**4. Vaccine research in NHP models**

The high degree of variability among HIV-1 strains and the lack of defined correlates of immune protection in HIV-1 infected individuals or SIV/SHIV NHP models have collectively posed a considerable challenge for the development of a vaccine that confers sterilizing immunity. Vaccine design thus far has focused heavily on the induction of T-cell immunity, since HIV-1 neutralizing antibodies have been difficult to induce and do not play a dominant role in the control of viral load. Early vaccines employing recombinant HIV-1 envelope glycoprotein were efficient in neutralizing lab-adapted HIV-1 strains but not primary isolates . Furthermore, several in vivo studies in macaques have demonstrated that while certain humoral-based vaccine candidates conferred partial protection to animals challenged with SHIV strains, no protection was observed against challenges with more pathogenic SIVmac strains . However, this does not entirely rule out a role for humoral immunity, since a prime boost vaccination approach in a study involving SIVmne gp160 was shown to be effective in protecting cynomolgus macaques against intrarectal challenges with uncloned SIVmne and protection was associated with the development of SIV-specific neutralizing antibodies [180]. Similar approaches involving a recombinant vaccinia virus or baculovirus expressing SIVmac239 gp160 did not protect rhesus macaques against intravenous challenges with homologous SIVmac239 or heterologous SIVmac251 strains [181].

Although it is evident that a skewed focus on eliciting broad neutralizing antibodies will not suffice, it has also become clear that while HIV-1 gag vaccines can be strongly immunogenic, a potent T-cell response does not necessarily translate to protection. This was especially highlighted by the Merck STEP clinical trial which showed that following administration of a replication defective recombinant adenovirus 5 (rAd5) expressing HIV-1 subtype B Gag/Pol/Nef, those vaccine recipients who were already seropositive for Ad5 had a higher incidence of HIV-1 infection [182]. This is in contrast to preceding preclinical studies in which rhesus macaques exhibited a high level of both the magnitude and duration of virusspecific immune responses following a DNA prime- rAd5 boost regimen, and were protected against challenges with SHIV89.6 [183]. Subsequent studies demonstrated that the Ad5 vaccine did not protect against challenge with SIVmac239, and reduced viral loads only in animals with the protective MHC class I allele Mamu A\*01 [184]. Furthermore, male rhesus macaques that were chronically infected with a host-range mutant Ad5 prior to immunization with an Ad5 vector expressing SIVmac239 Gag/Pol/Nef had a higher rate of infection following challenge with an escalating dose of SIVmac251 via penile exposure, recapitulating the outcome of the human clinical trial [179]. However unlike the Merck trial, the Ad5 immunized macaques showed a lower acute-phase viremia than the unimmunized animals.

The STEP Trial outcome led to a significant overhaul in the design and execution of vaccine studies. It was suggested that NHP models could not always be relied upon as a "gatekeeper" for determining go/no-go criteria. However NHP are the only animal models that best reflect many facets of HIV infection in humans, and therefore continue to a play a pivotal role in comparative and retrospective studies which can simultaneously inform vaccine strategies of both ongoing and future clinical trials.

334 Immunodeficiency

Ad5 seropositive individuals [179].

**4. Vaccine research in NHP models** 

well. The above rhesus macaque penile transmission study was used in an attempt to recapitulate the findings of the Merck Step trial which revealed enhanced HIV-1 infection in

The high degree of variability among HIV-1 strains and the lack of defined correlates of immune protection in HIV-1 infected individuals or SIV/SHIV NHP models have collectively posed a considerable challenge for the development of a vaccine that confers sterilizing immunity. Vaccine design thus far has focused heavily on the induction of T-cell immunity, since HIV-1 neutralizing antibodies have been difficult to induce and do not play a dominant role in the control of viral load. Early vaccines employing recombinant HIV-1 envelope glycoprotein were efficient in neutralizing lab-adapted HIV-1 strains but not primary isolates . Furthermore, several in vivo studies in macaques have demonstrated that while certain humoral-based vaccine candidates conferred partial protection to animals challenged with SHIV strains, no protection was observed against challenges with more pathogenic SIVmac strains . However, this does not entirely rule out a role for humoral immunity, since a prime boost vaccination approach in a study involving SIVmne gp160 was shown to be effective in protecting cynomolgus macaques against intrarectal challenges with uncloned SIVmne and protection was associated with the development of SIV-specific neutralizing antibodies [180]. Similar approaches involving a recombinant vaccinia virus or baculovirus expressing SIVmac239 gp160 did not protect rhesus macaques against intravenous

challenges with homologous SIVmac239 or heterologous SIVmac251 strains [181].

