**Part 2**

## **Prevention of Mother to Child Transmission of HIV (PMTCT)**

74 Understanding HIV/AIDS Management and Care – Pandemic Approaches in the 21st Century

Sidhu, I.K. (2008). *Business Coalitions Tackling AIDS: A Worldwide Review*. Retrieved from

Swartz, S., Van der Heijden, I., Makoae, M., Richter, L., Rozani, A., Runciman, T. &

UNAIDS. (2008). *2008 Report on the Global AIDS Epidemic*. Retrieved from www.unaids.org. UNAIDS. (2009). *Monitoring the Declaration of Commitment on HIV/AIDS: Guidelines on* 

Weston, M.D., Churchyard, G.J., Mametja, D., McIntyre, J.A., & Randera, F. (2007). Business

and AIDS: sectoral challenges and opportunities. AIDS, Vol. 21, Suppl 3, pp. S85-

*Construction of Core Indicators, 2010 Reporting*. UNAIDS, Geneva. UNAIDS. (2010). *UNAIDS Report on the Global Aids Epidemic 2010*. UNAIDS , Geneva UNAIDS. (2011). *Civil Society and Private Sector Division.* Retrieved from www.unaids.org. United Nations. (2010). *Millennium Development Goals Report: 2010*. United Nations, New

Education, 31 March). Retrieved from www.hsrc.ac.za.

Ndimande, N. (2009). *With a little help from my friends': exploring the impact of peer-led HIV intervention and psychosocial support groups for orphaned and vulnerable children in South Africa.* (Report produced for the Harvard Centre for the Support of Peer

http://data.unaids.org.

York.

S89.

**5** 

*Oman* 

**Antenatal Screening and HIV-Pregnancy:** 

*1Head of Immunology Division, Department of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat,* 

*3Department of Microbiology and Immunology, College of Medicine and Health Sciences,* 

More than half of all people living with HIV are women and girls (WHO & UNAIDS, 2010). In sub-Saharan Africa, more women than men are living with HIV, and young women aged 15–24 years are as much as eight times more likely than men to be HIV positive (WHO & UNAIDS, 2010). In developed countries, the women who are intravenous drug users, partners of drug users or bisexual men, or involved in sex work are more likely to be positive for HIV (WHO & UNAIDS, 1999). There are higher proportions of young women than young men who acquired HIV infection through sex. Their exposure to the virus at an earlier age, coupled with physiological and sociological factors increases their risk (WHO &

Globally, HIV is the leading cause of death in women of reproductive age. Since nearly all HIV infections in children are acquired from their mothers, the global epidemiology of HIV in children reflects that of HIV in women. Nearly all such infections can be prevented by programs providing highly effective antiretroviral therapy (ART) and antiretroviral (ARV)

There are tremendous efforts to control HIV/AIDS and reduce the mortality and morbidity rates by improving the accessibility to ART to all HIV- infected patients, trying to eradicate the virus from reservoirs of infection and designing an effective vaccine that can elicit protective antibody response as well as cell mediated response against HIV (Al-Jabri & Al-Enzi, 2009). An important step in the fight against HIV/AIDS is to perform antenatal

HIV testing of individuals should be undertaken only when they are informed about the test and should be entirely voluntary. HIV testing and counseling enable women to learn whether they are infected, understand their HIV status and make more informed choices for

**2. The importance of antenatal screening and counseling** 

**1. Introduction** 

UNAIDS, 1999).

prophylaxis interventions.

screening and counseling.

**Strategies for Treatment** 

Ali A. Al-Jabri1**,** Abdullah A. Balkhair2**,** 

*Sultan Qaboos University Hospital,Muscat,* 

*Sultan Qaboos University, Muscat,* 

Mohammed S. Al-Balosh3 and Sidgi S. Hasson3

*2Head of Infectious Diseases Unit, Department of Medicine,* 

## **Antenatal Screening and HIV-Pregnancy: Strategies for Treatment**

Ali A. Al-Jabri1**,** Abdullah A. Balkhair2**,** 

Mohammed S. Al-Balosh3 and Sidgi S. Hasson3

*1Head of Immunology Division, Department of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, 2Head of Infectious Diseases Unit, Department of Medicine, Sultan Qaboos University Hospital,Muscat, 3Department of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Oman* 

## **1. Introduction**

More than half of all people living with HIV are women and girls (WHO & UNAIDS, 2010). In sub-Saharan Africa, more women than men are living with HIV, and young women aged 15–24 years are as much as eight times more likely than men to be HIV positive (WHO & UNAIDS, 2010). In developed countries, the women who are intravenous drug users, partners of drug users or bisexual men, or involved in sex work are more likely to be positive for HIV (WHO & UNAIDS, 1999). There are higher proportions of young women than young men who acquired HIV infection through sex. Their exposure to the virus at an earlier age, coupled with physiological and sociological factors increases their risk (WHO & UNAIDS, 1999).

Globally, HIV is the leading cause of death in women of reproductive age. Since nearly all HIV infections in children are acquired from their mothers, the global epidemiology of HIV in children reflects that of HIV in women. Nearly all such infections can be prevented by programs providing highly effective antiretroviral therapy (ART) and antiretroviral (ARV) prophylaxis interventions.

There are tremendous efforts to control HIV/AIDS and reduce the mortality and morbidity rates by improving the accessibility to ART to all HIV- infected patients, trying to eradicate the virus from reservoirs of infection and designing an effective vaccine that can elicit protective antibody response as well as cell mediated response against HIV (Al-Jabri & Al-Enzi, 2009). An important step in the fight against HIV/AIDS is to perform antenatal screening and counseling.

