-92. Popovic M, Sarngadharan MG, Read E & Gallo RC. (1984). Detection, isolation, and

Rath M, Kalinovsky T & Niedzwiecki A. (2005). Reduction in the frequency of arrhythmic

Richman DD, Fischl MA, Grieco MH, Gottlieb MS, Volberding PA, Laskin OL, Leedom JM,

Roederer M. (1998). Getting to the HAART of T cell dynamics. *Nat Med*., Vol. 4, pp. 145-6. Roederer M, Staal FJ, Raju PA, Ela SW & Herzenberg LA. (1990). Cytokine-stimulated

Sappey C, Legrand-Poels S, Best Belpomme M, Favier A, Rentier B & Piette J. (1994).

oxidative stress. *AIDS Res. Hum. Retroviruses*, Vol. 10, No. 11, pp. 1451-61. Scrimshaw NS. (2003). Historical concepts of interactions, synergism and antagonism

Sleaseman JW and Goodenow MM. (2003). HIV-1 infection*. J Allergy Clin Immunol*., Vol. 111,

Suzuki YJ & Packer L. (1993). Inhibition of NF-κB activation by vitamin E derivatives. *Biochem. Biophys. Res. Commun.,* Vol. 193, No. 1, (May 28), pp. 277-83. Tang AM, Graham NM, Kirby AJ, McCall LD, Willett WC & Saah AJ. (1993). Dietary

Webb AL & Villamor E. (2007). Update: effects of antioxidant and non-antioxidant vitamin supplementation on immune function. *Nutr Rev*., Vol. 65, No. 5, pp. 181-217.

AIDS and pre-AIDS. *Science*, Vol. 224, No. 4648, (May 4), pp. 497-500. Rath M & Niedzwiecki A. (1996). Nutritional supplement program halts progression of early

redistribution and proliferation. *Nat Med.,* Vol. 4, No. 2, pp. 208-14.

antiretroviral therapy. *HIV Med*., Vol. 7, No. 5, pp. 323-30.

*Applied Nutrition*, Vol. 48, pp. 67-78.

Vol. 317, pp. 192-197.

No. 2 suppl, pp. S582-S592.

http://www.unaids.org/globalreport/

nutrient program. *JANA*, Vol. 8, pp. 21-25.

*Proc. Natl. Sci USA*, Vol. 87, No. 12, pp. 4884-8.

cells after triple combination therapy in HIV-1 infection: a composite of

survival and the CD4 count response in HIV-infected patients starting

CD. (2009). Global patterns of mortality in young people: a systematic analysis of

continuous production of cytopathic retroviruses (HTLV-III) from patients with

coronary atherosclerosis documented by ultrafast computed tomography. *Journal of* 

episodes in patients with paroxysmal atrial arrhythmia with a vitamin/essential

Groopman JE, Mildvan D, Hirsch MS, Jackson GG, Durack DT & Nusinoff-Lehrman S. The AZT collaborative working group. (1987). The toxicity of azidothymidine (AZT) in the treatment of patients with AIDS and AIDS related complex. A double-blind, placebo-controlled trial. *New England Journal of Medicine,* 

human immunodeficiency virus replication is inhibited by N-acetyl-L-cysteine.

Stimulation of glutathione peroxidase activity decreases HIV type 1 activation after

between nutrition and infection. *Journal of Nutrition*, Vol. 133, No. 1, pp. 316S-321S.

micronutrient intake and risk of progression to acquired immunodeficiency syndrome (AIDS) in human immunodeficiency virus type 1 (HIV-1)-infected homosexual men. *Am J Epidemiol*., Vol. 138, No. 11, (December 1), pp. 937-51. Turchenko LV, Voloshchuk EO, Ivanov V, Kalinovsky T, Niedzwiecki A & Rath M. (2008).

Clinical improvement of active tuberculosis patients with complex treatment and nutritional supplementation. *The Open Natural Products Journal*, Vol. 1, pp. 20-26. UNAIDS (2010). UNAIDS Report on the Global AIDS Epidemic, Retrieved from International guidelines have been developed for the use of medications in the treatment of substance use disorders (WHO, 2008; WHO, 2009). Medications used in the detoxification from drug abuse and dependence provide symptomatic relief of drug and alcohol withdrawal. For long term treatment or medical maintenance treatment, medications eliminate the physiological effects of drug use by blocking drug-receptor binding in the brain and are an important part of the recovery process. The use of medication assistant treatment (MAT) is part of a comprehensive treatment plan for drug and alcohol dependence that addresses the medical, social, and psychological needs of the patient (SAMHSA, 2005; SAMHSA, 2009). An effective long term treatment paradigm for the successful treatment of alcohol or opioid dependence is the concomitant use of medications that block the effects of drug use in concert with behavior change counseling and psychotherapy. Medications which have demonstrated effectiveness in the long term treatment of opioid dependence are methadone, buprenorphine (subutex®, suboxone®), and naltrexone (Revia®, Depade®) or extended release injectable naltrexone (vivitrol®). Pharmacotherapies used in the treatment of alcohol dependence include acamprosate (Campral®), disulfiram (antabuse®, antabus®) and naltrexone (Revia®, Depade®) or extended release injectable naltrexone (vivitrol®).

Time in treatment is a reliable indicator for successful treatment of drug dependence. Patients remain in treatment for longer periods of time when they perceive that their health care environment is supportive and non-stigmatizing, have a good patient-provider relationship, and feel that their needs are identified and met. Access to community-based substance abuse treatment that includes MAT is fundamental to achieving broad service coverage. Given that substance abuse treatment is Human Immunodeficiency Virus (HIV) prevention and the frequent co-morbidity of substance abuse and HIV infection, the provision of prevention, care and treatment for both need to be addressed in a coordinated manner for ideal patient outcomes. There are several models to achieve excellent patient outcomes for both HIV infection and the treatment of substance abuse (Proeschold-Bell et al 2010; Weiss et al, 2011). The highest level of coordinated care model has MAT and HIV services fully integrated with both the same medical record and health providers for both

Substance Abuse Treatment Utilizing Medication Assisted Treatment as HIV Prevention 529

Life Years and 3.2% of deaths, globally. The WHO estimates that about 2 billion individuals

Fig. 1. Interactions and linkages between drug and alcohol use and abuse in the course of

interventions are outpatient interventions that include some form of counseling.

Alcoholic beverage consumption can be described based on quantity. Abstainers or light/occasional drinkers comprise roughly 40% of a general population while moderate drinkers comprise about 35% of the general population. Both groups comprise approximately 55–75% of a general medical practice. At-risk drinkers, those with hazardous drinking patterns or quantities, and alcohol abusers, those with harmful drinking (meeting the required clinical criteria) comprise approximately 20% of the population and 20–35% of a general medical practice. At-risk drinkers are males who drink more than two drinks a day or greater than four drinks per occasion. For females and individuals over the age of 65, at-risk drinkers are those who drink greater than one drink per day or greater than three drinks per occasion. These individuals consume alcohol at levels that place them at-risk for alcohol-related social and/or medical problems (Dufour, 1999). These at-risk individuals are best managed through the use of brief interventions that can be provided by primary care physicians, health care providers or specialists, upon training. Usually these brief

worldwide consume alcoholic beverages.

HIV infection and AIDS.

services. Alternatively, MAT and HIV services can be separately managed but co-located to allow convenient utilization of both MAT and HIV services in another form of "one stop shopping". A third approach is coordinated care and treatment where MAT and HIV services are provided at distinct locations, and case managers, peer facilitators, or others promote coordination of referrals. This third model can pose significant barriers to substance users who are heavily stigmatized and medically disenfranchised and who have multiple competing medical, psychological and social needs that limit access to care. MAT programs that offer comprehensive services and care options can best contribute to improving the health of these individuals thereby reducing HIV infection in the community.

#### **2. Drug and alcohol use and their linkage to HIV infection**

Exposure to the HIV can result in a patent viral infection. HIV infection can occur via two transmission routes: direct injection of the virus through the use of injection equipment infected with HIV and through sexual contact with an infected individual. There is a direct linkage between these disparate behaviours and drug and alcohol use.

For people who inject drugs, there is a risk of HIV infection when the injection equipment is reused and not sterilized after use or when there is direct sharing of the injection equipment with individuals who may be infected with HIV. Drug users who are under the influence of drugs may engage in risk behaviours for HIV that they would not while sober. In addition, for drug users who develop dependence, withdrawal induced drug cravings may result in the exchange of sex for money or drugs or other behaviours that increase risk of HIV acquisition. Similarly, alcohol consumption increases sexual risk-taking including high risk behaviors (Figure 1). That includes sexual acts without the use of condoms and an increased number of sexual partners. Concurrent sexual partnerships are a significant risk factor for the transmission of HIV (Epstein & Morris, 2011). Alcohol can also be an important contributor in the progression of HIV infection to acquired immune deficiency syndrome (AIDS) (Hahn & Samet , 2010). Alcohol consumption is an important consideration in the medical management of patients with HIV infection, particularly those co-infected with the hepatitis C virus (HCV) (Edlin et al 2001). Studies have also shown that alcohol consumption can modify drug metabolism in the liver, and thereby potentially influence the effectiveness of HIV antiretroviral therapy. Alcohol-induced cirrhosis can result in changes in drug metabolism in the liver through compromised liver function. Research has shown that alcohol consumption greater than 50 g/day (4–5 drinks) is a risk factor for disease progression for patients with HIV/HCV co-infection.

All substance abuse, whether the use of opioids, stimulants, or excessive alcohol, can negatively influence the course of HIV disease progression when the use results in low antiretroviral adherence or facilitates missed medical appointments. Substance abuse has been associated with less access to antiretroviral medications, lower medication adherence, and increased mortality among HIV infected patients.

#### **2.1 Alcohol abuse, medication assisted treatment and HIV infection**

Alcohol abuse and dependence are global problems of major medical importance with high societal impact (WHO, 2010). In determining the global burden of disease, the World Health Organization (WHO) has noted that a leading cause of disability is alcohol and drug use disorders. Alcohol consumption is estimated to cause 4% of the total of Disability-Adjusted

services. Alternatively, MAT and HIV services can be separately managed but co-located to allow convenient utilization of both MAT and HIV services in another form of "one stop shopping". A third approach is coordinated care and treatment where MAT and HIV services are provided at distinct locations, and case managers, peer facilitators, or others promote coordination of referrals. This third model can pose significant barriers to substance users who are heavily stigmatized and medically disenfranchised and who have multiple competing medical, psychological and social needs that limit access to care. MAT programs that offer comprehensive services and care options can best contribute to improving the health of these individuals thereby reducing HIV infection in the community.

Exposure to the HIV can result in a patent viral infection. HIV infection can occur via two transmission routes: direct injection of the virus through the use of injection equipment infected with HIV and through sexual contact with an infected individual. There is a direct

For people who inject drugs, there is a risk of HIV infection when the injection equipment is reused and not sterilized after use or when there is direct sharing of the injection equipment with individuals who may be infected with HIV. Drug users who are under the influence of drugs may engage in risk behaviours for HIV that they would not while sober. In addition, for drug users who develop dependence, withdrawal induced drug cravings may result in the exchange of sex for money or drugs or other behaviours that increase risk of HIV acquisition. Similarly, alcohol consumption increases sexual risk-taking including high risk behaviors (Figure 1). That includes sexual acts without the use of condoms and an increased number of sexual partners. Concurrent sexual partnerships are a significant risk factor for the transmission of HIV (Epstein & Morris, 2011). Alcohol can also be an important contributor in the progression of HIV infection to acquired immune deficiency syndrome (AIDS) (Hahn & Samet , 2010). Alcohol consumption is an important consideration in the medical management of patients with HIV infection, particularly those co-infected with the hepatitis C virus (HCV) (Edlin et al 2001). Studies have also shown that alcohol consumption can modify drug metabolism in the liver, and thereby potentially influence the effectiveness of HIV antiretroviral therapy. Alcohol-induced cirrhosis can result in changes in drug metabolism in the liver through compromised liver function. Research has shown that alcohol consumption greater than 50 g/day (4–5 drinks) is a risk factor for disease

All substance abuse, whether the use of opioids, stimulants, or excessive alcohol, can negatively influence the course of HIV disease progression when the use results in low antiretroviral adherence or facilitates missed medical appointments. Substance abuse has been associated with less access to antiretroviral medications, lower medication adherence,

Alcohol abuse and dependence are global problems of major medical importance with high societal impact (WHO, 2010). In determining the global burden of disease, the World Health Organization (WHO) has noted that a leading cause of disability is alcohol and drug use disorders. Alcohol consumption is estimated to cause 4% of the total of Disability-Adjusted

**2. Drug and alcohol use and their linkage to HIV infection** 

linkage between these disparate behaviours and drug and alcohol use.

progression for patients with HIV/HCV co-infection.

and increased mortality among HIV infected patients.

**2.1 Alcohol abuse, medication assisted treatment and HIV infection** 

Life Years and 3.2% of deaths, globally. The WHO estimates that about 2 billion individuals worldwide consume alcoholic beverages.


Fig. 1. Interactions and linkages between drug and alcohol use and abuse in the course of HIV infection and AIDS.

Alcoholic beverage consumption can be described based on quantity. Abstainers or light/occasional drinkers comprise roughly 40% of a general population while moderate drinkers comprise about 35% of the general population. Both groups comprise approximately 55–75% of a general medical practice. At-risk drinkers, those with hazardous drinking patterns or quantities, and alcohol abusers, those with harmful drinking (meeting the required clinical criteria) comprise approximately 20% of the population and 20–35% of a general medical practice. At-risk drinkers are males who drink more than two drinks a day or greater than four drinks per occasion. For females and individuals over the age of 65, at-risk drinkers are those who drink greater than one drink per day or greater than three drinks per occasion. These individuals consume alcohol at levels that place them at-risk for alcohol-related social and/or medical problems (Dufour, 1999). These at-risk individuals are best managed through the use of brief interventions that can be provided by primary care physicians, health care providers or specialists, upon training. Usually these brief interventions are outpatient interventions that include some form of counseling.

Substance Abuse Treatment Utilizing Medication Assisted Treatment as HIV Prevention 531

Table 1. Approaches to Alcohol Use and Abuse in HIV/AIDS

disease in a substantial proportion of the population

the prevalent condition of alcohol abuse, assessment of the severity of the alcohol problem, and skills to intervene effectively to reduce the harm associated with alcohol use/abuse. New strategies to target alcohol use/ abuse in HIV populations need to be implemented in the context of existing recommended HIV clinical approaches. Addressing alcohol problems in HIV-infected persons has the potential to improve the overall management of HIV

Approximately 76.3 million individuals have a diagnosable alcohol use disorder. Alcohol and drug use disorders are defined clinically as alcohol/drug abuse or dependence (WHO, 2004). Diagnostic and Statistical Manual of Mental Disorders-4th edition (DSM-IV) definitions of abuse and dependence are maladaptive patterns of alcohol or drug use that result in clinically significant impairment or distress as well as significant behavior modifications. Individuals with alcohol dependence comprise approximately 5% of the population and around 5–10% of a general medical practice. Alcohol abusers and alcoholdependent individuals exhibit a varying degree of social and/or medical dysfunction. These individuals require intensive treatment, including structured counseling and/or pharmacotherapy (Fiellin et al., 2000). Severely involved dependent patients have been traditionally thought of requiring treatment in a specialty setting. However, studies (Fiellin et al., 2000a) have shown that primary care physicians may also have an important role in providing treatment to these individuals.

Pharmacotherapy for alcohol dependence is an important adjunct to behavioral therapies to reduce the risk of relapse to drinking after an initial period of abstinence (SAMHSA, 2009). Pharmacotherapy for alcohol consumption is also important for patients with co-occurring conditions such as patients with HIV and/or HCV infection(s) where alcohol consumption can augment disease progression. For these patients alcohol dependence treatment has been reported with either acamprosate, naltrexone, vivitrol or disulfiram (Collins et al, 2006). Acamprosate and naltrexone have different mechanisms of action and modify different behavioral aspects of alcohol dependence. Acamprosate is a long acting compound that prolongs periods of abstinence by normalizing glutamateric neurotransmission. Glutamateric neurotransmission in the brain is dysregulated during chronic alcohol consumption and withdrawal. Naltrexone is a fast acting opioid receptor antagonist that reduces heavy drinking through a decrease of the reward effects of ethanol. An evidencebased risk –benefits assessment can be used to inform health care providers on medication choice (Mason, 2003). However, the safety and efficacy of treatment using both medications for alcohol dependence has been shown in double blind studies (Kiefer & Wiedemann, 2004). Disulfiram, another pharmacotherpy option, blocks the oxidation of alcohol at the acetaldehyde stage of its metabolism. The increase in the levels of acetaldehyde resulting in a series of unpleasant symptoms (e.g., flushing, headache, and vomiting). Although disulfiram is widely used, particulalry is the setting of opioid dependence, superior data of studies support the use of naltrexone and acamprosate as pharmacologic treatments of alcoholism (Kiefer et al 2005). For resource limited settings, a series of factors acting synergistically may be creating the "perfect storm" promoting alcohol availability, alcohol consumption, and reducing alcohol control policies, thereby increasing the need for public health efforts (Table 1) to reduce alcohol consumption the beyond the use of medicationassisted treatment for alcohol abuse and dependence (Caetano & Laranjeira, 2006).