Although it is evident that a skewed focus on eliciting broad neutralizing antibodies will not suffice, it has also become clear that while HIV-1 gag vaccines can be strongly immunogenic, a potent T-cell response does not necessarily translate to protection. This was especially highlighted by the Merck STEP clinical trial which showed that following administration of a replication defective recombinant adenovirus 5 (rAd5) expressing HIV-1 subtype B Gag/Pol/Nef, those vaccine recipients who were already seropositive for Ad5 had a higher incidence of HIV-1 infection [182]. This is in contrast to preceding preclinical studies in which rhesus macaques exhibited a high level of both the magnitude and duration of virusspecific immune responses following a DNA prime- rAd5 boost regimen, and were protected against challenges with SHIV89.6 [183]. Subsequent studies demonstrated that the Ad5 vaccine did not protect against challenge with SIVmac239, and reduced viral loads only in animals with the protective MHC class I allele Mamu A\*01 [184]. Furthermore, male rhesus macaques that were chronically infected with a host-range mutant Ad5 prior to immunization with an Ad5 vector expressing SIVmac239 Gag/Pol/Nef had a higher rate of infection following challenge with an escalating dose of SIVmac251 via penile exposure, recapitulating the outcome of the human clinical trial [179]. However unlike the Merck trial, the Ad5 immunized macaques showed a lower acute-phase viremia than the unimmunized animals.

The STEP Trial outcome led to a significant overhaul in the design and execution of vaccine studies. It was suggested that NHP models could not always be relied upon as a Since the Merck STEP Trial, several NHP studies employing various types and combinations of HIV-1 antigen prime-boost vaccines have been conducted, with varying degrees of success. Rhesus macaques receiving a plasmid DNA prime and rAd5 vector expressing SIVmac239 env/gag/pol boost vaccine regimen, and challenged intrarectally for 12 weeks with either SIVmac251 or the heterogeneous SIVsmE660, exhibited 50% protection from infection with the latter virus strain [185]. In addition, among the SIVsmE660-infected animals, those expressing the Mamu-A\*01 MHC class I allele were found to have a log lower plasma peak viremia. The vaccinated Mamu-A\*01 negative animals in the SIVsmE660 group that were protected were also shown to express low levels of neutralizing antibodies and an envelope-specific CD4+ T cell response, highlighting roles for both humoral and cellular arms of the immune system. The presence of homozygous restrictive, allelic forms of the TRIM5alpha was shown to be associated with protection from infection [185]. The most recent, and perhaps most successful, vaccine study was the RV144 trial conducted in Thailand [186]. The vaccine candidates included a canarypox viral vector vaccine encoding clade B gag/pro and Clade E env as the prime (ALVAC-HIV vCP1521), and a boost with AIDSVAX gp120 B/E which is genetically engineered HIV-1 gp120 from both Clade B and E. Spanning over a six year period, this Phase IIb trial had an approximately 31% protection rate against HIV acquisition. While modest, this level of protection nonetheless re-energized the vaccine field, and several studies are underway in NHP models to recapitulate the results from the clinical trial, with the hope of identifying the specific immune response(s) that is responsible for protection.

The outcomes of HIV/SIV/SHIV vaccine trials thus far have made it apparent that the rate and level of virus acquisition and/or replication are at present, the only reliable factors when deciding the efficacy of a vaccine candidate, since the immune responses required for vaccine efficacy remain undefined. However, it is clear that NHP studies need to carefully account for the challenge virus as well as the genetic background of the macaque species, and perhaps standardize or implement more rigorous vaccine protocols to afford better predictive power and/or help in the identification and exclusion of confounding factors.