### **2. The importance of antenatal screening and counseling**

HIV testing of individuals should be undertaken only when they are informed about the test and should be entirely voluntary. HIV testing and counseling enable women to learn whether they are infected, understand their HIV status and make more informed choices for

Antenatal Screening and HIV-Pregnancy: Strategies for Treatment 79

There are a number of mechanisms through which zidovudine or other ARV drugs can reduce PT. One central mechanism is by decreasing maternal viral load in the blood and genital secretions via antenatal drug administration, particularly in women with high viral loads. However, ARV drugs have been shown to reduce the risk of transmission even among women with HIV RNA levels <1,000 copies/ml (Ioannidis et al. 2001). Additionally, the level of HIV RNA at delivery and receipt of antenatal ART are each independently associated with the risk of transmission, suggesting that ARV prophylaxis does not work

An additional mechanism of protection is pre-exposure infant prophylaxis provided by administration of ARV drugs that cross the placenta from the mother to the infant, resulting in adequate systemic drug levels in the infant. This mechanism of protection is particularly significant during the infant's passage through the birth canal, a time of rigorous exposure to maternal genital tract virus. Post-exposure infant prophylaxis is provided through administration of drug to the infant after birth. This mechanism protects the infant from cellfree or cell-associated virus that might have obtained access to the fetal/infant systemic circulation. This can occur through maternal-fetal transfusion during uterine contractions in labor or through systemic dissemination of virus swallowed by the infant during passage

It is predictable that efficacy of ARV drugs in reducing PT is multi-factorial, and each of these mechanisms is contributory. The efficacy of ARV regimens administered only during labor and/or to the newborn in reducing PT demonstrates the importance of the pre- and

In PACTG 076, antenatal maternal HIV RNA copy number was associated with HIV transmission in women receiving placebo. In women receiving zidovudine, the relationship was markedly attenuated and no longer statistically significant (Sperling et al. 1996). An HIV RNA threshold below which there was no risk of transmission was not identified; zidovudine was effective in reducing transmission regardless of maternal HIV RNA copy number (Shapiro et al. 1999). Other data from larger numbers of zidovudine-treated, HIVinfected pregnant women indicate that HIV RNA levels correlate with risk of transmission

Although the risk of PT in women with undetectable HIV RNA levels appears to be extremely low, transmission from mother to infant has been reported among women with all levels of maternal HIV RNA. Additionally, although HIV RNA may be an important risk factor for transmission, other factors also appear to play a role (Mock et al. 1999). Although there is a general correlation between viral load in plasma and in the genital tract, discordance has also been reported, particularly between HIV proviral load in blood and genital secretions, especially in the presence of other genital tract infections (Hart et al.

The use of ARV drugs during pregnancy for prevention of PT should be discussed with and offered to all infected pregnant women regardless of their HIV RNA level. Results of epidemiologic and clinical trials suggest that women receiving potent combinations of ARV

even in women treated with ARV agents (The European Collaborative Study, 1999).

post-exposure components of prophylaxis in reducing PT (Wade et al. 1998).

**5. Perinatal transmission of HIV and maternal viral load** 

**4. Mechanisms of action of ARV prophylaxis in reducing perinatal** 

solely through reduction in viral load (Sperling et al. 1996).

**transmission of HIV** 

through the birth canal.

1999).

the future. HIV testing and counseling also provide essential knowledge and support: they enable uninfected women to remain so, enable those infected with HIV to plan for the future and prevent HIV transmission to others. Those who are infected can also benefit from available care, treatment and support services. Knowledge of HIV infection leads to initiate ART for the long-term treatment of women living with HIV and intervenes with the transmission of HIV infections to infants and young children.

HIV testing in pregnancy is important for many reasons, but this must be balanced against the possible risks of stigmatization, discrimination and violence. Voluntary counseling and testing should be encouraged for pregnant women and couples. Post-test counseling is extremely important following a diagnosis of HIV and should include information about pregnancy-related issues and the risk of mother to child transmission (MTCT). Counseling is also important for HIV-negative women as it provides an opportunity for risk-reduction.

HIV-infected women who know their sero-status are able to make informed choices about their reproductive lives and, if pregnant, to access specific interventions, such as ARV drugs and infant-feeding counseling and support, which can significantly reduce the risk of MTCT of HIV. Currently, the majority of people infected with HIV unaware of their status and cannot receive the services they actually need.

A key factor limiting the scale-up of MTCT programs is lack of knowledge of HIV status (Al-Jabri et al. 2010). Increasing the availability and acceptability of HIV testing and counseling services, with no dough, will encourage more women to know their status, providing a gateway to MTCT interventions (Bolu et al*.* 2007). Key factors contributing to the scale-up of testing and counseling include a policy of provider-initiated testing and counseling with the right to refuse testing; group pretest counseling; rapid HIV testing; innovative staffing strategies; and community and male involvement. Integration of testing and counseling within the community and all maternal and child health settings are critical for scaling-up and for linking women and their families to care and treatment services (Bolu et al*.* 2007).

## **3. Strategies for treatment**

Three types of interventions should be tackled by any program concerned with prevention of MTCT: primary prevention of HIV in women; prophylaxis with ARV drugs in breastfeeding infants and prophylaxis with ARV drugs for lactating mothers.

Taking ARV treatment can reduce the risk of MTCT. There are two different ways in which drugs can act. First, they may reduce the viral load so the baby is exposed to less of the virus while in the uterine and during childbirth. The aim of HIV treatment is to decrease the viral load <50 copies/ml. Second, the drugs may cross the placenta and enter the baby's body, where they can prevent the virus from ever taking hold.

One of the key attainments in HIV research was the demonstration by the Pediatric AIDS Clinical Trials Group 076 (PACTG 076) that administration of zidovudine to the pregnant woman and her infant could reduce the risk of perinatal transmission (PT) by nearly 70% (Connor et al. 1994). Following the results of PACTG 076, implementation of the zidovudine regimen coupled with increased antenatal HIV testing and counseling rapidly resulted in significant declines in HIV transmission (CDC, 2006). Subsequent clinical trials and observational studies showed that combination ARV prophylaxis (initially dual and then triple combination therapy) given to the mother antenatally was associated with further declines in transmission to less than 2% (Cooper et al. 2002, WHO 2010).

the future. HIV testing and counseling also provide essential knowledge and support: they enable uninfected women to remain so, enable those infected with HIV to plan for the future and prevent HIV transmission to others. Those who are infected can also benefit from available care, treatment and support services. Knowledge of HIV infection leads to initiate ART for the long-term treatment of women living with HIV and intervenes with the

HIV testing in pregnancy is important for many reasons, but this must be balanced against the possible risks of stigmatization, discrimination and violence. Voluntary counseling and testing should be encouraged for pregnant women and couples. Post-test counseling is extremely important following a diagnosis of HIV and should include information about pregnancy-related issues and the risk of mother to child transmission (MTCT). Counseling is also important for HIV-negative women as it provides an opportunity for risk-reduction. HIV-infected women who know their sero-status are able to make informed choices about their reproductive lives and, if pregnant, to access specific interventions, such as ARV drugs and infant-feeding counseling and support, which can significantly reduce the risk of MTCT of HIV. Currently, the majority of people infected with HIV unaware of their status and

A key factor limiting the scale-up of MTCT programs is lack of knowledge of HIV status (Al-Jabri et al. 2010). Increasing the availability and acceptability of HIV testing and counseling services, with no dough, will encourage more women to know their status, providing a gateway to MTCT interventions (Bolu et al*.* 2007). Key factors contributing to the scale-up of testing and counseling include a policy of provider-initiated testing and counseling with the right to refuse testing; group pretest counseling; rapid HIV testing; innovative staffing strategies; and community and male involvement. Integration of testing and counseling within the community and all maternal and child health settings are critical for scaling-up and for linking women and their families to care and treatment services (Bolu et al*.* 2007).