Use of alcohol may impact the care and course of HIV infection for an individual patient (Baum et al, 2010; Hahn & Samet, 2010). Optimal management of HIV infected patients with alcohol problems requires recognition of the impact of alcohol on a number of issues: patient's linkage to medical care; adherence to anti-retroviral treatment, impact on comorbid conditions (such as HCV infection), liver function, and the stage of HIV disease. Due to its many ramifications, the clinical approach to the HIV infected patient with alcohol problems takes on a high priority, yet it is similar in many ways to the standard optimal approach to any medical patient (Bogart et al., 2000). It requires the effective screening for




Approximately 76.3 million individuals have a diagnosable alcohol use disorder. Alcohol and drug use disorders are defined clinically as alcohol/drug abuse or dependence (WHO, 2004). Diagnostic and Statistical Manual of Mental Disorders-4th edition (DSM-IV) definitions of abuse and dependence are maladaptive patterns of alcohol or drug use that result in clinically significant impairment or distress as well as significant behavior modifications. Individuals with alcohol dependence comprise approximately 5% of the population and around 5–10% of a general medical practice. Alcohol abusers and alcoholdependent individuals exhibit a varying degree of social and/or medical dysfunction. These individuals require intensive treatment, including structured counseling and/or pharmacotherapy (Fiellin et al., 2000). Severely involved dependent patients have been traditionally thought of requiring treatment in a specialty setting. However, studies (Fiellin et al., 2000a) have shown that primary care physicians may also have an important role in

Pharmacotherapy for alcohol dependence is an important adjunct to behavioral therapies to reduce the risk of relapse to drinking after an initial period of abstinence (SAMHSA, 2009). Pharmacotherapy for alcohol consumption is also important for patients with co-occurring conditions such as patients with HIV and/or HCV infection(s) where alcohol consumption can augment disease progression. For these patients alcohol dependence treatment has been reported with either acamprosate, naltrexone, vivitrol or disulfiram (Collins et al, 2006). Acamprosate and naltrexone have different mechanisms of action and modify different behavioral aspects of alcohol dependence. Acamprosate is a long acting compound that prolongs periods of abstinence by normalizing glutamateric neurotransmission. Glutamateric neurotransmission in the brain is dysregulated during chronic alcohol consumption and withdrawal. Naltrexone is a fast acting opioid receptor antagonist that reduces heavy drinking through a decrease of the reward effects of ethanol. An evidencebased risk –benefits assessment can be used to inform health care providers on medication choice (Mason, 2003). However, the safety and efficacy of treatment using both medications for alcohol dependence has been shown in double blind studies (Kiefer & Wiedemann, 2004). Disulfiram, another pharmacotherpy option, blocks the oxidation of alcohol at the acetaldehyde stage of its metabolism. The increase in the levels of acetaldehyde resulting in a series of unpleasant symptoms (e.g., flushing, headache, and vomiting). Although disulfiram is widely used, particulalry is the setting of opioid dependence, superior data of studies support the use of naltrexone and acamprosate as pharmacologic treatments of alcoholism (Kiefer et al 2005). For resource limited settings, a series of factors acting synergistically may be creating the "perfect storm" promoting alcohol availability, alcohol consumption, and reducing alcohol control policies, thereby increasing the need for public health efforts (Table 1) to reduce alcohol consumption the beyond the use of medication-

assisted treatment for alcohol abuse and dependence (Caetano & Laranjeira, 2006).

Use of alcohol may impact the care and course of HIV infection for an individual patient (Baum et al, 2010; Hahn & Samet, 2010). Optimal management of HIV infected patients with alcohol problems requires recognition of the impact of alcohol on a number of issues: patient's linkage to medical care; adherence to anti-retroviral treatment, impact on comorbid conditions (such as HCV infection), liver function, and the stage of HIV disease. Due to its many ramifications, the clinical approach to the HIV infected patient with alcohol problems takes on a high priority, yet it is similar in many ways to the standard optimal approach to any medical patient (Bogart et al., 2000). It requires the effective screening for

providing treatment to these individuals.



Table 1. Approaches to Alcohol Use and Abuse in HIV/AIDS

the prevalent condition of alcohol abuse, assessment of the severity of the alcohol problem, and skills to intervene effectively to reduce the harm associated with alcohol use/abuse. New strategies to target alcohol use/ abuse in HIV populations need to be implemented in the context of existing recommended HIV clinical approaches. Addressing alcohol problems in HIV-infected persons has the potential to improve the overall management of HIV disease in a substantial proportion of the population

Substance Abuse Treatment Utilizing Medication Assisted Treatment as HIV Prevention 533

Table 2. Elements of the Continuum of Care in the Treatment of Opioid Abuse/Dependence experienced and documented by opioid dependent individuals. Life priorities of opioid users have been reported as concern about HIV and treatment of infection with HIV,

Substance abuse is a complex medical disorder composed of multiple physiologic, social and behavioral problems often interrelated with psychological illness. Health care providers need to screen substance misusing patients for psychological illness (Schuckit, 2006). Although it can be difficult to ascertain whether substance abuse, psychological illness, or infectious comorbidities should be addressed first, an initial focus on the medical treatment of drug abuse is often necessary to create sufficient patient stability from which other treatments can begin. Stability is further increased with both mental health services and substance abuse treatment, subsequently enhancing the medical outcomes of treatment for

housing, money, and protection from violence (Mizuno et al, 2003).

comorbidities.

#### **2.2 Illicit opioid abuse, medication assisted treatment and HIV infection**

Based on the 2010 World Drug Report (UNODC, 2010) from the United Nations Office on Drugs and Crime (UNODC), it is estimated that between 175- 250 million people from almost every country, or 5 percent of the global population age 15-64, have used illicit drugs at least once in the last 12 months. Cannabis is by far the most widely used drug, followed by stimulants, such as amphetamines and ecstasy, then cocaine use and then opioids. While most individuals occasionally use or have casually tried illicit drugs, UNODC estimates that there are between 18-38 million problem drug users. These individuals consume most of the drugs and likely fulfill the criteria for a diagnosis of drug abuse or dependence.

These medical co-occurring conditions are specifically prevalent in injection drug users (IDU). Estimates for IDU's are available for at least 130 countries with approximately 78% of the 13.2 million IDU's living in developing or transitional countries (Aceijas et al 2004). Forty-one countries have reported a high prevalence (>5%) of HIV infection in this high-risk population. Globally, IDU's now account for at least 10% of all new HIV infections which are estimated at 5 million per year (IHRDP, 2006). In chronic HIV infection, AIDS has been reported as the leading cause of death in IDUs (Chin, 2007). Epidemiological data of HIV infection show that generalized HIV epidemics can result from diffusion transmission of HIV from high risk groups, such as IDUs. Thus, it is important for countries and regions to undertake surveillance studies to identify current alcohol and drug use patterns and develop best practices for the treatment of individuals who use and abuse alcohol and illicit drugs.

Drug dependence is a chronic, relapsing neurophysiological disease resulting from the prolonged effects of drug(s) on the brain. The neurochemical abnormalities resulting from chronic use are the underlying cause of many of the observed physical and behavioral aspects of abuse and dependence. The brain abnormalities associated with addiction are wide ranging, complex, and long lasting (Chana et al 2006; Goodkin et al 1998; Langford et al 2003). They can involve abnormal brain signaling pathways, psychological conditioning or stress and social factors that result in cravings leading to a predisposition to relapse even months or years after drug(s) use cessation. Thus, substance abuse/dependence can be most effectively addressed in a multifaceted medical-based paradigm that comprises a comprehensive program of interventions that are delivered through the course of long term treatment. Such comprehensive treatment programs include behavioral, social rehabilitative components, as well as biological (pharmacological) components Table 2. Behavioral therapy interventions have been extensively researched and are critical components of the treatment of all drug addictions. Social rehabilitative components are also important and may prove suited to certain treatment environments.

In the United States, opioid abuse/dependence can be treated in two differing medical paradigms. In the highly regulated and structured environment, methadone is dispensed daily at Opioid Treatment Programs (OTPs). These OTPs are increasingly providing "wraparound" services to address important patient needs, enhance time in treatment, and promote recovery. Alternatively, buprenorphine can be prescribed in a primary care health care setting similar to other illnesses to reduce the stigma/discrimination of drug dependence. Both medical paradigms need to address the reduced quality of life, physical and mental functioning, compared to the general population that is associated with drug abuse/dependence (Millson et al. 2006). In addition, multiple comorbidities are associated with substance abuse and dependence that also contribute to the lower quality of life

Based on the 2010 World Drug Report (UNODC, 2010) from the United Nations Office on Drugs and Crime (UNODC), it is estimated that between 175- 250 million people from almost every country, or 5 percent of the global population age 15-64, have used illicit drugs at least once in the last 12 months. Cannabis is by far the most widely used drug, followed by stimulants, such as amphetamines and ecstasy, then cocaine use and then opioids. While most individuals occasionally use or have casually tried illicit drugs, UNODC estimates that there are between 18-38 million problem drug users. These individuals consume most of the

These medical co-occurring conditions are specifically prevalent in injection drug users (IDU). Estimates for IDU's are available for at least 130 countries with approximately 78% of the 13.2 million IDU's living in developing or transitional countries (Aceijas et al 2004). Forty-one countries have reported a high prevalence (>5%) of HIV infection in this high-risk population. Globally, IDU's now account for at least 10% of all new HIV infections which are estimated at 5 million per year (IHRDP, 2006). In chronic HIV infection, AIDS has been reported as the leading cause of death in IDUs (Chin, 2007). Epidemiological data of HIV infection show that generalized HIV epidemics can result from diffusion transmission of HIV from high risk groups, such as IDUs. Thus, it is important for countries and regions to undertake surveillance studies to identify current alcohol and drug use patterns and develop best practices for the treatment of individuals who use and abuse alcohol and illicit

Drug dependence is a chronic, relapsing neurophysiological disease resulting from the prolonged effects of drug(s) on the brain. The neurochemical abnormalities resulting from chronic use are the underlying cause of many of the observed physical and behavioral aspects of abuse and dependence. The brain abnormalities associated with addiction are wide ranging, complex, and long lasting (Chana et al 2006; Goodkin et al 1998; Langford et al 2003). They can involve abnormal brain signaling pathways, psychological conditioning or stress and social factors that result in cravings leading to a predisposition to relapse even months or years after drug(s) use cessation. Thus, substance abuse/dependence can be most effectively addressed in a multifaceted medical-based paradigm that comprises a comprehensive program of interventions that are delivered through the course of long term treatment. Such comprehensive treatment programs include behavioral, social rehabilitative components, as well as biological (pharmacological) components Table 2. Behavioral therapy interventions have been extensively researched and are critical components of the treatment of all drug addictions. Social rehabilitative components are also important and

In the United States, opioid abuse/dependence can be treated in two differing medical paradigms. In the highly regulated and structured environment, methadone is dispensed daily at Opioid Treatment Programs (OTPs). These OTPs are increasingly providing "wraparound" services to address important patient needs, enhance time in treatment, and promote recovery. Alternatively, buprenorphine can be prescribed in a primary care health care setting similar to other illnesses to reduce the stigma/discrimination of drug dependence. Both medical paradigms need to address the reduced quality of life, physical and mental functioning, compared to the general population that is associated with drug abuse/dependence (Millson et al. 2006). In addition, multiple comorbidities are associated with substance abuse and dependence that also contribute to the lower quality of life

may prove suited to certain treatment environments.

**2.2 Illicit opioid abuse, medication assisted treatment and HIV infection** 

drugs and likely fulfill the criteria for a diagnosis of drug abuse or dependence.

drugs.

Table 2. Elements of the Continuum of Care in the Treatment of Opioid Abuse/Dependence

experienced and documented by opioid dependent individuals. Life priorities of opioid users have been reported as concern about HIV and treatment of infection with HIV, housing, money, and protection from violence (Mizuno et al, 2003).

Substance abuse is a complex medical disorder composed of multiple physiologic, social and behavioral problems often interrelated with psychological illness. Health care providers need to screen substance misusing patients for psychological illness (Schuckit, 2006). Although it can be difficult to ascertain whether substance abuse, psychological illness, or infectious comorbidities should be addressed first, an initial focus on the medical treatment of drug abuse is often necessary to create sufficient patient stability from which other treatments can begin. Stability is further increased with both mental health services and substance abuse treatment, subsequently enhancing the medical outcomes of treatment for comorbidities.

Substance Abuse Treatment Utilizing Medication Assisted Treatment as HIV Prevention 535

treatment and treatment outcomes (Fiellin et al 2003). Programs responsive to the severity of drug abuse during initial stages of drug treatment have been shown to produce positive treatment outcomes based on greater retention time in treatment and patient satisfaction with treatment services. Maximum retention time in methadone treatment is associated with comprehensive treatment, provision of frequent health service, as well as appropriate

In the United States and globally, primary care physicians can expand the accessibility of substance abuse treatment while mitigating the stigma associated with drug use and treatment through an outpatient treatment setting in primary care and the use of buprenorphine. However, in the United States, buprenorphine-only OTPs have been recently developed where buprenorphine is provided to opioid dependent patents under the highly regulated rules and regulations that apply to methadone. Buprenorphine, a partial mu-receptor opiate agonist (Ling & Smith 2002), differs significantly from full agonists. Most significantly, buprenorphine has a plateau of its agonist properties at higher doses. This results in an improved safety profile compared with a full agonist. Specifically, buprenorphine has a favorable 'ceiling effect' on respiratory depression precluding overdose potential (Walsh et al 1994). However, the abuse of other substances that may enhance respiratory depression (e.g., benzodiazepines) remains a contraindication with buprenorphine as with methadone. Improved safety and thrice weekly flexible dosing promotes patient acceptance. In addition, buprenorphine has two features that decrease street diversion. Buprenorphine can precipitate opiate withdrawal when buprenorphine is taken by an opiate dependent patient (Schuh et al. 1996) and buprenorphine can be marketed both alone (Subutex®) and in combination with naloxone (Suboxone®). In the latter formulation, if it is crushed and injected, acute opiate withdrawal symptoms will ocuur which are a potent disincentive for prescription opioid abuse (Yokell et al. 2011).

Naltrexone is a non-narcotic long-acting, opioid antagonist that blocks the euphoric effects of opioids binding the mu opioid receptor. Unlike methadone, there is no negative reinforcement (opioid withdrawal) upon discontinuation. Due to naltrexone's opioid antagonism, patients must abstain from opioids for a minimum of seven days prior to starting treatment to avoid the precipitation of opioid withdrawal. The effectiveness of naltrexone treatment depends upon patient motivation and social support system (Greenstein et al 1983). Thus, in cultures where there is strong family or social support for the patient in care, oral naltrexone has been shown to be effective in the prevention of relapse to heroin use (Krupitsky et al 2010). Because of a lack of positive reinforcing effects with naltrexone and low motivation on the part of many patients, as well as, poor clinician acceptability, it is not widely prescribed for the treatment of

Vivitrol is an injectable extended-release formulation of naltrexone that has recently been approved for the treatment of opioid abuse and dependence. Vivitrol addresses the concern of medication adherence as a monthly injectable formulation and has been shown to be more effective than oral naltrexone (Krupitskya & Blokhina, 2010). This was also shown in a recent Phase 3 clinical trial that confirmed vivitrol's safety and efficacy in the prevention of relapse to heroin use in a cohort of injection drug users. A higher retention in care and higher rates of

methadone dosing (Litwin et al 2001).

**2.2.2 Medication assited treatment utilizing buprenorphine** 

**2.2.3 Medication assited treatment utilizing naltrexone** 

opioid dependence in the United States.

In the United States, multiple pharmacological treatments, including both agonists and antagonists have been developed and approved by the Food and Drug Administration for specific drug dependence. Currently, medications and evidence-based treatment paridigms utilizing these pharmacotherapies are available for the treatment of nicotine, alcohol, and opioid substance use disorders. Although none are available for stimulants, such as cocaine and methamphetamine, many potential medications are now being developed for these drugs of abuse and are expected to be available over the next few years. An effective treatment strategy for drug abuse and dependence is to match a comprehensive treatment plan to the individual's particular substance abuse problems and needs. Desired treatment outcomes should: a) reduce dependence on drugs of abuse, b) reduce morbidity and mortality of and associated with drugs of abuse, and c) maximize the patients' abilities to access services and achieve social integration.

#### **2.2.1 Medication assited treatment utilizing methadone**

In most countries that utilize MAT for the treatment of opioid dependence, methadone is the pharmacotherapy of choice. Methadone is usually the least expensive medication and when used in evidence-based treatment paridigms is cost effective and can result in abstenence from illicit drug use over time and the achievement of recovery (Connock et al 2007; Skinner et al 2011). Methadone is a synthetic -opioid receptor agonist with pharmacological properties qualitatively similar to morphine and was originally used to treat the painful symptoms of withdrawal from heroin and other opioids (Gowing et al 2006; Payte & Zweben , 1998). Administered daily as an oral dose for the treatment of opioid dependence, an individual therapeutic dosage is determined to maintain an asymptomatic state and stabilize a patient, without episodes of opioid overmedication or withdrawal. The therapeutic dosage for a patient is a function of many factors including: absorption, metabolism, drug-drug interactions, physiology, diet and the use of alternative medications. Minimum retention time in treatment varies for residential and outpatient methadone treatment programs. The National Institutes of Health consensus panel on opioid-addiction treatment (NIH, 1997) concluded that individuals treated for fewer than three months with methadone do not show substantial medical gain. As time in treatment progresses, study outcomes have reported partial reductions of illicit opioid use progressing to abstinence. Relapse to opioid use is common when methadone is discontinued without further support or behavioral treatment. In the United States, OTPs or methadone maintenance treatment programs, MMTP, under the certification of the Substance Abuse and Mental Health Services Administration (SAMHSA), dispense methadone and can provide a comprehensive therapeutic milieu comprised of primary medical care, psychosocial counseling, vocational rehabilitation, HIV testing and counseling, hepatitis C education and testing and other vital medical and social services. Methadone treatment is effective as both primary and secondary HIV prevention (Kerr et al 2004) and cost-effective to society (Barnett et al 2001; Doran et al 2003). In addition to improving health outcomes, methadone treatment also substantially improves the quality of life of patients over the course of methadone treatment (Giacomuzzi et al 2005).