#### **5. The role of the NHP model for Pre-exposure Prophylaxis**

Microbicides are inhibitory compounds that when applied vaginally or rectally will prevent or reduce the likelihood of HIV transmission . PrEP is defined as the use of antiretroviral (ARV) drugs among HIV- negative individuals to prevent the acquisition of HIV. The successful use of ARVs in the treatment of HIV-infected individuals as post-exposure prophylaxis (PEP) and the associated knowledge gained on their safety have led the way for their use as PrEP agents. Various formulations of microbicides have been developed for rectal and vaginal application such as gels, films, suppositories (tablets) and intravaginal rings. It is necessary that a product that is destined for topical use is safe and widely acceptable, thereby promoting and enhancing adherence, cost effective and be able to deliver the drug at a high enough concentration locally to prevent the acquisition of HIV. The primary focus of microbicides has been placed on a coitally-dependent gel strategy, the method of choice for microbicide formulations, and intravaginal rings (IVR) that provide a sustained release of drugs over prolonged periods of time in a coitally-independent fashion.

Simian-Human Immunodeficiency Viruses and Their Impact on Non-Human Primate Models for AIDS 337

**Administration**

Botswana Oral TDF/

Oral i.TDF/

Oral TDF/

Oral TDF/

microbicide gel

i. Oral ii. Vaginal microbicide gel

TFV- Tenofovir, TDF-Tenofovir disproxil fumarate, a Both arms werehalted for futility. Final data from VOICE trial has

Several ARV based microbicides are currently under preclinical development in non-human primates to inhibit HIV replication at various stages of its lifecycle. The microbicides are classified based on the step that it inhibits in replication cycle of HIV such as entry, reverse transcription of its RNA genome, integration into the host chromosome, translation of new

Entry inhibitors - Binding of HIV gp120 to CD4 on T helper cells and macrophages triggers conformational changes in gp120 that allows binding to the CCR5 or CXCR4 co-receptor. Next, the gp41 ectodomain forms a six- helix bundle that allows close proximity of the viral and cell membranes leading to fusion . Small molecule inhibitors that bind gp120 and prevent attachment to CD4 such as BMS-378806 have been shown to be effective as a vaginal microbicide in rhesus macaques upon a high dose challenge. BMS-378806 in combination with C52L, a bacterially expressed gp41-mediated fusion inhibitor peptide, protected

**Table 4.** ARV based oral and topical PrEP trials among different populations: Adapted from [197].

**5.1. Antiretroviral inhibitors implemented in PrEP microbicide products** 

**Drug Effect** 

Emtricitabine ii. TDF

emtricitabine

emtricitabine

i. TFV

emtricitabine

1%TFV 39%

ii. 1%TFV Ineffectivea

**Size (95% CI)** 

73% (49-85)

62% (34-78)

63% (22-83)

44% (15-63)

(6-60)

0% (-69 -41)

**Country Route of** 

Kenya, Uganda

America, USA, South Africa, Thailand

Uganda, South

Africa, Zimbabwe

Kenya, Tanzania, South Africa

viral proteins, release and maturation of the progeny virions.

South Africa Vaginal

**Trial Study** 

Partners PrEP [197]

 CDC sponsored TDF2 [233]

CAPRISA-004 [198]

VOICE [234,

FEM-PrEP [200]

not been announced.

235]

**population** 

serodiscordant

Heterosexual men and women

Sexually active HIV uninfected

HIV-negative women

HIV uninfected

iPrEX [227] MSM South

women

women

HIV

couples

Though Sub-Saharan Africa is home to only 10% of the world's population it contains every two of three people living with HIV. More than 60% of the people living with HIV in Sub-Saharan Africa are women, and of these 75% are between the ages of 15 and 24 [187]. The increasing risk associated with women and their inability to negotiate consistent condom use or monogamy emphasizes the need for the development of female-controlled methods of prevention of HIV acquisition. It was predicted that a vaginal microbicide that is 50% efficacious may prevent 33% of HIV infections in a period of 25 years upon 75% usage [188].