Three types of interventions should be tackled by any program concerned with prevention of MTCT: primary prevention of HIV in women; prophylaxis with ARV drugs in

Taking ARV treatment can reduce the risk of MTCT. There are two different ways in which drugs can act. First, they may reduce the viral load so the baby is exposed to less of the virus while in the uterine and during childbirth. The aim of HIV treatment is to decrease the viral load <50 copies/ml. Second, the drugs may cross the placenta and enter the baby's body,

One of the key attainments in HIV research was the demonstration by the Pediatric AIDS Clinical Trials Group 076 (PACTG 076) that administration of zidovudine to the pregnant woman and her infant could reduce the risk of perinatal transmission (PT) by nearly 70% (Connor et al. 1994). Following the results of PACTG 076, implementation of the zidovudine regimen coupled with increased antenatal HIV testing and counseling rapidly resulted in significant declines in HIV transmission (CDC, 2006). Subsequent clinical trials and observational studies showed that combination ARV prophylaxis (initially dual and then triple combination therapy) given to the mother antenatally was associated with further

breastfeeding infants and prophylaxis with ARV drugs for lactating mothers.

declines in transmission to less than 2% (Cooper et al. 2002, WHO 2010).

where they can prevent the virus from ever taking hold.

transmission of HIV infections to infants and young children.

cannot receive the services they actually need.

**3. Strategies for treatment** 

## **4. Mechanisms of action of ARV prophylaxis in reducing perinatal transmission of HIV**

There are a number of mechanisms through which zidovudine or other ARV drugs can reduce PT. One central mechanism is by decreasing maternal viral load in the blood and genital secretions via antenatal drug administration, particularly in women with high viral loads. However, ARV drugs have been shown to reduce the risk of transmission even among women with HIV RNA levels <1,000 copies/ml (Ioannidis et al. 2001). Additionally, the level of HIV RNA at delivery and receipt of antenatal ART are each independently associated with the risk of transmission, suggesting that ARV prophylaxis does not work solely through reduction in viral load (Sperling et al. 1996).

An additional mechanism of protection is pre-exposure infant prophylaxis provided by administration of ARV drugs that cross the placenta from the mother to the infant, resulting in adequate systemic drug levels in the infant. This mechanism of protection is particularly significant during the infant's passage through the birth canal, a time of rigorous exposure to maternal genital tract virus. Post-exposure infant prophylaxis is provided through administration of drug to the infant after birth. This mechanism protects the infant from cellfree or cell-associated virus that might have obtained access to the fetal/infant systemic circulation. This can occur through maternal-fetal transfusion during uterine contractions in labor or through systemic dissemination of virus swallowed by the infant during passage through the birth canal.

It is predictable that efficacy of ARV drugs in reducing PT is multi-factorial, and each of these mechanisms is contributory. The efficacy of ARV regimens administered only during labor and/or to the newborn in reducing PT demonstrates the importance of the pre- and post-exposure components of prophylaxis in reducing PT (Wade et al. 1998).

## **5. Perinatal transmission of HIV and maternal viral load**

In PACTG 076, antenatal maternal HIV RNA copy number was associated with HIV transmission in women receiving placebo. In women receiving zidovudine, the relationship was markedly attenuated and no longer statistically significant (Sperling et al. 1996). An HIV RNA threshold below which there was no risk of transmission was not identified; zidovudine was effective in reducing transmission regardless of maternal HIV RNA copy number (Shapiro et al. 1999). Other data from larger numbers of zidovudine-treated, HIVinfected pregnant women indicate that HIV RNA levels correlate with risk of transmission even in women treated with ARV agents (The European Collaborative Study, 1999).

Although the risk of PT in women with undetectable HIV RNA levels appears to be extremely low, transmission from mother to infant has been reported among women with all levels of maternal HIV RNA. Additionally, although HIV RNA may be an important risk factor for transmission, other factors also appear to play a role (Mock et al. 1999). Although there is a general correlation between viral load in plasma and in the genital tract, discordance has also been reported, particularly between HIV proviral load in blood and genital secretions, especially in the presence of other genital tract infections (Hart et al. 1999).

The use of ARV drugs during pregnancy for prevention of PT should be discussed with and offered to all infected pregnant women regardless of their HIV RNA level. Results of epidemiologic and clinical trials suggest that women receiving potent combinations of ARV

Antenatal Screening and HIV-Pregnancy: Strategies for Treatment 81

with the development of nevirapine resistance in 15% of women with detectable HIV RNA postpartum (Cunningham et al. 2002). However, the number of women with detectable HIV RNA at delivery, and especially with HIV RNA >10,000 copies/ml, was small and may have been insufficient to allow assessment of a possible benefit of single-dose nevirapine in this subgroup. Given the risk of development of resistance and the lack of data to suggest added efficacy, addition of single-dose nevirapine when a woman has received antepartum drugs

All HIV-infected women who have not received antepartum ART should have intravenous zidovudine started immediately to prevent PT of HIV. Although intrapartum/neonatal ARTs will not prevent PT that occurs before labor, most transmission occurs near to or during labor and delivery. Pre-exposure prophylaxis for the fetus can be provided by giving the mother a drug that rapidly crosses the placenta to produce systemic ARV drug levels in the fetus during intensive exposure to HIV in maternal genital secretions and blood during birth. In general, zidovudine and other NRTI drugs as well as NNRTI drugs cross the