Barriers to retention in methadone treatment include the severity of drug, medical and social problems at initiation of treatment, as well as patient readiness for treatment and motivation. Integrating multiple components of the drug treatment program is fundamental to successful treatment outcomes. Treatment programs that offer a broader array of "wraparound" services and a greater frequency of services have reported improved retention in treatment and treatment outcomes (Fiellin et al 2003). Programs responsive to the severity of drug abuse during initial stages of drug treatment have been shown to produce positive treatment outcomes based on greater retention time in treatment and patient satisfaction with treatment services. Maximum retention time in methadone treatment is associated with comprehensive treatment, provision of frequent health service, as well as appropriate methadone dosing (Litwin et al 2001).

#### **2.2.2 Medication assited treatment utilizing buprenorphine**

534 Recent Translational Research in HIV/AIDS

In the United States, multiple pharmacological treatments, including both agonists and antagonists have been developed and approved by the Food and Drug Administration for specific drug dependence. Currently, medications and evidence-based treatment paridigms utilizing these pharmacotherapies are available for the treatment of nicotine, alcohol, and opioid substance use disorders. Although none are available for stimulants, such as cocaine and methamphetamine, many potential medications are now being developed for these drugs of abuse and are expected to be available over the next few years. An effective treatment strategy for drug abuse and dependence is to match a comprehensive treatment plan to the individual's particular substance abuse problems and needs. Desired treatment outcomes should: a) reduce dependence on drugs of abuse, b) reduce morbidity and mortality of and associated with drugs of abuse, and c) maximize the patients' abilities to

In most countries that utilize MAT for the treatment of opioid dependence, methadone is the pharmacotherapy of choice. Methadone is usually the least expensive medication and when used in evidence-based treatment paridigms is cost effective and can result in abstenence from illicit drug use over time and the achievement of recovery (Connock et al 2007; Skinner et al 2011). Methadone is a synthetic -opioid receptor agonist with pharmacological properties qualitatively similar to morphine and was originally used to treat the painful symptoms of withdrawal from heroin and other opioids (Gowing et al 2006; Payte & Zweben , 1998). Administered daily as an oral dose for the treatment of opioid dependence, an individual therapeutic dosage is determined to maintain an asymptomatic state and stabilize a patient, without episodes of opioid overmedication or withdrawal. The therapeutic dosage for a patient is a function of many factors including: absorption, metabolism, drug-drug interactions, physiology, diet and the use of alternative medications. Minimum retention time in treatment varies for residential and outpatient methadone treatment programs. The National Institutes of Health consensus panel on opioid-addiction treatment (NIH, 1997) concluded that individuals treated for fewer than three months with methadone do not show substantial medical gain. As time in treatment progresses, study outcomes have reported partial reductions of illicit opioid use progressing to abstinence. Relapse to opioid use is common when methadone is discontinued without further support or behavioral treatment. In the United States, OTPs or methadone maintenance treatment programs, MMTP, under the certification of the Substance Abuse and Mental Health Services Administration (SAMHSA), dispense methadone and can provide a comprehensive therapeutic milieu comprised of primary medical care, psychosocial counseling, vocational rehabilitation, HIV testing and counseling, hepatitis C education and testing and other vital medical and social services. Methadone treatment is effective as both primary and secondary HIV prevention (Kerr et al 2004) and cost-effective to society (Barnett et al 2001; Doran et al 2003). In addition to improving health outcomes, methadone treatment also substantially improves the quality of life of patients over the course of methadone treatment

Barriers to retention in methadone treatment include the severity of drug, medical and social problems at initiation of treatment, as well as patient readiness for treatment and motivation. Integrating multiple components of the drug treatment program is fundamental to successful treatment outcomes. Treatment programs that offer a broader array of "wraparound" services and a greater frequency of services have reported improved retention in

access services and achieve social integration.

(Giacomuzzi et al 2005).

**2.2.1 Medication assited treatment utilizing methadone** 

In the United States and globally, primary care physicians can expand the accessibility of substance abuse treatment while mitigating the stigma associated with drug use and treatment through an outpatient treatment setting in primary care and the use of buprenorphine. However, in the United States, buprenorphine-only OTPs have been recently developed where buprenorphine is provided to opioid dependent patents under the highly regulated rules and regulations that apply to methadone. Buprenorphine, a partial mu-receptor opiate agonist (Ling & Smith 2002), differs significantly from full agonists. Most significantly, buprenorphine has a plateau of its agonist properties at higher doses. This results in an improved safety profile compared with a full agonist. Specifically, buprenorphine has a favorable 'ceiling effect' on respiratory depression precluding overdose potential (Walsh et al 1994). However, the abuse of other substances that may enhance respiratory depression (e.g., benzodiazepines) remains a contraindication with buprenorphine as with methadone. Improved safety and thrice weekly flexible dosing promotes patient acceptance. In addition, buprenorphine has two features that decrease street diversion. Buprenorphine can precipitate opiate withdrawal when buprenorphine is taken by an opiate dependent patient (Schuh et al. 1996) and buprenorphine can be marketed both alone (Subutex®) and in combination with naloxone (Suboxone®). In the latter formulation, if it is crushed and injected, acute opiate withdrawal symptoms will ocuur which are a potent disincentive for prescription opioid abuse (Yokell et al. 2011).

#### **2.2.3 Medication assited treatment utilizing naltrexone**

Naltrexone is a non-narcotic long-acting, opioid antagonist that blocks the euphoric effects of opioids binding the mu opioid receptor. Unlike methadone, there is no negative reinforcement (opioid withdrawal) upon discontinuation. Due to naltrexone's opioid antagonism, patients must abstain from opioids for a minimum of seven days prior to starting treatment to avoid the precipitation of opioid withdrawal. The effectiveness of naltrexone treatment depends upon patient motivation and social support system (Greenstein et al 1983). Thus, in cultures where there is strong family or social support for the patient in care, oral naltrexone has been shown to be effective in the prevention of relapse to heroin use (Krupitsky et al 2010). Because of a lack of positive reinforcing effects with naltrexone and low motivation on the part of many patients, as well as, poor clinician acceptability, it is not widely prescribed for the treatment of opioid dependence in the United States.

Vivitrol is an injectable extended-release formulation of naltrexone that has recently been approved for the treatment of opioid abuse and dependence. Vivitrol addresses the concern of medication adherence as a monthly injectable formulation and has been shown to be more effective than oral naltrexone (Krupitskya & Blokhina, 2010). This was also shown in a recent Phase 3 clinical trial that confirmed vivitrol's safety and efficacy in the prevention of relapse to heroin use in a cohort of injection drug users. A higher retention in care and higher rates of

Substance Abuse Treatment Utilizing Medication Assisted Treatment as HIV Prevention 537

recovery from opioid abuse and dependence and social reintegration back into society. The individual in recovery is a functioning member of the community and contributes to the social fiber and health of the community. Thus, a foundation of MAT is the obtainment of

The recovery process is the individual way in which a person actively manages their substance use disorder with efforts to reclaim full functional and meaningful lives in the community. Recovery is personal process of growth and change which embraces hope, autonomy and the elements that result in establishing a satisfying and productive life. MAT is a recovery oriented system of care when integrated with other medical, social and rehabilitative services in support of the individual's and family's long term efforts to reclaim full and meaningful lives in the community. Important in recovery is the provision of comprehensive services in the context of MAT but also a supportive, enabling environment that fosters individual responsibility over one's health and empowerment to change to a

MAT, as a recovery orientated system of care, has four phases as shown in Table 4, along with a set of recovery oriented goals, strategies, and services (White & Mojer-Torres, 2010). An important consideration in phase four, long term sustained recovery, is the personal decision to continue with medical maintenance of pharmacotherapy or to taper the medication. In either case, the home or living environment is critical to the prevention of relapse to opioid use. To prevent relapse of opioid use the individual in recovery needs a drug free environment. While significant gains have been made through national prevention programs such as "Drug Free Communities", it remains a Herculean task to keep a community entirely free of illicit drug use. Thus, for long term recovery the home or living environment is where recovery is nucleated (Ashcraft et al, 2008). Local peer recovery programs as well as recovery oriented systems of care that link to or provide individualized, quality long term supportive care are critical (Jason & Ferrari, 2010). These settings provide a network of people to support abstinence as well as a low risk environment to support recovery. Receiving abstinence support, guidance and information from a recovery home, that is committed to long term sobriety, reduces the risk of relapse to illicit opioid use. These homes need to be considered as a fundamental component in the development and

Health service programs deliver MAT in a regulatory environment where both the federal government and state/local government provide a regulatory framework for the access to and delivery of medications that are controlled by international convention (Kresina et al, 2009). In the United States, state and local regulation can enhance the federal regulations but they can not negate the federal regulations. The MAT federal regulations can be found in the Code of Federal Regulations (CFR, 2002) and establish procedures to determine if a health practitioner is qualified to dispense methadone in the treatment of opioid abuse and dependence in opioid treatment programs, as well as, the quantity of methadone that can be provided for unsupervised use by patients. Thus, the federal regulations address the balance needed in the use of controlled medications for treatment versus the restrictions to limit

The MAT federal regulations do not regulate the health service models that can be use to maximize access to MAT as well as time in treatment. These are two important

Recovery from opioid abuse and dependence (Davidson & White , 2007).

healthy lifestyle (Sowers, 2005).

maintenance of the public health of communities.

**4. Service models for medication assisted treatment** 

diversion of the controlled medication (Yokell et al 2011).

opioid-free urine screens were observed along with a significant reduction in opioid craving compared to placebo. Currently, studies are underway to determine the most efficacious service model(s) for the use of vivitrol in the treatment of relapse prevention to heroin use.

#### **3. Medication assisted treatment: Stages of treatment and recovery**

The stages or phase of MAT are shown in Table 3. The patient travels through these three stages of treatment, sometimes linearly and sometimes with oscillations between phases. The ultimate goal upon entering MAT is a good clinical outcome which includes the

	- -
		-
		-
	-
	-
	-
	-
	-
	-
	-

Table 3. Stages or Phases of MAT

opioid-free urine screens were observed along with a significant reduction in opioid craving compared to placebo. Currently, studies are underway to determine the most efficacious service model(s) for the use of vivitrol in the treatment of relapse prevention to heroin use.

The stages or phase of MAT are shown in Table 3. The patient travels through these three stages of treatment, sometimes linearly and sometimes with oscillations between phases. The ultimate goal upon entering MAT is a good clinical outcome which includes the

**3. Medication assisted treatment: Stages of treatment and recovery** 

Table 3. Stages or Phases of MAT

recovery from opioid abuse and dependence and social reintegration back into society. The individual in recovery is a functioning member of the community and contributes to the social fiber and health of the community. Thus, a foundation of MAT is the obtainment of Recovery from opioid abuse and dependence (Davidson & White , 2007).

The recovery process is the individual way in which a person actively manages their substance use disorder with efforts to reclaim full functional and meaningful lives in the community. Recovery is personal process of growth and change which embraces hope, autonomy and the elements that result in establishing a satisfying and productive life. MAT is a recovery oriented system of care when integrated with other medical, social and rehabilitative services in support of the individual's and family's long term efforts to reclaim full and meaningful lives in the community. Important in recovery is the provision of comprehensive services in the context of MAT but also a supportive, enabling environment that fosters individual responsibility over one's health and empowerment to change to a healthy lifestyle (Sowers, 2005).

MAT, as a recovery orientated system of care, has four phases as shown in Table 4, along with a set of recovery oriented goals, strategies, and services (White & Mojer-Torres, 2010). An important consideration in phase four, long term sustained recovery, is the personal decision to continue with medical maintenance of pharmacotherapy or to taper the medication. In either case, the home or living environment is critical to the prevention of relapse to opioid use. To prevent relapse of opioid use the individual in recovery needs a drug free environment. While significant gains have been made through national prevention programs such as "Drug Free Communities", it remains a Herculean task to keep a community entirely free of illicit drug use. Thus, for long term recovery the home or living environment is where recovery is nucleated (Ashcraft et al, 2008). Local peer recovery programs as well as recovery oriented systems of care that link to or provide individualized, quality long term supportive care are critical (Jason & Ferrari, 2010). These settings provide a network of people to support abstinence as well as a low risk environment to support recovery. Receiving abstinence support, guidance and information from a recovery home, that is committed to long term sobriety, reduces the risk of relapse to illicit opioid use. These homes need to be considered as a fundamental component in the development and maintenance of the public health of communities.

#### **4. Service models for medication assisted treatment**

Health service programs deliver MAT in a regulatory environment where both the federal government and state/local government provide a regulatory framework for the access to and delivery of medications that are controlled by international convention (Kresina et al, 2009). In the United States, state and local regulation can enhance the federal regulations but they can not negate the federal regulations. The MAT federal regulations can be found in the Code of Federal Regulations (CFR, 2002) and establish procedures to determine if a health practitioner is qualified to dispense methadone in the treatment of opioid abuse and dependence in opioid treatment programs, as well as, the quantity of methadone that can be provided for unsupervised use by patients. Thus, the federal regulations address the balance needed in the use of controlled medications for treatment versus the restrictions to limit diversion of the controlled medication (Yokell et al 2011).

The MAT federal regulations do not regulate the health service models that can be use to maximize access to MAT as well as time in treatment. These are two important

Substance Abuse Treatment Utilizing Medication Assisted Treatment as HIV Prevention 539

characteristics to maximize as one designs model MAT programs to ensure good clinical and public health outcomes. Barrier free access to MAT is important for obtaining maximal public health impact and reaching all opioid abusing individuals seeking treatment. Research studies have shown that the more time in MAT the better the treatment outcome. Thus, MAT programs providing comprehensive services as part of the continuum of care (see Table 2) in an enabling environment result in quality and effective substance abuse treatment services that promote individual well-being and improved community health.

Health service models for MAT that provide comprehensive services interface substance abuse treatment services with primary medical care and social/rehabilitation services. That interface can be comprehensive through the integration of substance abuse treatment services, primary medical care, infectious disease prevention, care and treatment and social/rehabilitation services. An integrated care and treatment model, where MAT services are provided within primary care using a single medical record, minimizes the sigma and discrimination associated with drug treatment services while improving overall health

For OTPs dispensing methadone, primary medical care and social/rehabilitation services are integrated on-site in the structured environment where methadone is dispensed (Freidman et al 1999; Kresina et al 2008). Based on patient needs, various types of health services can be integrated into OTP MAT services including primary care, mental health, and infectious diseases. Specific limiting factors for the integration of services have been

Buprenorphine, a less regulated opioid agonist medication, is approved in the United States for office based opioid treatment. An office based setting provides enhanced treatment access to MAT using buprenorphine in a less stigmatized environment enabling integrated medical care of infectious diseases and co-morbid conditions (Gunderson & Fiellin , 2008). Multiple models have been piloted for the integration of MAT using buprenorphine within HIV primary care (Sullivan et al 2006). These include an on-site combination of addiction treatment/HIV specialist treatment; a HIV primary care physician prescribing buprenorphine; a non-physician health care provider integrating medical care and substance abuse treatment services using buprenorphine; and a community outreach model where buprenorphine is provided along with medical services in a mobile van. These pilot projects have uncovered barriers to integrating MAT using buprenorphine within HIV primary care that are both financial and regulatory. Regulatory challenges include licensing and training restrictions imposed by the Drug Addiction Treatment Act of 2000 and confidentiality regulations for alcohol and drug treatment records (Schackman et al 2006). A recent study has shown that in a primary care setting that used buprenorphine, prescription opioid dependent patients showed better clinical outcomes compared to patients who were

Naltrexone is a non-narcotic and therefore non-controlled medication for the treatment of opioid abuse as well as alcohol abuse. Naltrexone integrated with mental health services, particularly psychosocial treatment has been shown to be an effective maintenance treatment for reducing heroin use after detoxification (Minozzi et al. 2006). In addition, using clonidine and naltrexone together has been shown to be successfully integrated into a primary care setting (O'Connor et al. 1997). In this study retention in care and successful

**4.1 Integrated models of medication assisted treatment** 

outcomes in a cost-effective manner (Collins et al 2010).

shown to be the organizational structure of the OTP and cost.

dependent on heroin (Moore et al, 2007).

		-
		-
		-
		-
		-
		-
		-
		-
		-
		-
		-
		-
		-
		-
		-
		-

characteristics to maximize as one designs model MAT programs to ensure good clinical and public health outcomes. Barrier free access to MAT is important for obtaining maximal public health impact and reaching all opioid abusing individuals seeking treatment. Research studies have shown that the more time in MAT the better the treatment outcome. Thus, MAT programs providing comprehensive services as part of the continuum of care (see Table 2) in an enabling environment result in quality and effective substance abuse treatment services that promote individual well-being and improved community health.

#### **4.1 Integrated models of medication assisted treatment**

538 Recent Translational Research in HIV/AIDS

Table 4. Phases and Goals of MAT Recovery Oriented Systems of Care

Health service models for MAT that provide comprehensive services interface substance abuse treatment services with primary medical care and social/rehabilitation services. That interface can be comprehensive through the integration of substance abuse treatment services, primary medical care, infectious disease prevention, care and treatment and social/rehabilitation services. An integrated care and treatment model, where MAT services are provided within primary care using a single medical record, minimizes the sigma and discrimination associated with drug treatment services while improving overall health outcomes in a cost-effective manner (Collins et al 2010).

For OTPs dispensing methadone, primary medical care and social/rehabilitation services are integrated on-site in the structured environment where methadone is dispensed (Freidman et al 1999; Kresina et al 2008). Based on patient needs, various types of health services can be integrated into OTP MAT services including primary care, mental health, and infectious diseases. Specific limiting factors for the integration of services have been shown to be the organizational structure of the OTP and cost.