The effectiveness of PrEP in preventing mucosal infections with HIV will be influenced by the delivery of ARVs to a protective level at the mucosal site of transmission. The NHP model not only provides an experimentally controlled platform for the safety and pharmacokinetic evaluation of microbicides, but can also be used in the evaluation of efficacy in preventing mucosal transmission of HIV [189-192]. It is of utmost important for microbicides that are targeted for topical use in preventing sexually transmitted infections, such as HIV, to be tested in animal models prior to human trials. The sexual transmission of HIV involves a biologically complex milieu comprising initial infection among target cells at the port of entry (vaginal or rectal), the establishment of a small founder population, and local expansion to establish systemic infection . This poorly understood process cannot be properly evaluated in vitro. The recent failure and enhanced transmission observed in the first microbicide efficacy trials with nonoxynol-9 and Savvy [193, 194] warrant the need for controlled and careful investigation of topical products in animal models. A phase II/III trial with nonoxynol-9, an over the counter spermicide, in a vaginal gel formulation increased the risk of acquisition of HIV among users of the gel [195]. The detrimental effects associated with the multiple vaginal application of nonoxynol-9 such as epithelial disruption and inflammatory infiltration was also demonstrated in pig-tailed macaques [196].

The pharmacokinetic and pharmacodynamics evaluation of microbicides in animal models allows for the determination of not only the accumulation of ARVs in mucosal tissues , but also of the minimal effective dosing and the optimal timing with regards to the periods of virus exposure. The nonhuman primate model has been pivotal in producing preclinical data that can inform clinical trial design is this new and exciting field of prevention. There are several ARV PrEP candidates that have great potential for topical application and here we describe a few select ARVs and delivery methods that have progressed through initial preclinical evaluation in the NHP model. The implementation of ARVs for pre-exposure prophylaxis (PrEP) of HIV both as oral and topical applications is currently being investigated as outlined among the different trials in Table 4 [197-200].

rings. It is necessary that a product that is destined for topical use is safe and widely acceptable, thereby promoting and enhancing adherence, cost effective and be able to deliver the drug at a high enough concentration locally to prevent the acquisition of HIV. The primary focus of microbicides has been placed on a coitally-dependent gel strategy, the method of choice for microbicide formulations, and intravaginal rings (IVR) that provide a sustained release of drugs over prolonged periods of time in a coitally-independent fashion. Though Sub-Saharan Africa is home to only 10% of the world's population it contains every two of three people living with HIV. More than 60% of the people living with HIV in Sub-Saharan Africa are women, and of these 75% are between the ages of 15 and 24 [187]. The increasing risk associated with women and their inability to negotiate consistent condom use or monogamy emphasizes the need for the development of female-controlled methods of prevention of HIV acquisition. It was predicted that a vaginal microbicide that is 50% efficacious may prevent 33% of HIV infections in a period of 25 years upon 75% usage [188].

The effectiveness of PrEP in preventing mucosal infections with HIV will be influenced by the delivery of ARVs to a protective level at the mucosal site of transmission. The NHP model not only provides an experimentally controlled platform for the safety and pharmacokinetic evaluation of microbicides, but can also be used in the evaluation of efficacy in preventing mucosal transmission of HIV [189-192]. It is of utmost important for microbicides that are targeted for topical use in preventing sexually transmitted infections, such as HIV, to be tested in animal models prior to human trials. The sexual transmission of HIV involves a biologically complex milieu comprising initial infection among target cells at the port of entry (vaginal or rectal), the establishment of a small founder population, and local expansion to establish systemic infection . This poorly understood process cannot be properly evaluated in vitro. The recent failure and enhanced transmission observed in the first microbicide efficacy trials with nonoxynol-9 and Savvy [193, 194] warrant the need for controlled and careful investigation of topical products in animal models. A phase II/III trial with nonoxynol-9, an over the counter spermicide, in a vaginal gel formulation increased the risk of acquisition of HIV among users of the gel [195]. The detrimental effects associated with the multiple vaginal application of nonoxynol-9 such as epithelial disruption and

inflammatory infiltration was also demonstrated in pig-tailed macaques [196].

investigated as outlined among the different trials in Table 4 [197-200].