Epidemiologic data indicate that intravenous maternal intrapartum zidovudine followed by oral zidovudine for 6 weeks for the infant significantly reduces transmission compared to no treatment (Wade et al. 1998). In a New York State cohort study, transmission rates were 10% with intrapartum and neonatal zidovudine compared with 27% without zidovudine, a 62% reduction in risk (Wade et al. 1998). The PETRA study demonstrated that intrapartum prophylaxis alone, without an infant post-exposure prophylaxis component, is not effective

Whether the addition of other ARV drugs to the intravenous intrapartum/newborn zidovudine regimen when no maternal antepartum drugs have been received increases efficacy in preventing PT has not been directly studied. Several intrapartum/neonatal prophylaxis regimens have been found to be effective in international studies. These include oral zidovudine/lamivudine during labor followed by one week of oral zidovudine/lamivudine to the infant and single-dose intrapartum/newborn nevirapine (Petra Study Team, 2002). However, none of these regimens has been compared to

Studies need to address whether adding drugs to the intravenous intrapartum/newborn zidovudine regimen will enhance efficacy in reducing PT. In the absence of data, some experts feel additional drugs may be warranted. One option is to add the single-dose intrapartum/newborn nevirapine regimen to the intravenous/6-week infant zidovudine regimen. Although single-dose nevirapine did not provide additional efficacy when added to antepartum combination ARV regimens in PACTG 316, in this situation, no maternal antepartum therapy has been given. Theoretical advantages of combining the zidovudine and nevirapine intrapartum/neonatal regimens include the known short-term safety of each regimen alone, excellent tranplacental passage of both drugs, greater antiviral activity of nevirapine compared to zidovudine, as well as the activity of nevirapine against extracellular and intracellular virus (Musoke et al. 1999) and the known synergy of zidovudine and nevirapine in inhibiting HIV replication *in vitro* (Koup et al. 1993). However, single-dose nevirapine is associated with the development of nevirapine -resistant

intravenous zidovudine combined with 6 weeks of infant zidovudine prophylaxis.

is generally not recommended.

**6.3 Women who have not received antepartum ARV drugs** 

placenta well, although protease inhibitors drugs do not.

in reducing PT (Petra Study Team, 2002).

virus (Jourdain et al. 2004).

drugs that effectively reduce HIV RNA to <1,000 copies/ml or undetectable levels have very low rates of PT (Cooper et al. 2002). However, because transmission can occur even at low or undetectable HIV RNA copy numbers, HIV RNA levels should not be a determining factor when deciding whether to use ARV drugs for prevention of PT. Additionally, the efficacy of ARV drugs is not solely related to lowering viral load (Cooper et al. 2002; Ioannidis et al. 2001). Therefore, ARV prophylaxis should be given even to women who have a very low or undetectable viral load on no therapy.

## **6. Intrapartum ARV therapy/prophylaxis**

### **6.1 Women who have received antepartum ARV drugs**

The PACTG 076 results and subsequent epidemiologic studies have proven the efficacy of the three-part zidovudine chemoprophylaxis regimen alone or in combination with other ARV agents. The PACTG 076 zidovudine regimen includes a continuous intravenous infusion of zidovudine during labor (initial loading dose of 2 mg/kg intravenously over 1 hour, followed by continuous infusion of 1 mg/kg/hour until delivery). Therefore, intravenous zidovudine during the intrapartum period should be discussed with and recommended to all HIV-infected pregnant women. For a scheduled cesarean delivery, intravenous zidovudine should begin 3 hours before surgery, according to standard dosing recommendations. Women receiving fixed-dose combination regimens that include zidovudine (e.g., the zidovudine/lamivudine combination) should have zidovudine administered intravenously during labor while other ARV components are continued orally (e.g., if a woman is receiving zidovudine/lamivudine during pregnancy, zidovudine should be given intravenously and lamivudine should be given orally during labor).

If known or suspected zidovudine resistance or toxicity has precluded antenatal use of zidovudine, intrapartum zidovudine according to the PACTG 076 protocol should still be recommended unless a woman has a documented history of hypersensitivity. This intrapartum use of the drug is recommended due to the unique characteristics of zidovudine and its proven record in reducing PT.

There is a pharmacologic antagonism between zidovudine and stavudine, and therefore these drugs should not be co-administered during labor. Women who are receiving an antepartum stavudine-containing regimen should discontinue stavudine during labor while intravenous zidovudine is being administered, with other components of the regimen continued orally.

#### **6.2 Women who have received antepartum ARV drugs but have suboptimal viral suppression near delivery**

Women who have received ART may not achieve complete viral suppression by the time of delivery due to factors such as poor adherence, viral resistance, or late entry into care. Regardless of the reason, all women who have HIV RNA levels >1,000 copies/ml near the time of delivery should be offered a scheduled cesarean delivery at 38 weeks, which may significantly reduce the risk of transmission.

The addition of single-dose nevirapine during labor has not been shown to reduce PT of HIV in this group of women. The PACTG 316 study, conducted in women in the United States, Europe, Brazil, and the Bahamas who were receiving ARV drugs during pregnancy (primarily combination therapy), showed that the addition of single-dose nevirapine did not reduce the risk of MTCT of HIV even in the setting of maternal viremia but was associated

drugs that effectively reduce HIV RNA to <1,000 copies/ml or undetectable levels have very low rates of PT (Cooper et al. 2002). However, because transmission can occur even at low or undetectable HIV RNA copy numbers, HIV RNA levels should not be a determining factor when deciding whether to use ARV drugs for prevention of PT. Additionally, the efficacy of ARV drugs is not solely related to lowering viral load (Cooper et al. 2002; Ioannidis et al. 2001). Therefore, ARV prophylaxis should be given even to women who

The PACTG 076 results and subsequent epidemiologic studies have proven the efficacy of the three-part zidovudine chemoprophylaxis regimen alone or in combination with other ARV agents. The PACTG 076 zidovudine regimen includes a continuous intravenous infusion of zidovudine during labor (initial loading dose of 2 mg/kg intravenously over 1 hour, followed by continuous infusion of 1 mg/kg/hour until delivery). Therefore, intravenous zidovudine during the intrapartum period should be discussed with and recommended to all HIV-infected pregnant women. For a scheduled cesarean delivery, intravenous zidovudine should begin 3 hours before surgery, according to standard dosing recommendations. Women receiving fixed-dose combination regimens that include zidovudine (e.g., the zidovudine/lamivudine combination) should have zidovudine administered intravenously during labor while other ARV components are continued orally (e.g., if a woman is receiving zidovudine/lamivudine during pregnancy, zidovudine should