Buprenorphine, a less regulated opioid agonist medication, is approved in the United States for office based opioid treatment. An office based setting provides enhanced treatment access to MAT using buprenorphine in a less stigmatized environment enabling integrated medical care of infectious diseases and co-morbid conditions (Gunderson & Fiellin , 2008). Multiple models have been piloted for the integration of MAT using buprenorphine within HIV primary care (Sullivan et al 2006). These include an on-site combination of addiction treatment/HIV specialist treatment; a HIV primary care physician prescribing buprenorphine; a non-physician health care provider integrating medical care and substance abuse treatment services using buprenorphine; and a community outreach model where buprenorphine is provided along with medical services in a mobile van. These pilot projects have uncovered barriers to integrating MAT using buprenorphine within HIV primary care that are both financial and regulatory. Regulatory challenges include licensing and training restrictions imposed by the Drug Addiction Treatment Act of 2000 and confidentiality regulations for alcohol and drug treatment records (Schackman et al 2006). A recent study has shown that in a primary care setting that used buprenorphine, prescription opioid dependent patients showed better clinical outcomes compared to patients who were dependent on heroin (Moore et al, 2007).

Naltrexone is a non-narcotic and therefore non-controlled medication for the treatment of opioid abuse as well as alcohol abuse. Naltrexone integrated with mental health services, particularly psychosocial treatment has been shown to be an effective maintenance treatment for reducing heroin use after detoxification (Minozzi et al. 2006). In addition, using clonidine and naltrexone together has been shown to be successfully integrated into a primary care setting (O'Connor et al. 1997). In this study retention in care and successful

Substance Abuse Treatment Utilizing Medication Assisted Treatment as HIV Prevention 541

Coordinated MAT for patients seeking relapse prevention interventions after detoxification from opioid use can be provided by naltrexone or the recently approved vivitrol. As noted earlier, naltrexone it is not widely prescribed for the treatment of opioid dependence in the United States, but is provided as an office based treatment for opioid dependence after detoxification. In addition, studies have shown that the extended release formulations are effective in reducing opioid use and retaining patients in care after detoxification (Comer et al 2006; Kunoe et al. 2010). Fishman et al 2010 has shown good clinical outcomes (retention in care and reduced opioid use) for adolescents receiving vivitrol over a four month period. This study is important because of the limted use of controlled pharmacotherapies in

**5. Preventing HIV infection by integration of medication assisted treatment** 

Important HIV prevention interventions for people who inject drugs are the provision of clean needles and syringe through syringe service programs and associated HIV testing and counseling programs. These HIV prevention interventions, when integrated into MAT programs, maximize the enrollment in treatment programs for opioid and alcohol abuse, and thereby maximize HIV prevention efforts (Kidorf et al 2009; Lloyd et all 2005). Maximizing HIV prevention efforts targeting peple who use drugs and those dependent on opioids and alcohol are critcial to prevent HIV infection in these most-at –risk populations. Integrating drug abuse treatment and early HIV prevention interventions, particulaly HIV testing and counseling, are important as components of the newly emerging Seek,Test, Treat and Retain" strategy ( Crawford & Vlahov, 2010; Taege, 2011). This is an engagement and retention strategy that outreach workers can employ with injection drug users to reduce their risk for HIV infection. By utilizing outreach workers to seek out most-at-risk people who inject drugs, establish their HIV status through HIV testing, followed by sexual risk reduction councelling, HIV risk behaviors can be addressed with subsequent emphasis on

Unfortuately, there is not signifcant integration of HIV testing and counseling in OTPs. In the US, while approaximely 90% of opioid treatment programs provide some form of federally mandated HIV/AIDS education, only 74% of opiod treatment progams offered HIV testing (Kresina et al 2005). These services appear underutilized in that approximately one-in-three persons receiveving subtance abuse treatment also received HIV testing and counselling (Pollack & D'Aunno, 2010). Globally, although subtantial efforts are being made to increase the availability of HIV testing, most-at-risk populations remain underserved with regard to HIV prevention service utilization. It is estimated that only 10% of persons at-risk for HIV infection receive HIV testing. Thus, strategies such as opt-out testing, home-based testing, dor-to-door testing as well as providing dedicated HIV testings counselors at point-of-service locations are being utilized to enhance the uptake of HIV testing for people who use alcohol and inject drugs. Studies have shown that most-at-risk populations prefer point-of-service HIV testing, however, this intervention requires additional measures to support HIV positive individuals

**6. Preventing HIV transmission by integration of medication assisted** 

A significant factor in not reducing the global HIV epidemic is the lack of entrance into HIV care and treatment by most-at-risk populations. These populations, which include illicit

adolescent populations as part of national regulatory frameworks.

**into HIV prevention services** 

treatment for their substance use disorder.

entering into HIV care and treatment (Keller et al 2011).

**treatment into HIV care and treatment** 

detoxification from opioid abuse was observed with MAT using either naltrexone or buprenorphine. In other care settings, treatment of alcohol use disorders using naltrexone has been successfully integrated into the treatment of patients who have tuberculosis (Greenfield et al. 2010). Current efforts are determining the optimum conditions to integrate vivitrol (extended release naltrexone) into HIV primary care programs. Additionally, how to integrate vivitrol into an OTP setting and in primary medical care as a relapse prevention intervention for patients following their completion of maintenance treatment with either methadone or buprenorphine, is currently moving forward.

#### **4.2 Coordinated care models of medication assisted treament**

Health service models for MAT that provide comprehensive services can connect substance abuse treatment services with primary medical care and social/rehabilitation services in a non-integrated but coordinated fashion. Here, MAT services coordinate with primary medical care and social/rehabilitation services to promote good patient outcomes and enhance community health. MAT, health services and social/rehabilitation services can be separately managed with a different network of health care providers but colocated to allow convenient utilization of primary care, MAT and other services. An additional coordinated approach provides primary care, MAT and other services at distinct locations through a differing network of health care providers. As shown in a recent study where twice as many patients retained in MAT when the MAT services were provided at single location compared to referral of MAT to a distant location (Lucas et al 2010), providing needed health services at distinct locations is less than optimal. However, coordinated programs can be effective when case managers, peer facilitators, care navigators or others promote or support service utilization at the various locations. For example, a referral system intervention was modeled with linkages to treatment services for substance use, mental health and social services for HIV+ patients receiving HIV primary care (Zaller et al. 2007). Patients receiving the intervention were referred to MAT either at an OTP or in an office based setting that prescribed buprenorphine. An alternative model provided highly stable OTP patients with 28 days of methadone doses and required monthly check-ins. Successful patients were noted to have increased family and social activities and failed patients were provided stepped treatment intensification (King et al. 2006). Community –wide health service delivery programs also provide an alternative to integration through enhanced access to networked drug treatment and comorbidity health services (Neufeld et al 2010).

Unique to buprenorphine is the model that a substance abuse treatment specialist provides the initial treatment (induction) with buprenorphine until the patient is stabilized. Then the patient is transferred/referred to a primary care physician who can then provide maintenance buprenorphine treatment and medical primary care. This so called 'wheel and spoke model' allows for substance abuse treatment specialists to manage the more difficult portion of buprenorphine treatment (early treatment –or induction phase) while the primary care medical program manages the long term maintenance phase of buprenorphine treatment (BBI, 2008). This model is important in the United States since the Drug Addiction Treatment Act of 2000 limits the number of patients a qualified buprenorphine treatment provider can manage in their practice (DATA, 2000). This model has been adapted to HIV+ patients where the buprenorphine induction is performed by the substance abuse treatment specialist and then the patient is transferred/ referred to the HIV primary care physician (Basu et al. 2006).

detoxification from opioid abuse was observed with MAT using either naltrexone or buprenorphine. In other care settings, treatment of alcohol use disorders using naltrexone has been successfully integrated into the treatment of patients who have tuberculosis (Greenfield et al. 2010). Current efforts are determining the optimum conditions to integrate vivitrol (extended release naltrexone) into HIV primary care programs. Additionally, how to integrate vivitrol into an OTP setting and in primary medical care as a relapse prevention intervention for patients following their completion of maintenance treatment with either

Health service models for MAT that provide comprehensive services can connect substance abuse treatment services with primary medical care and social/rehabilitation services in a non-integrated but coordinated fashion. Here, MAT services coordinate with primary medical care and social/rehabilitation services to promote good patient outcomes and enhance community health. MAT, health services and social/rehabilitation services can be separately managed with a different network of health care providers but colocated to allow convenient utilization of primary care, MAT and other services. An additional coordinated approach provides primary care, MAT and other services at distinct locations through a differing network of health care providers. As shown in a recent study where twice as many patients retained in MAT when the MAT services were provided at single location compared to referral of MAT to a distant location (Lucas et al 2010), providing needed health services at distinct locations is less than optimal. However, coordinated programs can be effective when case managers, peer facilitators, care navigators or others promote or support service utilization at the various locations. For example, a referral system intervention was modeled with linkages to treatment services for substance use, mental health and social services for HIV+ patients receiving HIV primary care (Zaller et al. 2007). Patients receiving the intervention were referred to MAT either at an OTP or in an office based setting that prescribed buprenorphine. An alternative model provided highly stable OTP patients with 28 days of methadone doses and required monthly check-ins. Successful patients were noted to have increased family and social activities and failed patients were provided stepped treatment intensification (King et al. 2006). Community –wide health service delivery programs also provide an alternative to integration through enhanced access to networked drug treatment and co-

Unique to buprenorphine is the model that a substance abuse treatment specialist provides the initial treatment (induction) with buprenorphine until the patient is stabilized. Then the patient is transferred/referred to a primary care physician who can then provide maintenance buprenorphine treatment and medical primary care. This so called 'wheel and spoke model' allows for substance abuse treatment specialists to manage the more difficult portion of buprenorphine treatment (early treatment –or induction phase) while the primary care medical program manages the long term maintenance phase of buprenorphine treatment (BBI, 2008). This model is important in the United States since the Drug Addiction Treatment Act of 2000 limits the number of patients a qualified buprenorphine treatment provider can manage in their practice (DATA, 2000). This model has been adapted to HIV+ patients where the buprenorphine induction is performed by the substance abuse treatment specialist and then

the patient is transferred/ referred to the HIV primary care physician (Basu et al. 2006).

methadone or buprenorphine, is currently moving forward.

morbidity health services (Neufeld et al 2010).

**4.2 Coordinated care models of medication assisted treament** 

Coordinated MAT for patients seeking relapse prevention interventions after detoxification from opioid use can be provided by naltrexone or the recently approved vivitrol. As noted earlier, naltrexone it is not widely prescribed for the treatment of opioid dependence in the United States, but is provided as an office based treatment for opioid dependence after detoxification. In addition, studies have shown that the extended release formulations are effective in reducing opioid use and retaining patients in care after detoxification (Comer et al 2006; Kunoe et al. 2010). Fishman et al 2010 has shown good clinical outcomes (retention in care and reduced opioid use) for adolescents receiving vivitrol over a four month period. This study is important because of the limted use of controlled pharmacotherapies in adolescent populations as part of national regulatory frameworks.

#### **5. Preventing HIV infection by integration of medication assisted treatment into HIV prevention services**

Important HIV prevention interventions for people who inject drugs are the provision of clean needles and syringe through syringe service programs and associated HIV testing and counseling programs. These HIV prevention interventions, when integrated into MAT programs, maximize the enrollment in treatment programs for opioid and alcohol abuse, and thereby maximize HIV prevention efforts (Kidorf et al 2009; Lloyd et all 2005). Maximizing HIV prevention efforts targeting peple who use drugs and those dependent on opioids and alcohol are critcial to prevent HIV infection in these most-at –risk populations. Integrating drug abuse treatment and early HIV prevention interventions, particulaly HIV testing and counseling, are important as components of the newly emerging Seek,Test, Treat and Retain" strategy ( Crawford & Vlahov, 2010; Taege, 2011). This is an engagement and retention strategy that outreach workers can employ with injection drug users to reduce their risk for HIV infection. By utilizing outreach workers to seek out most-at-risk people who inject drugs, establish their HIV status through HIV testing, followed by sexual risk reduction councelling, HIV risk behaviors can be addressed with subsequent emphasis on treatment for their substance use disorder.

Unfortuately, there is not signifcant integration of HIV testing and counseling in OTPs. In the US, while approaximely 90% of opioid treatment programs provide some form of federally mandated HIV/AIDS education, only 74% of opiod treatment progams offered HIV testing (Kresina et al 2005). These services appear underutilized in that approximately one-in-three persons receiveving subtance abuse treatment also received HIV testing and counselling (Pollack & D'Aunno, 2010). Globally, although subtantial efforts are being made to increase the availability of HIV testing, most-at-risk populations remain underserved with regard to HIV prevention service utilization. It is estimated that only 10% of persons at-risk for HIV infection receive HIV testing. Thus, strategies such as opt-out testing, home-based testing, dor-to-door testing as well as providing dedicated HIV testings counselors at point-of-service locations are being utilized to enhance the uptake of HIV testing for people who use alcohol and inject drugs. Studies have shown that most-at-risk populations prefer point-of-service HIV testing, however, this intervention requires additional measures to support HIV positive individuals entering into HIV care and treatment (Keller et al 2011).

#### **6. Preventing HIV transmission by integration of medication assisted treatment into HIV care and treatment**

A significant factor in not reducing the global HIV epidemic is the lack of entrance into HIV care and treatment by most-at-risk populations. These populations, which include illicit

Substance Abuse Treatment Utilizing Medication Assisted Treatment as HIV Prevention 543

relationships. Both community outreach and peer –to-peer services can promote full service utilization. The national Ministries of Health need to embrace and support these health services and interventions through a supportive legal and policy framework validating their

place in the public health area and in society as they improve community health.

Table 5. Listing of Internationally Accepted Essential Interventions for HIV

incident infections in opioid and alcohol abusing populations.

vol 18 no 17 pp 2295-2303.

vol 28 no 5 pp 13-15.

pp1267-78.

5 pp 716-721

Substance abuse treatment is HIV prevention. The use of medication assisted treatment as a component of a comprehensive treatment plan for those individuals who abuse opioids and/or alcohol is an effective, evidence-based treatment paradigm that results in good medical outcomes including a reduction in HIV transmission as well as a reduction of

Aceijas, C., Stimson, G.V., Hickman, M., & Rhodes, T. (2004) Global overview of injecting

Altice, FL., Kamarulzaman, A., Soriano, V., Schechter, M., & Friedland, GH. (2010)

Barnett, P.G., Zaric, G.S.& Brandeau, M..L.(2001) The cost-effectiveness of buprenorphine

Basu, S., Smith-Rohrberg, D., Bruce, R.D.& Altice, F.L. (2006) Models for integrating

Baum, MK., Rafie, C., Lai, S., Sales, S., Page, JB., & Campa, A. (2010) Alcohol use accelerates HIV disease progression. *AIDS Res Hum Retroviruses* vol 26 no 5 pp 511-518.

infected with HIV who use drugs. *The Lancet* vol 376 no 9738 pp367-387. Ashcraft, L., Anthony, W.A,&, Martin C. (2008) Home is where recovery begins. *Behav Health*

drug use and HIV infection among injection drug users. *Acquir Immun Defic Syndr* 

Treatment of medical, psychiatric and substance use comorbidities in people

maintenance therapy for opiate addiction in the United States. *Addiction*. vol 96 no 9

buprenorphine therapy into the primary HIV care setting. Clin Infect Dis vol 42 no

**8. Conclusion** 

**9. References** 

drug users and alcohol abusers, encounter numerous barriers in accessing HIV care and treatment. In addition, once in treatment these individuals often suffer stigma and discrimination as they receive their needed medical care. The result is an increase in the prevalence of medical and psychiatric co-morbidities as well as social issues and high risk behaviors, in addition to worse clinical outcomes with a higher mortality rate compared to the non-drug and non-alcohol using populations infected with HIV (Altice et al 2010).

The increased mortally rate noted in people who inject drugs is related to their late presentation for HIV care. Patients who present late for care and treatment of HIV/AIDS are at a higher risk of significant clinical complications and are thus more difficult to clinically manage. Late presentation for treatment of HIV/AIDS is a common scenario leading to death (Moreno et al 2010). A recent study has documented a highly lethal neurological syndrome found in HIV-infected drug abusers (Newsome et al 2011). Although rare, the newly described syndrome is highly lethal with a mean survival time of 21 days after diagnosis. The authors suggest that access and initiation of antiretroviral therapy may provide a better outcome for these patients. In addition, substance abuse treatment, particularly MAT, which has been shown to enhance the health status, reduce mortality and quality of life of injection drug users, would be an important adjunct to anti-retroviral treatment for these patients. Thus, as noted earlier integrating both MAT with anti-retroviral treatment in a HIV primary care setting is a paradigm to optimize health outcomes and the health status of HIV-infected injection drug users.

How MAT is integrated in HIV primary care programs depends on the country's regulatory framework. In the United States, all medications accept methadone, can be prescribes to patients in a HIV primary care or outpatient HIV clinical care setting. The federal regulations in the United States require methadone to be dispenses in OTPs. However, in this setting studies have shown that HIV care and anti-retroviral treatment can be effectively prescribed either as directly observed therapy or as routine care. Other countries, such as Australia, have less stringent federal regulations for prescribing controlled medications and all medications comprising MAT can be provided in a primary care setting. In either case, the important aspect of providing integrated MAT and HIV primary care is the single location/clinic. In that case, the patient can receive all the needed services to support their recovery from drug/alcohol dependence as well as care and treatment for HIV infection.

#### **7. Essential health interventions for the prevention of HIV infection in people who inject opioids**

The WHO, UNODC and UNAIDS has approved and advocates for a package of essential interventions for the prevention, treatment and care of HIV for people who inject drugs (WHO, 2009a). These evidence based intervention, shown in Table 6, need three important characteristics in their implementation to maximize effectiveness. These interventions need to be part of a public health policy that is human rights based, gender responsive, and community owned.