The pharmacokinetic and pharmacodynamics evaluation of microbicides in animal models allows for the determination of not only the accumulation of ARVs in mucosal tissues , but also of the minimal effective dosing and the optimal timing with regards to the periods of virus exposure. The nonhuman primate model has been pivotal in producing preclinical data that can inform clinical trial design is this new and exciting field of prevention. There are several ARV PrEP candidates that have great potential for topical application and here we describe a few select ARVs and delivery methods that have progressed through initial preclinical evaluation in the NHP model. The implementation of ARVs for pre-exposure prophylaxis (PrEP) of HIV both as oral and topical applications is currently being


TFV- Tenofovir, TDF-Tenofovir disproxil fumarate, a Both arms werehalted for futility. Final data from VOICE trial has not been announced.

**Table 4.** ARV based oral and topical PrEP trials among different populations: Adapted from [197].

#### **5.1. Antiretroviral inhibitors implemented in PrEP microbicide products**

Several ARV based microbicides are currently under preclinical development in non-human primates to inhibit HIV replication at various stages of its lifecycle. The microbicides are classified based on the step that it inhibits in replication cycle of HIV such as entry, reverse transcription of its RNA genome, integration into the host chromosome, translation of new viral proteins, release and maturation of the progeny virions.

Entry inhibitors - Binding of HIV gp120 to CD4 on T helper cells and macrophages triggers conformational changes in gp120 that allows binding to the CCR5 or CXCR4 co-receptor. Next, the gp41 ectodomain forms a six- helix bundle that allows close proximity of the viral and cell membranes leading to fusion . Small molecule inhibitors that bind gp120 and prevent attachment to CD4 such as BMS-378806 have been shown to be effective as a vaginal microbicide in rhesus macaques upon a high dose challenge. BMS-378806 in combination with C52L, a bacterially expressed gp41-mediated fusion inhibitor peptide, protected macaques against vaginal challenge [201]. T1249, another fusion inhibitor, was also effective as a vaginal gel formulation against a variety of SHIV in macaques [202]. Cyanovirin, a cyanobacterial protein that binds non-competitively to gp120, was also effective in protecting pig-tailed macaques against vaginal infection [203]. CCR5 antagonists CMPD167 and maraviroc, and modified chemokines such as PSC-Rantes have shown protection in rhesus macaques against RLD vaginal challenge with SHIV [201, 204, 205].

Simian-Human Immunodeficiency Viruses and Their Impact on Non-Human Primate Models for AIDS 339

Vaginal combination gels containing zinc acetate dehydrate and the NNRTI MIV-150 provided complete protection in rhesus macaques against RT-SHIV up to 24 hours following 2 weeks of daily gel application. Partial protection was seen with formulations containing zinc acetate or MIV alone [214]. MC 1220 in a gel formulation also provided

Integrase inhibitors - HIV integrase is essential for incorporation of the viral genome into the host DNA and is an essential event for viral replication. Inhibitors that block this process are actively being developed for therapeutic applications and are just beginning to be investigated for PrEP. Because the integration step occurs later in the replication cycle than entry and reverse transcription, administration of integrase inhibitors may be effective when used as post-exposure prophylaxis (PEP). To address this question in the NHP model, topically applied L-870812 was evaluated for efficacy in preventing vaginal transmission of SHIV162P3 in a repeat low-dose macaque model. Pigtail macaques received 3 mL of a 0.2% L-870812 gel 30 minutes after intravaginal virus exposure with SHIV162P3 and partial efficacy was observed [216]. Further investigation of integrase inhibitors as sole PrEP agents and in combination with other PrEP agents is warranted given these encouraging results. A combination of ARVs that act at different stages of viral replication will theoretically

There are many different delivery platforms available for PrEP and these are being tested in NHP models. Many of the studies described above employ conventional gel formulations and are associated with problems such as leakage and the need to administer the gel shortly before every act of intercourse to prevent HIV acquisition. In addition there is also the lack of covertness with gel application which makes it difficult for women who need to use the microbicide without the knowledge of their partner. However, topical applications like gels, tablets, and films are administered directly to the site of transmission and very high local tissue levels can be achieved [217, 218]. Oral and injectable dosing is preferable in some settings, but the drug is delivered systemically, not locally, and therefore higher and more