If known or suspected zidovudine resistance or toxicity has precluded antenatal use of zidovudine, intrapartum zidovudine according to the PACTG 076 protocol should still be recommended unless a woman has a documented history of hypersensitivity. This intrapartum use of the drug is recommended due to the unique characteristics of

There is a pharmacologic antagonism between zidovudine and stavudine, and therefore these drugs should not be co-administered during labor. Women who are receiving an antepartum stavudine-containing regimen should discontinue stavudine during labor while intravenous zidovudine is being administered, with other components of the regimen

**6.2 Women who have received antepartum ARV drugs but have suboptimal viral** 

Women who have received ART may not achieve complete viral suppression by the time of delivery due to factors such as poor adherence, viral resistance, or late entry into care. Regardless of the reason, all women who have HIV RNA levels >1,000 copies/ml near the time of delivery should be offered a scheduled cesarean delivery at 38 weeks, which may

The addition of single-dose nevirapine during labor has not been shown to reduce PT of HIV in this group of women. The PACTG 316 study, conducted in women in the United States, Europe, Brazil, and the Bahamas who were receiving ARV drugs during pregnancy (primarily combination therapy), showed that the addition of single-dose nevirapine did not reduce the risk of MTCT of HIV even in the setting of maternal viremia but was associated

be given intravenously and lamivudine should be given orally during labor).

have a very low or undetectable viral load on no therapy.

**6.1 Women who have received antepartum ARV drugs** 

**6. Intrapartum ARV therapy/prophylaxis** 

zidovudine and its proven record in reducing PT.

significantly reduce the risk of transmission.

continued orally.

**suppression near delivery** 

with the development of nevirapine resistance in 15% of women with detectable HIV RNA postpartum (Cunningham et al. 2002). However, the number of women with detectable HIV RNA at delivery, and especially with HIV RNA >10,000 copies/ml, was small and may have been insufficient to allow assessment of a possible benefit of single-dose nevirapine in this subgroup. Given the risk of development of resistance and the lack of data to suggest added efficacy, addition of single-dose nevirapine when a woman has received antepartum drugs is generally not recommended.

#### **6.3 Women who have not received antepartum ARV drugs**

All HIV-infected women who have not received antepartum ART should have intravenous zidovudine started immediately to prevent PT of HIV. Although intrapartum/neonatal ARTs will not prevent PT that occurs before labor, most transmission occurs near to or during labor and delivery. Pre-exposure prophylaxis for the fetus can be provided by giving the mother a drug that rapidly crosses the placenta to produce systemic ARV drug levels in the fetus during intensive exposure to HIV in maternal genital secretions and blood during birth. In general, zidovudine and other NRTI drugs as well as NNRTI drugs cross the placenta well, although protease inhibitors drugs do not.

Epidemiologic data indicate that intravenous maternal intrapartum zidovudine followed by oral zidovudine for 6 weeks for the infant significantly reduces transmission compared to no treatment (Wade et al. 1998). In a New York State cohort study, transmission rates were 10% with intrapartum and neonatal zidovudine compared with 27% without zidovudine, a 62% reduction in risk (Wade et al. 1998). The PETRA study demonstrated that intrapartum prophylaxis alone, without an infant post-exposure prophylaxis component, is not effective in reducing PT (Petra Study Team, 2002).

Whether the addition of other ARV drugs to the intravenous intrapartum/newborn zidovudine regimen when no maternal antepartum drugs have been received increases efficacy in preventing PT has not been directly studied. Several intrapartum/neonatal prophylaxis regimens have been found to be effective in international studies. These include oral zidovudine/lamivudine during labor followed by one week of oral zidovudine/lamivudine to the infant and single-dose intrapartum/newborn nevirapine (Petra Study Team, 2002). However, none of these regimens has been compared to intravenous zidovudine combined with 6 weeks of infant zidovudine prophylaxis.

Studies need to address whether adding drugs to the intravenous intrapartum/newborn zidovudine regimen will enhance efficacy in reducing PT. In the absence of data, some experts feel additional drugs may be warranted. One option is to add the single-dose intrapartum/newborn nevirapine regimen to the intravenous/6-week infant zidovudine regimen. Although single-dose nevirapine did not provide additional efficacy when added to antepartum combination ARV regimens in PACTG 316, in this situation, no maternal antepartum therapy has been given. Theoretical advantages of combining the zidovudine and nevirapine intrapartum/neonatal regimens include the known short-term safety of each regimen alone, excellent tranplacental passage of both drugs, greater antiviral activity of nevirapine compared to zidovudine, as well as the activity of nevirapine against extracellular and intracellular virus (Musoke et al. 1999) and the known synergy of zidovudine and nevirapine in inhibiting HIV replication *in vitro* (Koup et al. 1993). However, single-dose nevirapine is associated with the development of nevirapine -resistant virus (Jourdain et al. 2004).

Antenatal Screening and HIV-Pregnancy: Strategies for Treatment 83

Intravenous zidovudine during the intrapartum period should be discussed with and

 HIV testing and counseling should be undertaken and should be entirely voluntary. Three types of interventions should be tackled by any program concerned with prevention of mother-to-child transmission: primary prevention of HIV in women; prophylaxis with antiretroviral drugs in breastfeeding infants and prophylaxis with

 Taking antiretroviral treatment can reduce the risk of mother-to-child transmission. Administration of zidovudine to the pregnant woman and her infant could significantly

 Combination antiretroviral prophylaxis (initially dual and then triple combination therapy) given to the mother antenatally can further declines the transmission

 Regardless of the reason, all women who have HIV RNA levels >1,000 copies/ml near the time of delivery should be offered a scheduled cesarean delivery at 38 weeks, which

Given the risk of development of resistance, addition of single-dose nevirapine when a

Antiretroviral Drugs used for the treatment of antiretroviral infections

Intravenous Occurring or introduced to within a vein or veins usually by means of injection

Mother to child transmission Is the transfer of an agent (e.g. HIV) from the mother to her unborn or born baby Perinatal Occurring during , or pertaining to, the periods before,

during, or after the time of birth i.e. before delivery from

woman has received antepartum drugs is generally not recommended.