As noted earlier, no single intervention alone will prevent or reverse to growing national HIV epidemics due to injection drug use and abuse. However, the greatest impact will be obtained when the interventions are provided through an integrated services platform in a comprehensive fashion. And in order to reach all of those seeking HIV prevention, care and treatment services, health service platforms need to provide an enabling environment that establishes confidentiality. In addition, they also need to develop patient –provider trusting relationships. Both community outreach and peer –to-peer services can promote full service utilization. The national Ministries of Health need to embrace and support these health services and interventions through a supportive legal and policy framework validating their place in the public health area and in society as they improve community health.

542 Recent Translational Research in HIV/AIDS

drug users and alcohol abusers, encounter numerous barriers in accessing HIV care and treatment. In addition, once in treatment these individuals often suffer stigma and discrimination as they receive their needed medical care. The result is an increase in the prevalence of medical and psychiatric co-morbidities as well as social issues and high risk behaviors, in addition to worse clinical outcomes with a higher mortality rate compared to the non-drug and non-alcohol using populations infected with HIV (Altice et al 2010). The increased mortally rate noted in people who inject drugs is related to their late presentation for HIV care. Patients who present late for care and treatment of HIV/AIDS are at a higher risk of significant clinical complications and are thus more difficult to clinically manage. Late presentation for treatment of HIV/AIDS is a common scenario leading to death (Moreno et al 2010). A recent study has documented a highly lethal neurological syndrome found in HIV-infected drug abusers (Newsome et al 2011). Although rare, the newly described syndrome is highly lethal with a mean survival time of 21 days after diagnosis. The authors suggest that access and initiation of antiretroviral therapy may provide a better outcome for these patients. In addition, substance abuse treatment, particularly MAT, which has been shown to enhance the health status, reduce mortality and quality of life of injection drug users, would be an important adjunct to anti-retroviral treatment for these patients. Thus, as noted earlier integrating both MAT with anti-retroviral treatment in a HIV primary care setting is a paradigm to optimize health outcomes and the

How MAT is integrated in HIV primary care programs depends on the country's regulatory framework. In the United States, all medications accept methadone, can be prescribes to patients in a HIV primary care or outpatient HIV clinical care setting. The federal regulations in the United States require methadone to be dispenses in OTPs. However, in this setting studies have shown that HIV care and anti-retroviral treatment can be effectively prescribed either as directly observed therapy or as routine care. Other countries, such as Australia, have less stringent federal regulations for prescribing controlled medications and all medications comprising MAT can be provided in a primary care setting. In either case, the important aspect of providing integrated MAT and HIV primary care is the single location/clinic. In that case, the patient can receive all the needed services to support their recovery from drug/alcohol dependence as well as care and treatment for HIV infection.

**7. Essential health interventions for the prevention of HIV infection in people** 

The WHO, UNODC and UNAIDS has approved and advocates for a package of essential interventions for the prevention, treatment and care of HIV for people who inject drugs (WHO, 2009a). These evidence based intervention, shown in Table 6, need three important characteristics in their implementation to maximize effectiveness. These interventions need to be part of a public health policy that is human rights based, gender responsive, and

As noted earlier, no single intervention alone will prevent or reverse to growing national HIV epidemics due to injection drug use and abuse. However, the greatest impact will be obtained when the interventions are provided through an integrated services platform in a comprehensive fashion. And in order to reach all of those seeking HIV prevention, care and treatment services, health service platforms need to provide an enabling environment that establishes confidentiality. In addition, they also need to develop patient –provider trusting

health status of HIV-infected injection drug users.

**who inject opioids** 

community owned.

Table 5. Listing of Internationally Accepted Essential Interventions for HIV

#### **8. Conclusion**

Substance abuse treatment is HIV prevention. The use of medication assisted treatment as a component of a comprehensive treatment plan for those individuals who abuse opioids and/or alcohol is an effective, evidence-based treatment paradigm that results in good medical outcomes including a reduction in HIV transmission as well as a reduction of incident infections in opioid and alcohol abusing populations.

#### **9. References**


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**27** 

*Spain* 

*than rigor than questionnaires."* 

*Edgar Morin 1*

**The Pertinence of Applying Qualitative** 

**Evaluation of HIV Prevention Policies** 

*National Centre for Tropical Medicine, Health Institute Carlos III* 

**Investigation Strategies in the Design and** 

Carmen Rodríguez, Teresa Blasco, Antonio Vargas and Agustín Benito

*"We think that there still exists a vast sector of science in which we are no further than in taylorian stages of intellectual work rationalization and which can do nothing but contribute to scientific rigor. Rigor when reasoning is more important that rigor when calculating. Questions is more important* 

In the last decade, the incidence of HIV has globally diminished by 19% (Joint United Nations Programme on HIV/Aids, [Unaids], 2010). Likewise, new cases of children infected by HIV have diminished due to the spread of vertical mother-child prevention among pregnant women. The percentage of people who received anti-retroviral treatment increased by 30% due to improvements in the accessibility of therapeutic treatment. Also, the annual percentage of death caused by Aids has decreased (Unaids, 2010). Nevertheless, in spite of this progress, HIV/Aids pandemic continues to be one of the main threats to global health. On a world scale, pandemic epidemiological data shows alarming numbers: there are more than 33 million people around the world living with HIV. Approximately, 7,000 new cases of infection arise every day. Every year, an average of 1,800,000 people die as a consequence

of Aids. It is estimated that 12 million children have been orphaned (Unaids, 2010).

However, such numbers are distributed in unequal proportions throughout the world. Thus, since not every country presents the same level of prevalence, big differences can be found. Currently, most of the countries suffering from HIV pandemic are developing countries. Also, these countries show low or medium levels of human development. The Table 1 shows the relation between Human Development Index in some countries with a

As seen on table 1, the distribution of the pandemic tendency throughout these countries reveals a clear correlation: The less socio-economically developed a country is, the higher the HIV prevalence is. This being so, it is hardly surprising that sub-Saharan African or Asian countries present widespread epidemic HIV levels. On a world scale, these countries hold the highest Multidimensional Poverty Index (United Nations Development Programme, [Unpd], 2010). For instance, the worst Human Development Index in the world belongs to

**1. Introduction** 

higher pandemic level.

1 Morin, Eder. (2006). *The method* (ed. Cátedra).


### **The Pertinence of Applying Qualitative Investigation Strategies in the Design and Evaluation of HIV Prevention Policies**

Carmen Rodríguez, Teresa Blasco, Antonio Vargas and Agustín Benito *National Centre for Tropical Medicine, Health Institute Carlos III Spain* 

#### **1. Introduction**

548 Recent Translational Research in HIV/AIDS

Schuh, K.J., Walsh, S.L., Bigelow, G.E., Preston, K.L. & Stitzer, M.L. (1996) Buprenorphine,

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Zaller, N., Gillani, F.S.& Rich, J.D.(2007) A model of integrated primary care for HIV-positve

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the Great Lakes Addiction Technology Transfer Center, the Philadelphia Department of Behavioral Health and Mental Retardation Services, and the

*Global Status Report on Alcohol 2004.* Department of Mental Health and Substance Abuse. World Health Organization Publishers. ISBN 92 4 156272 2 Geneva pp9-15*.* WHO (World Health Organization) (2008). *Guidelines for the Psychosocially assisted* 

*pharmacological treatment of opioid dependence.* World Health Organization, WHO

*substance use disorders*. mhGAP Mental Health Gap Action Programme. World Health Organization, WHO Press ISBN 978 92 4 159620 6 Geneva, Switzerland WHO (World Health Organization) (2009a). *WHO,UNODC, UNAIDS Technical guide for* 

*countries to set targets for universal access to HIV prevention, treatment and care for injection drug users*. World Health Organization, WHO Press ISBN 978 92 4 159776 0

buprenorphine/naloxone diversion, misue and illicit use: An international review.

patients with underlying substance use and metal illness. *AIDS Care* vol 19 no 9 pp

*"We think that there still exists a vast sector of science in which we are no further than in taylorian stages of intellectual work rationalization and which can do nothing but contribute to scientific rigor. Rigor when reasoning is more important that rigor when calculating. Questions is more important than rigor than questionnaires." Edgar Morin 1*

In the last decade, the incidence of HIV has globally diminished by 19% (Joint United Nations Programme on HIV/Aids, [Unaids], 2010). Likewise, new cases of children infected by HIV have diminished due to the spread of vertical mother-child prevention among pregnant women. The percentage of people who received anti-retroviral treatment increased by 30% due to improvements in the accessibility of therapeutic treatment. Also, the annual percentage of death caused by Aids has decreased (Unaids, 2010). Nevertheless, in spite of this progress, HIV/Aids pandemic continues to be one of the main threats to global health.

On a world scale, pandemic epidemiological data shows alarming numbers: there are more than 33 million people around the world living with HIV. Approximately, 7,000 new cases of infection arise every day. Every year, an average of 1,800,000 people die as a consequence of Aids. It is estimated that 12 million children have been orphaned (Unaids, 2010).

However, such numbers are distributed in unequal proportions throughout the world. Thus, since not every country presents the same level of prevalence, big differences can be found. Currently, most of the countries suffering from HIV pandemic are developing countries. Also, these countries show low or medium levels of human development. The Table 1 shows the relation between Human Development Index in some countries with a higher pandemic level.

As seen on table 1, the distribution of the pandemic tendency throughout these countries reveals a clear correlation: The less socio-economically developed a country is, the higher the HIV prevalence is. This being so, it is hardly surprising that sub-Saharan African or Asian countries present widespread epidemic HIV levels. On a world scale, these countries hold the highest Multidimensional Poverty Index (United Nations Development Programme, [Unpd], 2010). For instance, the worst Human Development Index in the world belongs to

<sup>1</sup> Morin, Eder. (2006). *The method* (ed. Cátedra).

The Pertinence of Applying Qualitative

response to the HIV problem.

MDG (United Nations, [Nu], 2000).

which they will be implemented.

(PUND, 2001).

Investigation Strategies in the Design and Evaluation of HIV Prevention Policies 551

survey revealed that 61% of these families moved to cheaper places to live, 39% lost their access to drinking water and also 21% of girls and 17% of boys gave up their school studies

Finally, the spread of the epidemic in these countries is increasing, deepening existing poverty and social inequality, and reversing the trend towards their level of human development. Hence, HIV/Aids epidemic has become one of the main key aspects in national policies of Poverty Reduction Strategies (PRS) (PNUD, 2002) Such strategies are becoming the main national planning instrument in many countries. That is why HIV/Aids plays a central role in the processes of national development planning and in the budgetary allocation of these States. This contributes to the creation of adequate policies and providing the necessary resources, in order to give a wide multisectorial

Generally, a higher prevalence of the pandemic in countries with low human development index increases social injustice and emphasizes the north-south divide. So that the fight against HIV/Aids together with the fight poverty has become two parts of the same parts of the same battle. Therefore, regarding poverty in certain countries, there will be no possibility to achieve the Millenium Development Goals (MDGs) unless HIV/Aids is efficiently treated. That is why the fight against HIV/Aids has been chosen as the sixth

After three decades fighting against the epidemic, in the field of public health there is no doubt that the intervention from prevention policies is the most efficient weapon in order to eliminate and combat the epidemic (World Health Organization, [Who], 2010). In this sense, competent international organizations struggle more and more to make governments and other institutions aware of the importance of implementing efficient actions in order to prevent HIV. So, the last UNAIDS world report has included measurement indexes for different aspects of prevention processes carried out in different countries as evaluative

All over the world, the high number of new cases of HIV in 2010 corroborate the pressing need for intervention in order to stop the development of the epidemic. There again, a higher incidence of HIV cases in developing countries has established prevention as a priority in the national policies of these countries. In connection with this, it is worth knowing that 97% of the new infections produced every day are to be found in people who live in countries with medium or low human development index (Unaids, 2010). Specifically, it is in the African context where the cases of incidence occur more frequently. As for the development of HIV prevention policies, experience reveals that there are no universal formulae for success that might be applied to all countries. Therefore, certain strategies of prevention proved to be successful in a given country may not constitute any guarantee in another, due to the multiple factors that interfere in the development of the different policies. For instance, each context presents a set of specific needs which must be taken into account for the design and implementation of such policies. Among other things, exclusive qualities of the social and cultural elements also play a part when defining the context in which the intervention is going to take place. That is why it is advised to take into account all of these aspects and adapt the design of HIV prevention policies to the context in

**2. The importance of prevention in the response to the epidemic** 

indicators of the current state of the epidemic (Unaids, 2010).


Table 1. Relation between HIV and HDI prevalence Self-made

belongs to Zimbabwe (0.291) and, at the same time, the highest levels of HIV prevalence can be found there – around 20% and 25%-, (UNPD, 2010). With the one before, in countries like Sweden, where the Human Development Index is very high, (0.773) such prevalence varies between 1% and 5% (UNPD, 2010).

Therefore, the highest number of cases are to be found in lesser developed countries, bearing almost 90% of the whole HIV prevalence worldwide. In these countries, the HIV epidemic is one of the largest National Health problems faced by their Governments. As a consequence, strategic plans of prevention, together with the provision of welfare coverage in the treatment of HIV, are given top priority by the National policies. Nonetheless, HIV epidemic does not only mean a challenge at political level, it also entails a menace to entails a menace to democracy and governability in these countries' political systems. Regarding this last statement, the South African Institute for Democracy has published a report highlighting the negative effect of HIV/Aids in the electoral processes of these countries.

HIV epidemic in these countries does not only mean a medical and sanitary problem, it also constitutes one of the main obstacles for their socio-economic development. Given the huge percentage of adults who die as a consequence of Aids, these countries lose their young people, those who could help with their economic development to a higher degree. In addition, economic productivity also decreases due to the fact that the number of people infected by Aids or those who take care of them must quit their jobs. Besides, their educational systems are affected due to the percentage of teachers who die as a consequence of Aids. This situation brings about an important loss of highly educated inhabitants. Likewise, medical expenses generated out of the provision of health services to people who live with HIV (PLHIV) and Aids, involve budgetary restrictions in the investment of public expenses in other sectors, with the aim of promoting the economic and social development of these countries. In connection with this, the UNPD estimates that in Bostwana the State Revenue dropped by 20% in 2010 as a direct result of HIV/Aids (PNUD, 2010).

Regarding families, the consequences of this epidemic in the domestic economy of these countries are also devastating. When they lose the "head of the family" –the one who has to meet the economic needs of the family– they lose their income, their nutrition worsens, agricultural production falls, medical expenses increase, savings turn into debts, funeral expenses multiply, children leave schools, people's health deteriorates, and so on. In fact, a survey carried out in Zambia shows that two thirds of urban homes which have lost the head of the family as a consequence of Aids have seen their income drop by 80%. The same

belongs to Zimbabwe (0.291) and, at the same time, the highest levels of HIV prevalence can be found there – around 20% and 25%-, (UNPD, 2010). With the one before, in countries like Sweden, where the Human Development Index is very high, (0.773) such prevalence varies

Therefore, the highest number of cases are to be found in lesser developed countries, bearing almost 90% of the whole HIV prevalence worldwide. In these countries, the HIV epidemic is one of the largest National Health problems faced by their Governments. As a consequence, strategic plans of prevention, together with the provision of welfare coverage in the treatment of HIV, are given top priority by the National policies. Nonetheless, HIV epidemic does not only mean a challenge at political level, it also entails a menace to entails a menace to democracy and governability in these countries' political systems. Regarding this last statement, the South African Institute for Democracy has published a report highlighting the negative effect of HIV/Aids in the electoral processes of these countries. HIV epidemic in these countries does not only mean a medical and sanitary problem, it also constitutes one of the main obstacles for their socio-economic development. Given the huge percentage of adults who die as a consequence of Aids, these countries lose their young people, those who could help with their economic development to a higher degree. In addition, economic productivity also decreases due to the fact that the number of people infected by Aids or those who take care of them must quit their jobs. Besides, their educational systems are affected due to the percentage of teachers who die as a consequence of Aids. This situation brings about an important loss of highly educated inhabitants. Likewise, medical expenses generated out of the provision of health services to people who live with HIV (PLHIV) and Aids, involve budgetary restrictions in the investment of public expenses in other sectors, with the aim of promoting the economic and social development of these countries. In connection with this, the UNPD estimates that in Bostwana the State

Revenue dropped by 20% in 2010 as a direct result of HIV/Aids (PNUD, 2010).

Regarding families, the consequences of this epidemic in the domestic economy of these countries are also devastating. When they lose the "head of the family" –the one who has to meet the economic needs of the family– they lose their income, their nutrition worsens, agricultural production falls, medical expenses increase, savings turn into debts, funeral expenses multiply, children leave schools, people's health deteriorates, and so on. In fact, a survey carried out in Zambia shows that two thirds of urban homes which have lost the head of the family as a consequence of Aids have seen their income drop by 80%. The same

**Adults and children HDI 2010** 

**Countries 2009 HIV estimates in** 

Table 1. Relation between HIV and HDI prevalence

between 1% and 5% (UNPD, 2010).

Self-made

Nigeria 3,300,000 0.432 India 2,400,0000 0.59 United Republic Of Tanzania 140,000 0.398 Zimbawe 120,000 0.140 Uganda 120,000 0.422 Malawi 920,000 0.385 Zambia 98,000 0.395 survey revealed that 61% of these families moved to cheaper places to live, 39% lost their access to drinking water and also 21% of girls and 17% of boys gave up their school studies (PUND, 2001).