Alternative delivery platforms are being investigated in the NHP model to overcome some of the problems encountered with conventional dosing methods. For instance, intravaginal rings (IVR), such as those commercially available for contraception, can help overcome some of the barriers associated with conventional gel formulations and delivery. IVRs are torus shaped flexible drug delivery devices that are self –inserted and when placed is located close to the cervix in the upper two-thirds of the vagina and provide sustained release of one or more drugs for mucosal and possibly systemic effects. The advantageous properties of IVRs such as the capacity to provide sustained and controlled release of drugs over extended periods of time, non-coital dependency, and the need for a single application in women of only once a month or every few months, are beginning to be exploited in the field

partial protection against RT-SHIV in rhesus macaques [215].

provide broader protection.

of microbicides [219-223].

**5.2. Drug delivery vehicles** 

frequent dosing may be required for protection.

Reverse transcriptase inhibitors - Nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTI) block reverse transcriptase activity. The NRTIs such as zidovudine, lamivudine, and Tenofovir (TFV) require phosphorylation by host cell enzymes to their pharmacologically active triphosphate (TP) anabolite [206-208]. The TPs are analogs of endogenous 2'-deoxynucleotides (dNTPs) and compete for incorporation into the growing HIV DNA chain by HIV reverse transcriptase leading to chain termination .

The potential of TFV for preventing acquisition of HIV derives from its capacity to prevent SIV infection in rhesus macaques. After four weeks subcutaneous administration of once daily TFV beginning 48 hours before and up to 24 hours after intravenous inoculation of SIV, macaques were protected against systemic infection. The macaques showed no evidence of virus in the plasma or PBMC for 56 weeks. Lymphoid tissues and major organs obtained from healthy euthanized animals 40 weeks post inoculation were also free of SIV. The efficacy of TFV and Truvada (TDF and emtricitabine) as PrEP agents was proven with repeated exposures to physiological equivalents of SHIV162P3 in rhesus macaques [153, 209, 210]. Intermittent dosing with an oral pre-exposure dose 1, 3 or 7 days before virus exposure followed by a dose of TDF/emtricitabine 2 hours after exposure was associated with a 16.7, 15.3, and 9.4 factor reduction respectively in comparison to the controls against rectal SHIV162P3 protection. No protection against rectal SHIV162P3 exposure was observed if the first dose was delayed up to 24 hours after exposure emphasizing the need for interdicting the initial replication events [211].

TFV alone (1%) or in combination with emtricitabine (5%) in a vaginal gel formulation was also effective in protecting pig-tailed macaques against a repeat low-dose exposure to SHIV162P3 [212]. The correlation of intracellular TFV-DP levels in vaginal tissue lymphocytes at the time of vaginal exposure and reduced efficacy in protecting pigtail macaques was demonstrated recently with intermittent application of a 1%TFV gel once per week and virus exposures occurring twice weekly. It was estimated that the median TFV-DP concentrations were 1810 fmol/106 cells at 4 hours and above 1000 fmol/106 cells in the vaginal lymphocytes that were obtained from animals necropsied at 1 and 2 days after gel application. However, the median TFV-DP concentrations dropped to 252 fmol/106 cells 3 days after gel application which correlated to 74% efficacy [213]. This study was therefore able to find a direct correlate between intracellular TFV-DP levels and efficacy in the nonhuman primate model.

NNRTIs differ from NRTIs in binding to the reverse transcriptase outside of the active site and have been shown to be efficacious in the vaginal SHIV challenge models in macaques. Vaginal combination gels containing zinc acetate dehydrate and the NNRTI MIV-150 provided complete protection in rhesus macaques against RT-SHIV up to 24 hours following 2 weeks of daily gel application. Partial protection was seen with formulations containing zinc acetate or MIV alone [214]. MC 1220 in a gel formulation also provided partial protection against RT-SHIV in rhesus macaques [215].