Antiretroviral Therapy Drugs used against retroviruses such as HIV Intrapartum During labour and delivery or childbirth

undetectable viral load on no therapy.

antiretroviral drugs for lactating mothers.

reduce the risk of perinatal transmission.

Antenatal Before birth

may significantly reduce the risk of transmission.

**11. Definitions and explanations of words and terms** 

Antepartum Before labour or child birth

Labour The process of child birth

Monotherapy Single drug treatment

Microbicides An agent destructive to microbes

**10. Summary points of chapter** 

significantly.

recommended to all HIV-infected pregnant women.

HIV is the leading cause of death in women of reproductive age.

single-dose intrapartum nevirapine is generally not recommended for women who are receiving the standard recommended antenatal antiretroviral prophylaxis regimens. Antiretroviral prophylaxis should be given to women who have a very low or

Studies have shown that nevirapine resistance after intrapartum administration of singledose nevirapine can be substantially reduced (but not eliminated) by using a short postpartum course of ARV agents from alternate classes (a "tail"). There is no current consensus about the exact duration or composition of the ARV tail. Several trials in Africa have found 3 to 7 days of maternal/infant postpartum zidovudine/lamivudine to be effective (Chaix et al. 2006; McIntyre et al. 2009). Development of resistance to zidovudine or lamivudine given for a short period in this setting is rare (Mandelbrot et al. 2001).

More recent studies have found that 7 days of tenofovir/emtricitabine (TEmAA ANRS 12109 Study Group, 2009), 7 days of zidovudine/didanosine/lopinavir-ritonavir (Van Dyke et al. 2009), and 30 days of zidovudine/didanosine or zidovudine/didanosine/lopinavirritonavir (Lallemant et al. 2010) all appear to be effective at reducing the development of nevirapine resistance.

## **7. The efficiency of current anti-HIV treatments**

The efficacy of ARV drugs in preventing MTCT of HIV varies with the type of regimen used and the duration over which it is given. Combination regimens which include different types of ARV drugs are more efficacious than monotherapies as discussed above. It is well known that monotherapies can lead to ARV resistance in the virus, which may limit future therapeutic options when treatment is needed. According to the 2010 WHO treatment guidelines it is recommended that pregnant women living with HIV and their exposed infants receive combination therapy rather than single-dose nevirapine. ARV prophylaxis is also recommended during breastfeeding in settings where breastfeeding is judged to be the safest infant feeding option. In addition, all women eligible for treatment under WHO guidelines should receive an appropriate combination therapy for their own health (WHO, 2010).

## **8. Practice and procedures**

Three types of interventions should be tackled by any program concerned with prevention of mother-to-child transmission of HIV: primary prevention of HIV-1 in women; prophylaxis with ARV drugs in breastfeeding infants and prophylaxis with ARV drugs for lactating mothers. Administration of zidovudine to the pregnant woman and her infant could reduce the risk of perinatal transmission by nearly 70%. Combination ARV prophylaxis given to the mother antenatally is associated with further declines in transmission to <2%. Combination regimens are more effective than single-drug regimens in reducing perinatal transmission.

## **9. Chapter key facts**


single-dose intrapartum nevirapine is generally not recommended for women who are receiving the standard recommended antenatal antiretroviral prophylaxis regimens.


## **10. Summary points of chapter**

82 Understanding HIV/AIDS Management and Care – Pandemic Approaches in the 21st Century

Studies have shown that nevirapine resistance after intrapartum administration of singledose nevirapine can be substantially reduced (but not eliminated) by using a short postpartum course of ARV agents from alternate classes (a "tail"). There is no current consensus about the exact duration or composition of the ARV tail. Several trials in Africa have found 3 to 7 days of maternal/infant postpartum zidovudine/lamivudine to be effective (Chaix et al. 2006; McIntyre et al. 2009). Development of resistance to zidovudine or

More recent studies have found that 7 days of tenofovir/emtricitabine (TEmAA ANRS 12109 Study Group, 2009), 7 days of zidovudine/didanosine/lopinavir-ritonavir (Van Dyke et al. 2009), and 30 days of zidovudine/didanosine or zidovudine/didanosine/lopinavirritonavir (Lallemant et al. 2010) all appear to be effective at reducing the development of

The efficacy of ARV drugs in preventing MTCT of HIV varies with the type of regimen used and the duration over which it is given. Combination regimens which include different types of ARV drugs are more efficacious than monotherapies as discussed above. It is well known that monotherapies can lead to ARV resistance in the virus, which may limit future therapeutic options when treatment is needed. According to the 2010 WHO treatment guidelines it is recommended that pregnant women living with HIV and their exposed infants receive combination therapy rather than single-dose nevirapine. ARV prophylaxis is also recommended during breastfeeding in settings where breastfeeding is judged to be the safest infant feeding option. In addition, all women eligible for treatment under WHO guidelines should receive an appropriate combination therapy for their own health (WHO,

Three types of interventions should be tackled by any program concerned with prevention of mother-to-child transmission of HIV: primary prevention of HIV-1 in women; prophylaxis with ARV drugs in breastfeeding infants and prophylaxis with ARV drugs for lactating mothers. Administration of zidovudine to the pregnant woman and her infant could reduce the risk of perinatal transmission by nearly 70%. Combination ARV prophylaxis given to the mother antenatally is associated with further declines in transmission to <2%. Combination regimens are more effective than single-drug regimens in

Zidovudine administration to the pregnant woman and her infant can reduce the risk of

 Combination regimens are more effective than single-drug regimens in reducing perinatal transmission. A longer three-part regimen given antenatally, intrapartum, and postpartum is superior in preventing perinatal transmission than a shorter two-part

 The standard recommendation for infant prophylaxis in the absence of maternal antenatal and intrapartum therapy is six weeks of infant zidovudine. The addition of

antepartum/intrapartum or intrapartum/postpartum regimen.

lamivudine given for a short period in this setting is rare (Mandelbrot et al. 2001).

**7. The efficiency of current anti-HIV treatments** 

nevirapine resistance.

**8. Practice and procedures** 

reducing perinatal transmission.

perinatal transmission by nearly 70%.

**9. Chapter key facts** 

2010).