Finally, the spread of the epidemic in these countries is increasing, deepening existing poverty and social inequality, and reversing the trend towards their level of human development. Hence, HIV/Aids epidemic has become one of the main key aspects in national policies of Poverty Reduction Strategies (PRS) (PNUD, 2002) Such strategies are becoming the main national planning instrument in many countries. That is why HIV/Aids plays a central role in the processes of national development planning and in the budgetary allocation of these States. This contributes to the creation of adequate policies and providing the necessary resources, in order to give a wide multisectorial response to the HIV problem.

Generally, a higher prevalence of the pandemic in countries with low human development index increases social injustice and emphasizes the north-south divide. So that the fight against HIV/Aids together with the fight poverty has become two parts of the same parts of the same battle. Therefore, regarding poverty in certain countries, there will be no possibility to achieve the Millenium Development Goals (MDGs) unless HIV/Aids is efficiently treated. That is why the fight against HIV/Aids has been chosen as the sixth MDG (United Nations, [Nu], 2000).

#### **2. The importance of prevention in the response to the epidemic**

After three decades fighting against the epidemic, in the field of public health there is no doubt that the intervention from prevention policies is the most efficient weapon in order to eliminate and combat the epidemic (World Health Organization, [Who], 2010). In this sense, competent international organizations struggle more and more to make governments and other institutions aware of the importance of implementing efficient actions in order to prevent HIV. So, the last UNAIDS world report has included measurement indexes for different aspects of prevention processes carried out in different countries as evaluative indicators of the current state of the epidemic (Unaids, 2010).

All over the world, the high number of new cases of HIV in 2010 corroborate the pressing need for intervention in order to stop the development of the epidemic. There again, a higher incidence of HIV cases in developing countries has established prevention as a priority in the national policies of these countries. In connection with this, it is worth knowing that 97% of the new infections produced every day are to be found in people who live in countries with medium or low human development index (Unaids, 2010). Specifically, it is in the African context where the cases of incidence occur more frequently.

As for the development of HIV prevention policies, experience reveals that there are no universal formulae for success that might be applied to all countries. Therefore, certain strategies of prevention proved to be successful in a given country may not constitute any guarantee in another, due to the multiple factors that interfere in the development of the different policies. For instance, each context presents a set of specific needs which must be taken into account for the design and implementation of such policies. Among other things, exclusive qualities of the social and cultural elements also play a part when defining the context in which the intervention is going to take place. That is why it is advised to take into account all of these aspects and adapt the design of HIV prevention policies to the context in which they will be implemented.

The Pertinence of Applying Qualitative

prevention area:

community actions.

adapting actions to contexts.

improve the management of the intervention.

diagnosis and treatment, respectively.

care services.

Investigation Strategies in the Design and Evaluation of HIV Prevention Policies 553

regards their evaluation processes (Campbell et al., 2000). In order to do so, it is advisable to take into account the adaptation of the process to the local circumstances before being implemented once it has been completely standardized. Therefore, for an optimal implementation, it is currently considered as necessary to have the adequate knowledge to

In the African context, a low specific applicability of these criteria can be found. That is why a low level of key targets for prevention has been achieved. It is worth mentioning that only 32% of the African population knows their serological status. Likewise, only 45% of pregnant women receive proper care in order to prevent their children from contracting the disease. (Who, 2010). As regards the type of epidemic, actions carried out in the African context must have plenty of characteristics unique to the generalized epidemic. This is the most common epidemiological situation in most countries of the continent. The fact of presenting generalized epidemic levels determines the monitoring of intervention priorities within the

 Using strategies that would cover all risk behaviour of contracting the disease. Besides, these strategies must be as accessible as possible to the population in need of help. Decentralizing the provision of services by incorporating primary care actions as well as

Integrating prevention services together with treatment and care services into primary

 Giving priority to actions aimed at tertiary prevention making it possible to interrupt the epidemiological chain of the disease, diminishing the appearance of new cases. Recommending the diagnostic sifting of every person who makes use of health care

The existence of a whole multiplicity of cultural components characteristic of every area and unique to every social group is something that, for the time being, has not been sufficiently reflected in the adaptation of the general frameworks to each context. The recommendation that second generation epidemiological vigilance (Grulich & Kaldor, 2002) (Who, 2000) should be integrated into the tracking and monitoring processes of the disease makes it possible to count on a broader knowledge of people's attitudes and practices. Moreover, adaptation to strategies has improved. The use of qualitative methodologies in order to generate this knowledge is still very recent and, in some contexts, almost nonexistent. This is shown as a tool that supplies key elements, having a deeper impact on health when

Weakness in health systems which implement actions and policies is a very common factor all around Africa. Such weakness does not always derive from the low budget allocated to its development. In the last years, poor systems and limited resources, together with important obstacles that eliminate the possibility to improve these systems, demand the need to search for evidence-based knowledge in order to determine which actions are the most cost-effective in HIV. This knowledge would help to establish criteria that could

Out of all this generated knowledge, taking into account the target context of the chapter as well as efficiency criteria previously mentioned, we wanted to pay special attention to the value of implementing associated measures of secondary and tertiary prevention, like

The initial intervention model in the fight against HIV implemented only tertiary prevention measures, in spite of its low efficiency when trying to reduce the progression of the disease.

services and to all pregnant women or those in a lactation period.

orientate this process of adaptability of the standard theoretical framework.

Currently, there is a big concern about how to adapt prevention policies to African countries. On the one hand, it is due to the urgent need to change the development of the epidemic considering its magnitude and, on the other hand, to the ineffectiveness shown by prevention policies to date, considering the high rates of incidence. As a consequence, the epidemic stabilization has not yet been reached in some countries.

#### **2.1 Prevention policies in the African context**

HIV prevention actions developed in Africa are key to slow down the great development of this disease. Besides, prevention actions have an added value since either they must be linked to or they must incorporate values, such as justice, fairness and the promotion of dignity. At the same time, it constitutes basic principles which help to improve the African context.

Now, some of the international recommendations on public health as well as some of the existing evidence on HIV prevention in the African context to date are described.

Prevention actions must be aware of the main HIV routes of transmission in the continent. In Africa, the HIV epidemic spreads mainly through sexual intercourse. The percentage of people who became infected by this disease through routes other than the sexual intercourse is low. Nevertheless, mother-to-child transmission and hemoderivatives transfusion are the other two relevant aspects when assessing prevention measures in countries where, like in the African context, the investment in public health is not enough.

It is advisable to develop HIV prevention policies that interact with the different levels of prevention. According to the World Organization of Family Doctors (Wonca), there are four grades of prevention. Primary prevention is the one that involves action before the disease appears and, among these actions are the ones in charge of promoting health, those focused on environmental hygiene or those like vaccination or chemical prophylaxis. Secondary prevention deals with actions aimed at identifying ill patients within the population, implementing strategies of population sifting, enabling the early detection of diseases. Through implemented actions, tertiary prevention involves easing or avoiding the effects of the disease once it has been contracted. Finally, in 1986 Marc Jamoulle defined quaternary prevention as *"those actions designed to restrict unnecessary damage produced as a consequence of health-care activity".* 

Within the current priority of international recommendations on health-HIV, five guidelines for intervention have been developed. These guidelines are especially relevant in countries with higher prevalence, as in most of the African countries (Who, 2009). They mainly include preventive actions in the three first levels:


In general, as a maxim of intervention in public health, the development of HIV-related actions, including all actions and not only preventive ones, should be adapted to each and every context in three determining factors. Firstly, they should adapt to the specific characteristics of the epidemic in question, like the particular context of each country as well as that of its community. Secondly, they should pay attention to the cultural context and, thirdly, to the level of provision of services and resources set aside for health. Related to this last aspect and in order to strengthen the adaptation of international frameworks at local level, actions developed to prevent HIV are considered to be complex interventions as

Currently, there is a big concern about how to adapt prevention policies to African countries. On the one hand, it is due to the urgent need to change the development of the epidemic considering its magnitude and, on the other hand, to the ineffectiveness shown by prevention policies to date, considering the high rates of incidence. As a consequence, the

HIV prevention actions developed in Africa are key to slow down the great development of this disease. Besides, prevention actions have an added value since either they must be linked to or they must incorporate values, such as justice, fairness and the promotion of dignity. At the same time, it constitutes basic principles which help to improve the African context. Now, some of the international recommendations on public health as well as some of the

Prevention actions must be aware of the main HIV routes of transmission in the continent. In Africa, the HIV epidemic spreads mainly through sexual intercourse. The percentage of people who became infected by this disease through routes other than the sexual intercourse is low. Nevertheless, mother-to-child transmission and hemoderivatives transfusion are the other two relevant aspects when assessing prevention measures in countries where, like in

It is advisable to develop HIV prevention policies that interact with the different levels of prevention. According to the World Organization of Family Doctors (Wonca), there are four grades of prevention. Primary prevention is the one that involves action before the disease appears and, among these actions are the ones in charge of promoting health, those focused on environmental hygiene or those like vaccination or chemical prophylaxis. Secondary prevention deals with actions aimed at identifying ill patients within the population, implementing strategies of population sifting, enabling the early detection of diseases. Through implemented actions, tertiary prevention involves easing or avoiding the effects of the disease once it has been contracted. Finally, in 1986 Marc Jamoulle defined quaternary prevention as *"those actions designed to restrict unnecessary damage produced as a consequence of* 

Within the current priority of international recommendations on health-HIV, five guidelines for intervention have been developed. These guidelines are especially relevant in countries with higher prevalence, as in most of the African countries (Who, 2009). They mainly

In general, as a maxim of intervention in public health, the development of HIV-related actions, including all actions and not only preventive ones, should be adapted to each and every context in three determining factors. Firstly, they should adapt to the specific characteristics of the epidemic in question, like the particular context of each country as well as that of its community. Secondly, they should pay attention to the cultural context and, thirdly, to the level of provision of services and resources set aside for health. Related to this last aspect and in order to strengthen the adaptation of international frameworks at local level, actions developed to prevent HIV are considered to be complex interventions as

1. Strengthening actions in primary prevention of the disease from the health sector.

2. Making it possible for the population to know its serological status. 3. Accelerating the spread of the treatment as well as the HIV/Aids care.

existing evidence on HIV prevention in the African context to date are described.

epidemic stabilization has not yet been reached in some countries.

the African context, the investment in public health is not enough.

**2.1 Prevention policies in the African context** 

include preventive actions in the three first levels:

5. Increasing knowledge in order to improve response.

4. Strengthening health systems capacities.

*health-care activity".* 

regards their evaluation processes (Campbell et al., 2000). In order to do so, it is advisable to take into account the adaptation of the process to the local circumstances before being implemented once it has been completely standardized. Therefore, for an optimal implementation, it is currently considered as necessary to have the adequate knowledge to orientate this process of adaptability of the standard theoretical framework.

In the African context, a low specific applicability of these criteria can be found. That is why a low level of key targets for prevention has been achieved. It is worth mentioning that only 32% of the African population knows their serological status. Likewise, only 45% of pregnant women receive proper care in order to prevent their children from contracting the disease. (Who, 2010).

As regards the type of epidemic, actions carried out in the African context must have plenty of characteristics unique to the generalized epidemic. This is the most common epidemiological situation in most countries of the continent. The fact of presenting generalized epidemic levels determines the monitoring of intervention priorities within the prevention area:


The existence of a whole multiplicity of cultural components characteristic of every area and unique to every social group is something that, for the time being, has not been sufficiently reflected in the adaptation of the general frameworks to each context. The recommendation that second generation epidemiological vigilance (Grulich & Kaldor, 2002) (Who, 2000) should be integrated into the tracking and monitoring processes of the disease makes it possible to count on a broader knowledge of people's attitudes and practices. Moreover, adaptation to strategies has improved. The use of qualitative methodologies in order to generate this knowledge is still very recent and, in some contexts, almost nonexistent. This is shown as a tool that supplies key elements, having a deeper impact on health when adapting actions to contexts.

Weakness in health systems which implement actions and policies is a very common factor all around Africa. Such weakness does not always derive from the low budget allocated to its development. In the last years, poor systems and limited resources, together with important obstacles that eliminate the possibility to improve these systems, demand the need to search for evidence-based knowledge in order to determine which actions are the most cost-effective in HIV. This knowledge would help to establish criteria that could improve the management of the intervention.

Out of all this generated knowledge, taking into account the target context of the chapter as well as efficiency criteria previously mentioned, we wanted to pay special attention to the value of implementing associated measures of secondary and tertiary prevention, like diagnosis and treatment, respectively.

The initial intervention model in the fight against HIV implemented only tertiary prevention measures, in spite of its low efficiency when trying to reduce the progression of the disease.

The Pertinence of Applying Qualitative

Investigation Strategies in the Design and Evaluation of HIV Prevention Policies 555

biomedical model. Concerning this subject, most of the investigations to be found on scientific literature are prevalence and/or ecological studies. In this sense and in general terms, knowledge based on data that mainly describe the way this epidemic is distributed throughout the population according to certain factors predominate (Caitlin et al., 2010). However, the relevance and significance that HIV epidemic has gained in the social sphere nowadays constitute evidence. This fact can be clearly seen, for instance, when paying attention to the social stigma generated around HIV (Skinner & Mfecane, 2004). That is why most of the interventions being carried out in the field of HIV take into account the social dimension of the problem. In this sense, epidemic research beyond simple observation of

Currently, the HIV epidemic represents a social phenomenon that mainly affects the area of public health. The HIV epidemic as a social phenomenon acquires different meanings depending on the kind of society in which it is found, since cultural and social contexts play an important role in all countries. For example, although HIV is seen as a cronical disease in the collective unconscious of most developed countries, this virus is considered to be fatal and lethal in the developing countries (Conde, 1997). There again, as HIV is meddled in social constructs like life, health, dead and disease, it is also steeped in specific connotations

So that HIV acquires meanings, representations, perceptions, values and erelated to social and cultural contexts. Such contexts give meaning and guide people's behaviour and actions to confront HIV. These distinctive values, meanings and so forth, will have an effect on preventive actions that are being taken in order to face the HIV phenomenon. As can be seen, qualitative aspects that are also important to know in order to prevent and eliminate

By applying the methodologies which have developed the traditional approaches to HIV study, the knowledge of HIV qualitative aspects is difficult to achieve. Nowadays, with the aim of setting out a deeper and more holistic HIV knowledge, other perspectives and theoretic approaches belonging to disciplines other than health are applied. For instance, approaches to the epidemic phenomenon have been carried out by different sciences -like hermeneutics, phenomenology or ethnography- in which their theoretical frameworks from disciplines like Anthropology or Sociology (Arachu & Pau, 2003). The efforts made by UNESCO in order to develop and promote a cultural approach to the HIV epidemic are especially worthy of notice (United Nations Educational, Scientific and Cultural

Theoretical approaches in the field of social sciences have incorporated HIV study methodologies different from the quantitative, which is traditionally applied by clinic epidemiology. Among these methodologies, the use of qualitative methodology prevails. Therefore, qualitative methodology is no longer a method exclusively used in disciplines connected to the social sphere. In this sense, it appears more and more frequently in health related studies, whose scientific expansion displays as much on publications and seminars

On an international level, different authors have placed particular emphasis on the necessity and in the advantages of using this methodology in the field of public health (Bryman, 1984). Also, its relevance to social epidemiology has been highlighted. For instance, it has been pointed out its importance when used in order to evaluate peoples' health care from a

how it is distributed throughout a given population is required.

that every culture attributes to these social values.

as on medicine and public health related conferences.

the epidemic mark the HIV phenomenon.

Organozation, [Unicef ], 2003).

The target was to reduce the high rate of mortality caused by this disease at that time. Nowadays, the antiretroviral treatment has evolved and it is considered to be highly active (HAART). Cohort studies in serodiscordant couples and pregnant women living with HIV have proved that patients who receive good treatment and have an undetectable viral load are less likely to transmit the disease (Quinn et al., 2000). This ART has proved to be a very good method to reduce the transmission of this disease and that is the reason why, currently, on a global scale, all countries are advised to reach universal coverage (Granich et al, 2009) of the treatment. The reason is not only that the rate of mortality decreases but also its effect on the transmission chain.

There is a great variability when it comes to value the cost-effectiveness of HIV strategies depending, above all, on the country where the action is implemented (Andrew, 2002). Following this criteria, actions of prevention that cause a deeper impact and those enjoying a better cost-effectiveness criteria are meant to limit mother-to-child transmission of the disease.

Regarding the diagnosis of the disease in the population, the HIV strategies meet the requirements defined by Frame and Carslon (Frame & Carslon, 1975) in order to be able to carry out actions of secondary prevention and, therefore, make quite an impact both on the health of the patient and on the health of the whole population. The necessary criteria for this applicability are the following:


In the last years, the scientific community issued an appeal for the innovation of developed prevention strategies (Piot,2008). Current recommendations in order to cause a greater impact on health in the African context suggest that actions of secondary prevention, such as diagnosis, and actions of tertiary prevention, such as ARV treatment, are joined (Dood, 2010). It is important to bear in mind that without diagnosis there is no treatment and, as seen before, preventing this disease from being transmitted becomes a limited task.

#### **3. Application of qualitative methodologies in HIV prevention**

*"Aids has proved that epidemics take place at different levels: biological event, social perception, collective response and individual phenomenon, both existential and moral [...].Each disease, as social phenomenon, is a unique configuration of events and responses both in the biological sphere and in the social sphere"( Mariano Bronfman2).* 

In the scientific field, HIV epidemic has traditionally been investigated by clinical epidemiology. For this reason, the predominant theoretical development has been the

<sup>2</sup> Mariano Bronfman: Social Sciences and Aids. Magazine of Public Health and Mexico 1999;Vol.41(2):83- 84.