Integrase inhibitors - HIV integrase is essential for incorporation of the viral genome into the host DNA and is an essential event for viral replication. Inhibitors that block this process are actively being developed for therapeutic applications and are just beginning to be investigated for PrEP. Because the integration step occurs later in the replication cycle than entry and reverse transcription, administration of integrase inhibitors may be effective when used as post-exposure prophylaxis (PEP). To address this question in the NHP model, topically applied L-870812 was evaluated for efficacy in preventing vaginal transmission of SHIV162P3 in a repeat low-dose macaque model. Pigtail macaques received 3 mL of a 0.2% L-870812 gel 30 minutes after intravaginal virus exposure with SHIV162P3 and partial efficacy was observed [216]. Further investigation of integrase inhibitors as sole PrEP agents and in combination with other PrEP agents is warranted given these encouraging results. A combination of ARVs that act at different stages of viral replication will theoretically provide broader protection.

#### **5.2. Drug delivery vehicles**

338 Immunodeficiency

chain termination .

interdicting the initial replication events [211].

nonhuman primate model.

macaques against vaginal challenge [201]. T1249, another fusion inhibitor, was also effective as a vaginal gel formulation against a variety of SHIV in macaques [202]. Cyanovirin, a cyanobacterial protein that binds non-competitively to gp120, was also effective in protecting pig-tailed macaques against vaginal infection [203]. CCR5 antagonists CMPD167 and maraviroc, and modified chemokines such as PSC-Rantes have shown protection in

Reverse transcriptase inhibitors - Nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTI) block reverse transcriptase activity. The NRTIs such as zidovudine, lamivudine, and Tenofovir (TFV) require phosphorylation by host cell enzymes to their pharmacologically active triphosphate (TP) anabolite [206-208]. The TPs are analogs of endogenous 2'-deoxynucleotides (dNTPs) and compete for incorporation into the growing HIV DNA chain by HIV reverse transcriptase leading to

The potential of TFV for preventing acquisition of HIV derives from its capacity to prevent SIV infection in rhesus macaques. After four weeks subcutaneous administration of once daily TFV beginning 48 hours before and up to 24 hours after intravenous inoculation of SIV, macaques were protected against systemic infection. The macaques showed no evidence of virus in the plasma or PBMC for 56 weeks. Lymphoid tissues and major organs obtained from healthy euthanized animals 40 weeks post inoculation were also free of SIV. The efficacy of TFV and Truvada (TDF and emtricitabine) as PrEP agents was proven with repeated exposures to physiological equivalents of SHIV162P3 in rhesus macaques [153, 209, 210]. Intermittent dosing with an oral pre-exposure dose 1, 3 or 7 days before virus exposure followed by a dose of TDF/emtricitabine 2 hours after exposure was associated with a 16.7, 15.3, and 9.4 factor reduction respectively in comparison to the controls against rectal SHIV162P3 protection. No protection against rectal SHIV162P3 exposure was observed if the first dose was delayed up to 24 hours after exposure emphasizing the need for

TFV alone (1%) or in combination with emtricitabine (5%) in a vaginal gel formulation was also effective in protecting pig-tailed macaques against a repeat low-dose exposure to SHIV162P3 [212]. The correlation of intracellular TFV-DP levels in vaginal tissue lymphocytes at the time of vaginal exposure and reduced efficacy in protecting pigtail macaques was demonstrated recently with intermittent application of a 1%TFV gel once per week and virus exposures occurring twice weekly. It was estimated that the median TFV-DP concentrations were 1810 fmol/106 cells at 4 hours and above 1000 fmol/106 cells in the vaginal lymphocytes that were obtained from animals necropsied at 1 and 2 days after gel application. However, the median TFV-DP concentrations dropped to 252 fmol/106 cells 3 days after gel application which correlated to 74% efficacy [213]. This study was therefore able to find a direct correlate between intracellular TFV-DP levels and efficacy in the

NNRTIs differ from NRTIs in binding to the reverse transcriptase outside of the active site and have been shown to be efficacious in the vaginal SHIV challenge models in macaques.

rhesus macaques against RLD vaginal challenge with SHIV [201, 204, 205].