## **11. Definitions and explanations of words and terms**


Antenatal Screening and HIV-Pregnancy: Strategies for Treatment 85

Cunningham, C.K. and M.L. Chaix, C. Rekacewicz, P. Britto, C. Rouzioux, R.D. Gelber, A.

Hart, C.E. and J.L. Lennox, M. Pratt-Palmore, T.C. Wright, R.F. Schinazi, T. Evans-

Ioannidis, J.P. and E.J. Abrams, A. Ammann, M. Bulterys, J.J. Goedert, L. Gray, B.T. Korber,

Jourdain, G. and N. Ngo-Giang-Huong S. Le Coeur, C. Bowonwatanuwong, P. Kantipong,

Koup, R.A and F. Brewster, P. Grob and J.L. Sullivan. 1993. Nevirapine synergistically

Lallemant, M. and N. Ngo-Giang-Huong G. Jourdain, P. Traisaithit, T.R. Cressey, I.J. Collins,

Mandelbrot, L. and A. Landreau-Mascaro, C. Rekacewicz, A. Berrebi, J.L. Bénifla, M.

McIntyre, J.A. and M. Hopley, D. Moodley, M. Eklund, G.E. Gray, D.B. Hall, P. Robinson, D.

Mock, P.A. and N. Shaffer, C. Bhadrakom, W. Siriwasin, T. Chotpitayasunondh, S.

intrapartum single-dose nevirapine. Clin. Infect. Dis. 50:898-908.

randomized clinical trial. PLoS Med. 6:e1000172.

Transmission Study Group. AIDS. 13:407-414.

trials group protocol 316. J. Infect. Dis. 186:181-188.

and the female genital tract. J. Infect. Dis. 179:871-882.

Engl. J. Med. 351:229-240.

JAMA. 285:2083-2093.

immunoadhesin. AIDS. 7:1181-1184.

Dorenbaum, J.F. Delfraissy, B. Bazin, L. Mofenson and J.L. Sullivan. 2002. Development of resistance mutations in women receiving standard antiretroviral therapy who received intrapartum nevirapine to prevent perinatal human immunodeficiency virus type 1 transmission: a substudy of pediatric AIDS clinical

Strickfaden, T.J. Bush, C. Schnell, L.J. Conley, K.A. Clancy and T.V. Ellerbrock. 1999. Correlation of human immunodeficiency virus type 1 RNA levels in blood

M.J. Mayaux, L.M. Mofenson, M.L. Newell, D.E. Shapiro, J.P. Teglas and C.M. Wilfert. 2001. Perinatal transmission of human immunodeficiency virus type 1 by pregnant women with RNA virus loads <1000 copies/ml. J. Infect. Dis. 183:539-545.

P. Leechanachai, S. Ariyadej, P. Leenasirimakul, S. Hammer, M. Lallemant; Perinatal HIV Prevention Trial Group. 2004. Intrapartum exposure to nevirapine and subsequent maternal responses to nevirapine-based antiretroviral therapy. N.

inhibits HIV-1 replication in combination with zidovudine, interferon or CD4

T. Jarupanich, T. Sukhumanant, J. Achalapong, P. Sabsanong, N. Chotivanich, N. Winiyakul, S. Ariyadej, A. Kanjanasing, J. Ratanakosol, J. Hemvuttiphan, K. Kengsakul, W. Wannapira, V. Sittipiyasakul, W. Pornkitprasarn, P. Liampongsabuddhi, K. McIntosh, R.B. Van Dyke, L.M. Frenkel, S. Koetsawang, S. Le Coeur, S. Kanchana; PHPT-4 Study Team. 2010. Efficacy and safety of 1-month postpartum zidovudine-didanosine to prevent HIV-resistance mutations after

Burgard, E. Lachassine, B. Barret, M.L. Chaix, A. Bongain, N. Ciraru-Vigneron, C. Crenn-Hébert, J.F. Delfraissy, C. Rouzioux, M.J. Mayaux, S. Blanche; Agence Nationale de Recherches sur le SIDA (ANRS) 075 Study Group. 2001. Lamivudinezidovudine combination for prevention of maternal-infant transmission of HIV-1.

Mayers and N.A. Martinson. 2009. Efficacy of short-course AZT plus 3TC to reduce nevirapine resistance in the prevention of mother-to-child HIV transmission: a

Chearskul, N.L. Young, A. Roongpisuthipong, P. Chinayon, M.L. Kalish, B. Parekh and T.D. Mastro. 1999. Maternal viral load and timing of mother-to-child HIV transmission, Bangkok, Thailand. Bangkok Collaborative Perinatal HIV


#### **12. List of abbreviations**


#### **13. References**


Viral Resistance The development of mutation within the virus genome

Al-Jabri, A.A. and F.Q. Alenzi. 2009. Vaccines, virucides and drugs against HIV/AIDS: Hopes and optimisms for the future. The Open AIDS Journal 3: 1-3. Al-Jabri A.A. Al-Muharrmi Z.K., Balkhair A.A. and Ganguly S. 2010. The importance of

Bolu, O.O. and V. Allread, T. Creek, E. Stringer, F. Forna, M. Bulterys and N. Shaffer. 2007.

Cooper, E.R. and M. Charurat, L. Mofenson, C. Hanson, J. Pitt, C. Diaz, K. Hayani, E.

Connor, E.M. and R.S. Sperling, R. Gelber, P. Kiselev, G. Scott, M.J. O'Sullivan, R. Van Dyke,

prevention of perinatal HIV-1-transmission. JAIDS. 29:484-494.

1 with Zidovudine treatment. N. Engl. J. Med. 331:1173-1180.

settings in resource-limited countries. Am J Obstet Gynecol. 197:S83-9. CDC. 2006. Achievements in public health. Reduction in perinatal transmission of HIV infection--United States, 1985-2005. MMWR Morb Mortal Wkly Rep. 55:592-597. Chaix, M.L. and D.K. Ekouevi, F. Rouet, B. Tonwe-Gold, I. Viho, L. Bequet, G. Peytavin, H.

antenatal screening of HIV among pregnant women. Saudi Medical Journal 31:64-8.

Approaches for scaling up human immunodeficiency virus testing and counseling in prevention of mother-to-child human immunodeficiency virus transmission

Toure, H. Menan, V. Leroy, F. Dabis, C. Rouzioux; Agence Nationale de Recherches sur le SIDA Ditrame Plus Study Group. 2006. Low risk of nevirapine resistance mutations in the prevention of mother-to-child transmission of HIV-1: Agence Nationale de Recherches sur le SIDA Ditrame Plus, Abidjan, Cote d'Ivoire. J. Infect.