The target was to reduce the high rate of mortality caused by this disease at that time. Nowadays, the antiretroviral treatment has evolved and it is considered to be highly active (HAART). Cohort studies in serodiscordant couples and pregnant women living with HIV have proved that patients who receive good treatment and have an undetectable viral load are less likely to transmit the disease (Quinn et al., 2000). This ART has proved to be a very good method to reduce the transmission of this disease and that is the reason why, currently, on a global scale, all countries are advised to reach universal coverage (Granich et al, 2009) of the treatment. The reason is not only that the rate of mortality decreases but also

There is a great variability when it comes to value the cost-effectiveness of HIV strategies depending, above all, on the country where the action is implemented (Andrew, 2002). Following this criteria, actions of prevention that cause a deeper impact and those enjoying a better cost-effectiveness criteria are meant to limit mother-to-child transmission of the disease. Regarding the diagnosis of the disease in the population, the HIV strategies meet the requirements defined by Frame and Carslon (Frame & Carslon, 1975) in order to be able to carry out actions of secondary prevention and, therefore, make quite an impact both on the health of the patient and on the health of the whole population. The necessary criteria for

1. The disease must be an important health problem, having a clear effect on the quality of

2. The disease must develop through an asymptomatic initial stage and its natural history

3. There must be an effective treatment accepted by the population in the case that the

4. There must be a sifting test which has to be quick, safe, easy-to-do, highly sensitive, highly specific, of high positive predictive value and well accepted by doctors and patients.

6. The early detection of the disease and its treatment during the asymptomatic period

In the last years, the scientific community issued an appeal for the innovation of developed prevention strategies (Piot,2008). Current recommendations in order to cause a greater impact on health in the African context suggest that actions of secondary prevention, such as diagnosis, and actions of tertiary prevention, such as ARV treatment, are joined (Dood, 2010). It is important to bear in mind that without diagnosis there is no treatment and, as

*"Aids has proved that epidemics take place at different levels: biological event, social perception, collective response and individual phenomenon, both existential and moral [...].Each disease, as social phenomenon, is a unique configuration of events and responses both in the biological sphere and in* 

In the scientific field, HIV epidemic has traditionally been investigated by clinical epidemiology. For this reason, the predominant theoretical development has been the

2 Mariano Bronfman: Social Sciences and Aids. Magazine of Public Health and Mexico 1999;Vol.41(2):83-

*the social sphere"( Mariano Bronfman2).* 

must diminish morbidity as well as global or each of them separately.

seen before, preventing this disease from being transmitted becomes a limited task.

**3. Application of qualitative methodologies in HIV prevention** 

its effect on the transmission chain.

this applicability are the following:

disease is detected in its initial state.

5. The sifting test must keep a good cost-effectiveness balance.

life and life expectancy.

must be known.

84.

biomedical model. Concerning this subject, most of the investigations to be found on scientific literature are prevalence and/or ecological studies. In this sense and in general terms, knowledge based on data that mainly describe the way this epidemic is distributed throughout the population according to certain factors predominate (Caitlin et al., 2010).

However, the relevance and significance that HIV epidemic has gained in the social sphere nowadays constitute evidence. This fact can be clearly seen, for instance, when paying attention to the social stigma generated around HIV (Skinner & Mfecane, 2004). That is why most of the interventions being carried out in the field of HIV take into account the social dimension of the problem. In this sense, epidemic research beyond simple observation of how it is distributed throughout a given population is required.

Currently, the HIV epidemic represents a social phenomenon that mainly affects the area of public health. The HIV epidemic as a social phenomenon acquires different meanings depending on the kind of society in which it is found, since cultural and social contexts play an important role in all countries. For example, although HIV is seen as a cronical disease in the collective unconscious of most developed countries, this virus is considered to be fatal and lethal in the developing countries (Conde, 1997). There again, as HIV is meddled in social constructs like life, health, dead and disease, it is also steeped in specific connotations that every culture attributes to these social values.

So that HIV acquires meanings, representations, perceptions, values and erelated to social and cultural contexts. Such contexts give meaning and guide people's behaviour and actions to confront HIV. These distinctive values, meanings and so forth, will have an effect on preventive actions that are being taken in order to face the HIV phenomenon. As can be seen, qualitative aspects that are also important to know in order to prevent and eliminate the epidemic mark the HIV phenomenon.

By applying the methodologies which have developed the traditional approaches to HIV study, the knowledge of HIV qualitative aspects is difficult to achieve. Nowadays, with the aim of setting out a deeper and more holistic HIV knowledge, other perspectives and theoretic approaches belonging to disciplines other than health are applied. For instance, approaches to the epidemic phenomenon have been carried out by different sciences -like hermeneutics, phenomenology or ethnography- in which their theoretical frameworks from disciplines like Anthropology or Sociology (Arachu & Pau, 2003). The efforts made by UNESCO in order to develop and promote a cultural approach to the HIV epidemic are especially worthy of notice (United Nations Educational, Scientific and Cultural Organozation, [Unicef ], 2003).

Theoretical approaches in the field of social sciences have incorporated HIV study methodologies different from the quantitative, which is traditionally applied by clinic epidemiology. Among these methodologies, the use of qualitative methodology prevails. Therefore, qualitative methodology is no longer a method exclusively used in disciplines connected to the social sphere. In this sense, it appears more and more frequently in health related studies, whose scientific expansion displays as much on publications and seminars as on medicine and public health related conferences.

On an international level, different authors have placed particular emphasis on the necessity and in the advantages of using this methodology in the field of public health (Bryman, 1984). Also, its relevance to social epidemiology has been highlighted. For instance, it has been pointed out its importance when used in order to evaluate peoples' health care from a

The Pertinence of Applying Qualitative

Investigation Strategies in the Design and Evaluation of HIV Prevention Policies 557

HIV/Aids. Results related to the execution of HIV diagnosis show that almost ¾ of the people who participated in the survey had never had the tests done (Ministry of Health and Social Welfare of Malabo, 2006). The fact that an important percentage of the population is not aware of their serological status aggravates the magnitude of the epidemic due to the existing risk of exponential growth, as a consequence of HIV transmission through people unaware of their seropositive status. Likewise, this study also stresses the vulnerability of this society towards the epidemic, since evidence shows that being aware of one's

serological status is the first step to be taken in order to prevent and treat the disease.

by 73% of those men who had sex with prostitutes either.

knowledge about the epidemic phenomenon in the country.

implemented prevention strategies was non-existent.

knowledge about the epidemic phenomenon in Equatorial Guinea".

regarding HIV prevention.

**4.2 Justification for the study** 

in the area of HIV prevention.

Regarding the use of male condoms among the Guinean population, the CAP survey produced alarming results. Sexual intercourse is the main HIV transmission route in Equatorial Guinea. That is why, in this country, different social agents competent in HIV prevention have made tremendous efforts to extend and promote the use of male condoms when having sex in order to control this transmission route. In spite of the efforts made, the results of the CAP survey show that the use of the male condom has not been one of the prevention methods used by a vast majority of the Guinean population. Although 73% of those polled point out that they do know how to use male condoms as an HIV preventive measure, almost seven out of ten declared that they had not used them in the last twelve months when having occasional sex with different partners. The male condom was not used

Generally, CAP results reveal that the Guinean population has taken few measures

No sociological investigation considering HIV as a social phenomenon had been previously carried out in Equatorial Guinea. That is why there was no holistic or hermeneutic

Until the completion of the ESEVIGUE study, there was no qualitative data about the epidemic. In this sense, the qualitative aspects of the epidemic constitute key elements to take into account when designing HIV prevention policies. For instance, there was no information about the meaning or possible meanings that the epidemic had acquired in society. Moreover, there was no information about the elements –whatever their nature: cultural, social, political, economic…-that could be interfering with the results of the implemented prevention strategies, acting like barriers and/or facilitators. In general, explanatory information about people's practices and behaviours related to the

Given the absence of these data, the possibility to carry out an investigation in order to announce this situation arises. In the last analysis, the reasons why the preventive measures developed in the country got such results will be evaluated and explained. In this context, the ESEVIGUE comes into being with the general aim of: "understanding and generating

This study targeted the use of generated knowledge as support in order to direct the decision taking process, regarding the different strategies and measures to be implemented

This research started in Bata in August 2009 and has been led jointly by MINSABBS, in Malabo, and the National Centre for Tropical Medicine of the Carlos III Health Institute, (Referential Centre for the Control of Endemic Diseases [CRCE], 2009) in Spain. This

more dynamic and comprehensive perspective. It has also been stated the necessity to know both the adaptation of qualitative methodology for its study and the socio-cultural background together with people's values as health determining factors.

As compared to other diseases or to other health related subjects, qualitative methodology has been widely used in the study of the HIV. However, qualitative investigations performed in this field are still few. But there are still fewer qualitative research aimed at putting into practice the necessary knowledge in order to evaluate and design HIV prevention policies.

Nevertheless, the results of the studies carried out show the adaptation and benefits of this methodology to the understanding and comprehension of the different factors that intervene in the HIV epidemic phenomenon. So that the adaptation of qualitative strategies to the study of the HIV phenomenon constitutes a potential instrument of support to design effective preventive strategies and, therefore, carry out effective and efficient policies in the intervention of this epidemic.

#### **4. Application of a case: Equatorial Guinea**

#### **4.1 Epidemiological context**

The HIV/Aids epidemic is also severely affecting Guinean people's health. On the basis of parameters established by the WHO, Equatorial Guinea suffers from generalized epidemic (Unaids, 2010 b). The HIV prevalence among people between 15 and 49 years of age is 3.2% (IC 95% 2.0 -4.4%). Likewise, Aids represents the main cause of individual mortality (Who, 2008).

In a context of generalized epidemic, competent international organizations warn of the urgent need for these countries to set appropriate measures in motion in order to reverse the epidemic curve. They also emphasize the importance of carrying out prevention policies with the aim of diminishing and eliminating the magnitude of the epidemic. At the same time, they urge the governments of these countries to establish and coordinate effective strategic plans for the prevention of the HIV. In this regard, one of the preventive measures recommended by the WHO is to spread the HIV diagnosis tests throughout the whole population, regardless whether there is clinical suspicion or not, and suggests having the test done at least once a year (Who, 2007) given the positive results that this action would carry with it in both individual level and community level. On an individual level: it initiates and holds preventive behaviour towards the HIV acquisition and transmission, immediate access to care, treatment and support of people living with HIV, major efficacy on interventions in order to prevent motherto-child transmission, better planning to improve the future life. On community level: it diminishes denial, stigma and discrimination associated to the HIV and demands aid for an adequate answer. However, evidence suggests that, in terms of cost-effectiveness, everyone having the test done becomes profitable on the long term, regardless whether they take part in high risk behaviour or not. (Patiel, 2005).

As for HIV preventive measures carried out by the Guinean population, there is almost no information that describes and explains this aspect due to the lack of research in this field of study. Nevertheless, there is some data extracted from a transversal study conducted by ISCIII in collaboration with MINSABS. In this study, people aged between 15 and 50 were given a questionnaire – the CAP survey3 – about different aspects concerning sexual life and

<sup>3 &</sup>quot;Knowledge, attitudes and practices about nuptiality, sexual activity, HIV/Aids and STD"

HIV/Aids. Results related to the execution of HIV diagnosis show that almost ¾ of the people who participated in the survey had never had the tests done (Ministry of Health and Social Welfare of Malabo, 2006). The fact that an important percentage of the population is not aware of their serological status aggravates the magnitude of the epidemic due to the existing risk of exponential growth, as a consequence of HIV transmission through people unaware of their seropositive status. Likewise, this study also stresses the vulnerability of this society towards the epidemic, since evidence shows that being aware of one's serological status is the first step to be taken in order to prevent and treat the disease.

Regarding the use of male condoms among the Guinean population, the CAP survey produced alarming results. Sexual intercourse is the main HIV transmission route in Equatorial Guinea. That is why, in this country, different social agents competent in HIV prevention have made tremendous efforts to extend and promote the use of male condoms when having sex in order to control this transmission route. In spite of the efforts made, the results of the CAP survey show that the use of the male condom has not been one of the prevention methods used by a vast majority of the Guinean population. Although 73% of those polled point out that they do know how to use male condoms as an HIV preventive measure, almost seven out of ten declared that they had not used them in the last twelve months when having occasional sex with different partners. The male condom was not used by 73% of those men who had sex with prostitutes either.

Generally, CAP results reveal that the Guinean population has taken few measures regarding HIV prevention.

#### **4.2 Justification for the study**

556 Recent Translational Research in HIV/AIDS

more dynamic and comprehensive perspective. It has also been stated the necessity to know both the adaptation of qualitative methodology for its study and the socio-cultural

As compared to other diseases or to other health related subjects, qualitative methodology has been widely used in the study of the HIV. However, qualitative investigations performed in this field are still few. But there are still fewer qualitative research aimed at putting into practice the necessary knowledge in order to evaluate and design HIV

Nevertheless, the results of the studies carried out show the adaptation and benefits of this methodology to the understanding and comprehension of the different factors that intervene in the HIV epidemic phenomenon. So that the adaptation of qualitative strategies to the study of the HIV phenomenon constitutes a potential instrument of support to design effective preventive strategies and, therefore, carry out effective and efficient policies in the

The HIV/Aids epidemic is also severely affecting Guinean people's health. On the basis of parameters established by the WHO, Equatorial Guinea suffers from generalized epidemic (Unaids, 2010 b). The HIV prevalence among people between 15 and 49 years of age is 3.2% (IC 95% 2.0 -4.4%). Likewise, Aids represents the main cause of individual mortality (Who,

In a context of generalized epidemic, competent international organizations warn of the urgent need for these countries to set appropriate measures in motion in order to reverse the epidemic curve. They also emphasize the importance of carrying out prevention policies with the aim of diminishing and eliminating the magnitude of the epidemic. At the same time, they urge the governments of these countries to establish and coordinate effective strategic plans for the prevention of the HIV. In this regard, one of the preventive measures recommended by the WHO is to spread the HIV diagnosis tests throughout the whole population, regardless whether there is clinical suspicion or not, and suggests having the test done at least once a year (Who, 2007) given the positive results that this action would carry with it in both individual level and community level. On an individual level: it initiates and holds preventive behaviour towards the HIV acquisition and transmission, immediate access to care, treatment and support of people living with HIV, major efficacy on interventions in order to prevent motherto-child transmission, better planning to improve the future life. On community level: it diminishes denial, stigma and discrimination associated to the HIV and demands aid for an adequate answer. However, evidence suggests that, in terms of cost-effectiveness, everyone having the test done becomes profitable on the long term, regardless whether they take part in

As for HIV preventive measures carried out by the Guinean population, there is almost no information that describes and explains this aspect due to the lack of research in this field of study. Nevertheless, there is some data extracted from a transversal study conducted by ISCIII in collaboration with MINSABS. In this study, people aged between 15 and 50 were given a questionnaire – the CAP survey3 – about different aspects concerning sexual life and

3 "Knowledge, attitudes and practices about nuptiality, sexual activity, HIV/Aids and STD"

background together with people's values as health determining factors.

prevention policies.

intervention of this epidemic.

**4.1 Epidemiological context** 

high risk behaviour or not. (Patiel, 2005).

2008).

**4. Application of a case: Equatorial Guinea** 

No sociological investigation considering HIV as a social phenomenon had been previously carried out in Equatorial Guinea. That is why there was no holistic or hermeneutic knowledge about the epidemic phenomenon in the country.

Until the completion of the ESEVIGUE study, there was no qualitative data about the epidemic. In this sense, the qualitative aspects of the epidemic constitute key elements to take into account when designing HIV prevention policies. For instance, there was no information about the meaning or possible meanings that the epidemic had acquired in society. Moreover, there was no information about the elements –whatever their nature: cultural, social, political, economic…-that could be interfering with the results of the implemented prevention strategies, acting like barriers and/or facilitators. In general, explanatory information about people's practices and behaviours related to the implemented prevention strategies was non-existent.

Given the absence of these data, the possibility to carry out an investigation in order to announce this situation arises. In the last analysis, the reasons why the preventive measures developed in the country got such results will be evaluated and explained. In this context, the ESEVIGUE comes into being with the general aim of: "understanding and generating knowledge about the epidemic phenomenon in Equatorial Guinea".

This study targeted the use of generated knowledge as support in order to direct the decision taking process, regarding the different strategies and measures to be implemented in the area of HIV prevention.

This research started in Bata in August 2009 and has been led jointly by MINSABBS, in Malabo, and the National Centre for Tropical Medicine of the Carlos III Health Institute, (Referential Centre for the Control of Endemic Diseases [CRCE], 2009) in Spain. This

The Pertinence of Applying Qualitative

was taken into account.

dimensions are shown:

HIV

the interviewee.

experience:

people living with the HIV.

ANALYTICAL DIMENSION

Social Perception of the

Knowledge about the HIV What is the HIV?

Table 2. Analytical Dimensions of the ESEVIGUE research

Investigation Strategies in the Design and Evaluation of HIV Prevention Policies 559

purpose of this research, the sex and the gender of the individuals, their education level, etc.

Information gathering techniques applied in order to produce and collect information were: semi-structured individual interview and discussion group. These techniques are generically named qualitative techniques. In order to develop both techniques, the script item was outlined. The script item is a gathering information tool that contains qualitative techniques. This script item was drawn up through open questions that explored analytical dimensions related to the object of study. Now, in table 2, some of the analytical

CATEGORIES AND QUESTIONS Health meaning and value What are the important things in your life?

HIV?

design as regards the content of the questions, the format and the language.