There are many different delivery platforms available for PrEP and these are being tested in NHP models. Many of the studies described above employ conventional gel formulations and are associated with problems such as leakage and the need to administer the gel shortly before every act of intercourse to prevent HIV acquisition. In addition there is also the lack of covertness with gel application which makes it difficult for women who need to use the microbicide without the knowledge of their partner. However, topical applications like gels, tablets, and films are administered directly to the site of transmission and very high local tissue levels can be achieved [217, 218]. Oral and injectable dosing is preferable in some settings, but the drug is delivered systemically, not locally, and therefore higher and more frequent dosing may be required for protection.

Alternative delivery platforms are being investigated in the NHP model to overcome some of the problems encountered with conventional dosing methods. For instance, intravaginal rings (IVR), such as those commercially available for contraception, can help overcome some of the barriers associated with conventional gel formulations and delivery. IVRs are torus shaped flexible drug delivery devices that are self –inserted and when placed is located close to the cervix in the upper two-thirds of the vagina and provide sustained release of one or more drugs for mucosal and possibly systemic effects. The advantageous properties of IVRs such as the capacity to provide sustained and controlled release of drugs over extended periods of time, non-coital dependency, and the need for a single application in women of only once a month or every few months, are beginning to be exploited in the field of microbicides [219-223].

The initial safety and size guidelines to develop ring devices that are suitable for use in pig-tailed and rhesus macaques came from the administration of different sized rings and the close monitoring of the safety of these devices. Non-medicated silicone elastomer vaginal rings of 3 different sizes were administered to pig-tailed and Chinese rhesus macaques for a 28 day period [224]. No signs of inflammation or irritation were observed on colposcopic examinations and the animals showed no behavioral changes or other problems following insertion of the rings. Mucosal proinflammatory cytokines were unchanged in the presence of the rings (for 4 weeks) or upon removal (4 weeks post removal). Safety analyses of macaque-sized elastomeric silicone and polyurethane intravaginal rings (IVRs) loaded with candidate ARV drugs were tested in pig-tailed macaques in four studies ranging in duration from 49 to 73 days with retention of the IVR being 28 days in each study. The presence of IVRs not only made of silicone but other polymers such as, polyurethane in pig-tailed macaques does not cause an alteration longitudinally in the levels of the proinflammatory cytokines locally or systemically and in the vaginal microbiological patterns [225, 226]. Efficacy studies in the NHP model with IVRs are just beginning, but preliminary pharmacokinetic studies are very promising [227-235].

Simian-Human Immunodeficiency Viruses and Their Impact on Non-Human Primate Models for AIDS 341

*Laboratory Branch, Division of HIV/AIDs Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD, TB Prevention, Centers for Disease Control and Prevention, Atlanta,* 

We would like to thank Dr. Michael Hendry and Dr. Janet McNicholl for valuable input. Work performed at the Centers for Disease Control and Prevention was supported in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Interagency

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**Author details** 

*LRRI, Albuquerque, NM, USA* 

**Acknowledgement** 

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#### **6. Summary and outlook**

As we move forward in our endeavors to prevent HIV infections, it is clear that having viable animal models are a vital component of a comprehensive approach to develop and test biomedical preventions. The field of HIV treatment and prevention has broadened to include not only vaccine discovery and treatment of infected individuals to PEP, PrEP, combination therapies, and discussions of eradication and cure. The pharmaceutical discoveries of recent years have increased our options for PEP and PrEP, and vaccine designs are becoming much more sophisticated. As the prevention field moves forward we are constantly modifying the macaque model to accommodate new combinations of interventions. The new SHIVs will have to incorporate the elements necessary to evaluate vaccines and other prevention modalities both singly and in combination. The likelihood that future clinical vaccine trials will be conducted in concert with PrEP trials is very high, and the recombinant viruses we use in the NHP models have to keep pace in evaluating promising candidates in the most rigorous way possible. The nonhuman primate model has adapted to aid researchers in answering ever more complex questions surrounding the interaction of the virus, host, and antiretroviral drugs. The coming years will be very interesting and fruitful as we move towards our common goal; to make HIV and AIDS a disease of our past, not of our future.

#### **Disclaimer**

The findings and conclusions are those of the author and do not necessarily represent the official position of the Centers for Disease Control and Prevention.