**4.3.1 The reasons why semi-structured interviews are applied** 

Before the fieldwork was performed, the script item was tested following preliminary interviews. The aim of testing this instrument was to check the validity of its technical

Semi-structured interviews were done to people living with the HIV. The application of this technique, in opposition to other group qualitative techniques like discussion groups, has the advantage of preserving anonymity and confidentiality about the serological status of

Semi-structured interview is performed on the basis of a face-to-face conversation between the interviewer and the interviewee. That involves immediate and personal norms of verbal interaction generating, therefore, a subjective knowledge of the observed reality (Alonso, 1994). The interview is a communicative tool that sets out to grasp meanings that are influenced by the constructions made by the individuals themselves according to their

*"Personal interview results very productive to the study of extreme or typical cases in which the attitudes of certain people embody, in every sense, the ideal model of a certain attitude much less* 

So that this technique was carried out with the aim of getting to know the experiences and meanings felt by people living with the HIV. Therefore, its application in the ESEVIGUE resulted in the knowledge of the subjective aspect of suffering HIV. Also, this technique provided information about the phenomenon of the epidemic from the point of view of the

*crystallized in the average of the group of reference (Ortí, 1986)".* 

What does health mean to you?

What do you think about the HIV? What do people say about the HIV?

Are the HIV and Aids the same disease? How can a person become infected by the

investigation has been carried out by a research team made up of professionals belonging to both institutions.

#### **4.3 The basis of the methodological design**

Although epidemiological methods have been traditionally regarded as the standard reference for the study of public health, these methodologies are based on a reductionist view of the world in which simple causality standards are established through statistical processes (Baum, 1997). Health and disease are the result of a complex interrelation of social, economical, political and environmental factors. Interpretative methods based on qualitative techniques are generally well prepared for the analysis of complex situations and contribute to a large extent to the study of public health (Baum, 1997).

The aim of the research was to observe the epidemic as a social phenomenon so that, considering biological investigative or medical approaches, a social perspective of the HIV study was adopted. The design of the investigation has been a qualitative one. The decision of carrying out a qualitative methological design was backed up by the adaptation that qualitative strategies have shown in order to generate holistic, deep and comprehensive knowledge of the real situation under study. Nowadays, in the area of public health, the effective use of qualitative techniques for the understanding and comprehension of factors and processes affecting health and disease is also sufficiently verified. To be more specific, many examples of qualitative methodology applied to the study of the HIV and its context can be found in scientific literature (Medicus Mundi, 2007). In this respect, it is worth mentioning that, confronting epidemiological and quantitative HIV medical study, this type of investigations have proliferated over the last decades.

The sample used in the investigation was a structural one. As in quantitative investigations, the sample design consitutes one of the first methodological aspects to be defined. However, its theoretical basis differs from the sample design in quantitative research. Whereas sample design in quantitative research is based on the concept of statistic representation, in qualitative research it is based on the social signification criteria of the individuals (Valles, 1997). In this sense, sample validity is not given by the number of individuals that make up the sample but by the pertinence of selected individuals in connection with the aims of the study.

Regarding the object of the study, a first general criterion of sample inclusion was established: Individuals aged between 13 and 60. Defining this section of age span became relevant due to the fact that the main HIV route of transmission is sexual intercourse. Therefore, in view of prevention, getting to know the meaning of the HIV epidemic phenomenon and other related aspects within the sexually active population in Guinea was considered to be pertinent.

In the second place, the sample was segmented according to certain variables of interest: being infected by HIV or not, sex, age, education level and place of residence. This segmentation was aimed at identifying and gathering different perceptions and experiences of the HIV. For instance, the initial hypothesis was that, in general terms, there would be differences between PLWHIV and those who do not, as regards perceptions, meanings and practices. Likewise, it was also considered to be pertinent to pay attention to the thoughts of different social groups so as to know the various conceptions of HIV that there may be throughout the whole variety of social groups that form the Guinean society. The objective was to adequate prevention strategies to the specific needs of each social group. For the

investigation has been carried out by a research team made up of professionals belonging to

Although epidemiological methods have been traditionally regarded as the standard reference for the study of public health, these methodologies are based on a reductionist view of the world in which simple causality standards are established through statistical processes (Baum, 1997). Health and disease are the result of a complex interrelation of social, economical, political and environmental factors. Interpretative methods based on qualitative techniques are generally well prepared for the analysis of complex situations and contribute

The aim of the research was to observe the epidemic as a social phenomenon so that, considering biological investigative or medical approaches, a social perspective of the HIV study was adopted. The design of the investigation has been a qualitative one. The decision of carrying out a qualitative methological design was backed up by the adaptation that qualitative strategies have shown in order to generate holistic, deep and comprehensive knowledge of the real situation under study. Nowadays, in the area of public health, the effective use of qualitative techniques for the understanding and comprehension of factors and processes affecting health and disease is also sufficiently verified. To be more specific, many examples of qualitative methodology applied to the study of the HIV and its context can be found in scientific literature (Medicus Mundi, 2007). In this respect, it is worth mentioning that, confronting epidemiological and quantitative HIV medical study, this type

The sample used in the investigation was a structural one. As in quantitative investigations, the sample design consitutes one of the first methodological aspects to be defined. However, its theoretical basis differs from the sample design in quantitative research. Whereas sample design in quantitative research is based on the concept of statistic representation, in qualitative research it is based on the social signification criteria of the individuals (Valles, 1997). In this sense, sample validity is not given by the number of individuals that make up the sample but by the pertinence of selected individuals in connection with the aims of the

Regarding the object of the study, a first general criterion of sample inclusion was established: Individuals aged between 13 and 60. Defining this section of age span became relevant due to the fact that the main HIV route of transmission is sexual intercourse. Therefore, in view of prevention, getting to know the meaning of the HIV epidemic phenomenon and other related aspects within the sexually active population in Guinea was

In the second place, the sample was segmented according to certain variables of interest: being infected by HIV or not, sex, age, education level and place of residence. This segmentation was aimed at identifying and gathering different perceptions and experiences of the HIV. For instance, the initial hypothesis was that, in general terms, there would be differences between PLWHIV and those who do not, as regards perceptions, meanings and practices. Likewise, it was also considered to be pertinent to pay attention to the thoughts of different social groups so as to know the various conceptions of HIV that there may be throughout the whole variety of social groups that form the Guinean society. The objective was to adequate prevention strategies to the specific needs of each social group. For the

both institutions.

study.

considered to be pertinent.

**4.3 The basis of the methodological design** 

to a large extent to the study of public health (Baum, 1997).

of investigations have proliferated over the last decades.

purpose of this research, the sex and the gender of the individuals, their education level, etc. was taken into account.

Information gathering techniques applied in order to produce and collect information were: semi-structured individual interview and discussion group. These techniques are generically named qualitative techniques. In order to develop both techniques, the script item was outlined. The script item is a gathering information tool that contains qualitative techniques. This script item was drawn up through open questions that explored analytical dimensions related to the object of study. Now, in table 2, some of the analytical dimensions are shown:


Table 2. Analytical Dimensions of the ESEVIGUE research

Before the fieldwork was performed, the script item was tested following preliminary interviews. The aim of testing this instrument was to check the validity of its technical design as regards the content of the questions, the format and the language.

#### **4.3.1 The reasons why semi-structured interviews are applied**

Semi-structured interviews were done to people living with the HIV. The application of this technique, in opposition to other group qualitative techniques like discussion groups, has the advantage of preserving anonymity and confidentiality about the serological status of the interviewee.

Semi-structured interview is performed on the basis of a face-to-face conversation between the interviewer and the interviewee. That involves immediate and personal norms of verbal interaction generating, therefore, a subjective knowledge of the observed reality (Alonso, 1994). The interview is a communicative tool that sets out to grasp meanings that are influenced by the constructions made by the individuals themselves according to their experience:

*"Personal interview results very productive to the study of extreme or typical cases in which the attitudes of certain people embody, in every sense, the ideal model of a certain attitude much less crystallized in the average of the group of reference (Ortí, 1986)".* 

So that this technique was carried out with the aim of getting to know the experiences and meanings felt by people living with the HIV. Therefore, its application in the ESEVIGUE resulted in the knowledge of the subjective aspect of suffering HIV. Also, this technique provided information about the phenomenon of the epidemic from the point of view of the people living with the HIV.

The Pertinence of Applying Qualitative

research team was quite difficult.

Ministry of Science and Innovation of Spain.

*Social Anthropology*, Vol.14, pp.29-47

**6. Acknowledgment** 

people and institutions.

Madrid

**7. References** 

effectiveness in results.

Investigation Strategies in the Design and Evaluation of HIV Prevention Policies 561

account when designing prevention strategies in order to achieve maximal efficiency and

Nevertheless, the development of the methodology has not been exempt from difficulties and inconveniences. The first drawback was brought about by the application of this type of methodology in a context of low involvement of the population in social movements and slender culture of social discussion forums. In spite of counting on the collaboration and participation of MINSABS, occasionally people did not want to participate in group sessions for fear of attending politically oriented meetings. Likewise, this fear was also revealed through some of the participants' concerns regarding the confidentiality of the provided

Difficulties were also revealed due to the lack of tradition in qualitative research in the country. That is why human resources educated and/or qualified in this methodology can scarcely be found. As a consequence, identifying and integrating Guinean personnel into the

To sum up, the HIV epidemic is a complex social phenomenon in the Guinean society. In this sense, the HIV epidemic phenomenon has acquired particular connotations that require specific interventions in this field. Moreover, it also reveals that working frameworks and

To the people who took part in interviews and discussion groups, for sharing their time and HIV experiences with the research team. To Jesús Nzang for the generosity that he showed during the development of fieldwork. His dedication and collaboration made it possible to overcome the problems found during the data collection. To Catalina Mangue, Gaspar Nkoni and Luis Mbonio from the CRCE in Equatorial Guinea, for the support they gave me during the recruitment of participants. To Doctor Nemesio Abeso, Director of the MINSABS National Plan of Fight against Aids in Malabo, and Pilar Aparicio, for his support and confidence in the study for his support and confidence in the study. To Jose Luís Cenal, Leticia Díaz and Alberto Hedo from the Foundation for International Health and Cooperation of the National Health System of Spain, for his assistance in the logistic management of the project. And To the Spanish Agency for International Development Cooperation, for financing the study. The study was co-funded by the Instituto de Salud Carlos III, Spanish Research Network of Tropical Diseases (RICET;RD06/0021/0000),

It has been possible to write these pages thanks to the contributions made by all these

Alonso, LE. (1994). Subject and Discourse: The place of the open interview in the practices of

Arachu & Pau. (2003). Aids and structural violence: The blaming of the victim. *Notebooks on* 

qualitative sociology. In: *The Qualitative look on sociology,* Síntesís, pp. 144-53,

information related to personal assessment and opinions about the country.

multidisciplinary action is required in order to prevent and treat the epidemic.

#### **4.3.2 The reason why the discussion group is applied to the study**

The discussion group was performed among the population segment of participants who did not know if they were seropositive and/or those whose blood tests results were negative. Since it is a group technique, it has the advantage of confronting and gathering discourse from the different social groups in one session. At the same time, in economical terms, this technique becomes more profitable

The different points of view of the studied reality regarding the position occupied by the individual in the social framework can be known through the application of discussion groups (ref.). This used to be a key aspect of the technical selection since it allowed the gathering of different HIV related discourses in order to produce and adapt prevention strategies to the specificities of each social group. Therefore, the application of such technique created diverse knowledge about the different social aspects regarding the HIV.

#### **4.3.3 Fieldwork**

Finally, fieldwork based on qualitative methodological strategies was done with the aim of generating knowledge about the HIV phenomenon. Prior to the phase of gathering information, the research team applied the non-participant observation technique. It was aimed at validating different aspects of the study protocol such as sample design or instruments to collect information. It was also aimed at later development of fieldwork.

Geographically, this research took place in Bata between January and April 2010 and it was carried out by a multidisciplinary working group made up of sociologists, doctors, nurses and lab assistants.

#### **5. Conclusions**

In terms of results achieved, the application of qualitative methodology has turned out to be very adequate and valid in order to generate holistic and comprehensive information of the HIV epidemic phenomenon in Equatorial Guiena. So that data has been generated in order to allow both evaluating some of the prevention strategies in the country and showing some of the key aspects to take into account in decision-making processes in the strategy to be implemented.

For instance, results show the inadequacy of massive HIV/Aids information and prevention campaigns carried out in Equatorial Guinea. The association established between the HIV, Aids and death has caused much alarm and fear among the population. Nowadays, it constitutes one of the main barriers for the population to have diagnostic tests done of their own free will. The conducted campaigns played a decisive role in the HIV social construction, which does not favour the integration of people living with Aids into society. Such social construction about people living with the HIV having abandoned the services of the HIV treatment and diagnosis, influenced the reasons given in order to explain why they decided to give up their treatment.

Results have also revealed the importance and necessity of implementing and carrying out prevention strategies considering already existing specificities and needs among different social groups. In this sense, interviewed women's and men's different ways of understanding health and facing the disease are shown. These aspects must be taken into account when designing prevention strategies in order to achieve maximal efficiency and effectiveness in results.

Nevertheless, the development of the methodology has not been exempt from difficulties and inconveniences. The first drawback was brought about by the application of this type of methodology in a context of low involvement of the population in social movements and slender culture of social discussion forums. In spite of counting on the collaboration and participation of MINSABS, occasionally people did not want to participate in group sessions for fear of attending politically oriented meetings. Likewise, this fear was also revealed through some of the participants' concerns regarding the confidentiality of the provided information related to personal assessment and opinions about the country.

Difficulties were also revealed due to the lack of tradition in qualitative research in the country. That is why human resources educated and/or qualified in this methodology can scarcely be found. As a consequence, identifying and integrating Guinean personnel into the research team was quite difficult.

To sum up, the HIV epidemic is a complex social phenomenon in the Guinean society. In this sense, the HIV epidemic phenomenon has acquired particular connotations that require specific interventions in this field. Moreover, it also reveals that working frameworks and multidisciplinary action is required in order to prevent and treat the epidemic.

#### **6. Acknowledgment**

560 Recent Translational Research in HIV/AIDS

The discussion group was performed among the population segment of participants who did not know if they were seropositive and/or those whose blood tests results were negative. Since it is a group technique, it has the advantage of confronting and gathering discourse from the different social groups in one session. At the same time, in economical

The different points of view of the studied reality regarding the position occupied by the individual in the social framework can be known through the application of discussion groups (ref.). This used to be a key aspect of the technical selection since it allowed the gathering of different HIV related discourses in order to produce and adapt prevention strategies to the specificities of each social group. Therefore, the application of such technique created diverse knowledge about the different social aspects regarding the

Finally, fieldwork based on qualitative methodological strategies was done with the aim of generating knowledge about the HIV phenomenon. Prior to the phase of gathering information, the research team applied the non-participant observation technique. It was aimed at validating different aspects of the study protocol such as sample design or instruments to collect information. It was also aimed at later development of fieldwork. Geographically, this research took place in Bata between January and April 2010 and it was carried out by a multidisciplinary working group made up of sociologists, doctors, nurses

In terms of results achieved, the application of qualitative methodology has turned out to be very adequate and valid in order to generate holistic and comprehensive information of the HIV epidemic phenomenon in Equatorial Guiena. So that data has been generated in order to allow both evaluating some of the prevention strategies in the country and showing some of the key aspects to take into account in decision-making processes in the strategy to be

For instance, results show the inadequacy of massive HIV/Aids information and prevention campaigns carried out in Equatorial Guinea. The association established between the HIV, Aids and death has caused much alarm and fear among the population. Nowadays, it constitutes one of the main barriers for the population to have diagnostic tests done of their own free will. The conducted campaigns played a decisive role in the HIV social construction, which does not favour the integration of people living with Aids into society. Such social construction about people living with the HIV having abandoned the services of the HIV treatment and diagnosis, influenced the reasons given in order to explain why they

Results have also revealed the importance and necessity of implementing and carrying out prevention strategies considering already existing specificities and needs among different social groups. In this sense, interviewed women's and men's different ways of understanding health and facing the disease are shown. These aspects must be taken into

**4.3.2 The reason why the discussion group is applied to the study** 

terms, this technique becomes more profitable

HIV.

**4.3.3 Fieldwork** 

and lab assistants.

**5. Conclusions** 

implemented.

decided to give up their treatment.

To the people who took part in interviews and discussion groups, for sharing their time and HIV experiences with the research team. To Jesús Nzang for the generosity that he showed during the development of fieldwork. His dedication and collaboration made it possible to overcome the problems found during the data collection. To Catalina Mangue, Gaspar Nkoni and Luis Mbonio from the CRCE in Equatorial Guinea, for the support they gave me during the recruitment of participants. To Doctor Nemesio Abeso, Director of the MINSABS National Plan of Fight against Aids in Malabo, and Pilar Aparicio, for his support and confidence in the study for his support and confidence in the study. To Jose Luís Cenal, Leticia Díaz and Alberto Hedo from the Foundation for International Health and Cooperation of the National Health System of Spain, for his assistance in the logistic management of the project. And To the Spanish Agency for International Development Cooperation, for financing the study. The study was co-funded by the Instituto de Salud Carlos III, Spanish Research Network of Tropical Diseases (RICET;RD06/0021/0000), Ministry of Science and Innovation of Spain.

It has been possible to write these pages thanks to the contributions made by all these people and institutions.

#### **7. References**


The Pertinence of Applying Qualitative

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### *Edited by Yi-Wei Tang*

The collective efforts of HIV/AIDS research scientists from over 16 countries in the world are included in the book. This 27-chapter Open Access book well covers HIV/ AIDS translational researches on pathogenesis, diagnosis, treatment, prevention, and also those beyond conventional fields. These are by no means inclusive, but they do offer a good foundation for the development of clinical patient care. The translational model forms the basis for progressing HIV/AIDS clinical research. When linked to the care of the patients, translational researches should result in a direct benefit for HIV/ AIDS patients.

Recent Translational Research in HIV/AIDS

Recent Translational Research

in HIV/AIDS

*Edited by Yi-Wei Tang*

Photo by Rost-9D / iStock