Handelsman, V. Smeruglio, R. Hoff and W. Blattner. 2002. Combination antiretroviral strategies for the treatment of pregnant HIV-1-infected women and

M. Bey, W. Shearer, R.L. Jacobson, E. Jimenez, E.O'Neill, B. Bazin, J-F. Delfraissy, M. Culnane, R. Coombs, M. Elkins, J. Moye, P. Stratton and J. Balsley. 1994. Reduction of maternal-infant transmission of human immunodeficiency virus type

delivery

treatment

Prophylaxis Protection treatment against disease

Pharmacologic Antagonism Opposition in action of drugs

NRTI Nucleoside reverse transcriptase inhibitors NNRTI Non-nucleoside reverse transcriptase inhibitors

**12. List of abbreviations** 

ARV Antiretroviral

Dis. 193:482-487.

**13. References** 

ART Antiretroviral Therapy

PT Perinatal Transmission RNA Ribonucleic acid UNAIDS United Nations AIDS

the 28th week of gestation through the first 7 days after

that leads to the virus becoming resistance to the drug


**6** 

**Effectiveness of the Regular Implementation of the Mother to Child Transmission Plus (MTCT-**

**Plus) Program in Burkina Faso, West Africa** 

Fabio Buelli1,2,5, Virginio Pietra2,3, Richard Fabian Schumacher2,4, Jacques Simpore3, Salvatore Pignatelli2,3, Francesco Castelli1,2

*1Institute for Infectious and Tropical Diseases, University of Brescia, Brescia,* 

*5Ph.D. in Appropriate Methods and Technologies for International Development* 

Since the 90s, many developing countries have introduced health strategies aimed at reducing Mother-to-Child transmission rate of HIV. These strategies (PMTCT – Prevention of Mother-to-Child Transmission) are based on (i) adequate counselling for HIV voluntary testing during antenatal care visits, (ii) single dose or a short antiretroviral therapy treatment to the mother and the newly-born baby (WHO, 2001; WHO, 2004; WHO, 2006) and (iii) formula feeding or (iv) exclusive breast-feeding with early weaning (WHO, 2003a;

During the last years, thanks to the increase of the PMTCT program coverage, the aim of the program was enlarged to address the needs of all the family members of the HIV-positive women detected by the program. The World Health Organization (WHO) then introduced the Mother-to-Child Transmission Plus (MTCT-plus) program aimed at promoting health, eventually including the use of Highly Active Antiretroviral Treatment (HAART) for the infected mothers, their children, even those whose father is different from the current

Aims of the program are, therefore, (i) to reduce HIV vertical transmission rates through specific antiretroviral therapies for the HIV-infected pregnant women, (ii) to involve a larger number of children and partners in the early stage of the disease and (iii) to increase the children survival rates by improving the HIV-positive mothers' life expectancy. (Berer, 1999; Brahmbatt et al, 2006). Psychosocial and nutritional supports and family planning are also integral components of MTCT-plus activities. Programs also focus on health education,

including best breastfeeding practices to reduce transmission risk and nutrition.

**1. Introduction** 

WHO, 2006).

partner, and the partners themselves (WHO, 2003b).

and the ESTHER-Brescia Study Group

*3St. Camille Medical Center, Ouagadougou, 4University Children's Hospital, Brescia,* 

 *Co-operation, University of Brescia (A. Archetti Fund),* 

*2Medicus Mundi Italy, Brescia,* 

*1,2,4,5Italy 3Burkina Faso* 


http://www.who.int/hiv/pub/mtct/antiretroviral2010/en/index.html

## **Effectiveness of the Regular Implementation of the Mother to Child Transmission Plus (MTCT-Plus) Program in Burkina Faso, West Africa**

Fabio Buelli1,2,5, Virginio Pietra2,3, Richard Fabian Schumacher2,4, Jacques Simpore3, Salvatore Pignatelli2,3, Francesco Castelli1,2 and the ESTHER-Brescia Study Group *1Institute for Infectious and Tropical Diseases, University of Brescia, Brescia, 2Medicus Mundi Italy, Brescia, 3St. Camille Medical Center, Ouagadougou, 4University Children's Hospital, Brescia, 5Ph.D. in Appropriate Methods and Technologies for International Development Co-operation, University of Brescia (A. Archetti Fund), 1,2,4,5Italy 3Burkina Faso* 

## **1. Introduction**

86 Understanding HIV/AIDS Management and Care – Pandemic Approaches in the 21st Century

Musoke, P. and L. Guay, D. Bagenda, M. Mirochnick, C. Nakabiito, T. Fleming, T. Elliott, S.

Petra Study Team. 2002. Efficacy of three short-course regimens of zidovudine and

Shapiro, D.E. and R.S. Sperling and R.W. Coombs. 1999. Effect of zidovudine on perinatal HIV-1 transmission and maternal viral load. Lancet 354:156; author reply 157-158. Sperling R.S. and D.E. Shapiro, R.W. Coombs, J.A. Todd, S.A. Herman, G.D. McSherry, M.J.

their neonates (HIVNET 006). AIDS. 13:479-486.

HIV-1. AIDS 23:825-833.

Abstract 95aLB.

Engl. J. Med. 339:1409-1414.

blind, placebo-controlled trial. Lancet 359:1178-1186.

Horton, K. Dransfield, J.W. Pav, A. Murarka, M. Allen, M.G. Fowler, L. Mofenson, D. Hom, F. Mmiro and J.B. Jackson. 1999. A phase I/II study of the safety and pharmacokinetics of nevirapine in HIV-1-infected pregnant Ugandan women and

lamivudine in preventing early and late transmission of HIV-1 from mother to child in Tanzania, South Africa, and Uganda (Petra study): a randomised, double-

O'Sullivan, R.B. Van Dyke, E. Jimenez, C. Rouzioux, P.M. Flynn and J.L. Sullivan. 1996. Maternal viral load, zidovudine treatment, and the risk of transmission of human immunodeficiency virus type 1 from mother to infant. Pediatric AIDS Clinical Trials Group Protocol 076 Study Group. N Engl J Med. 335:1621-1629. TEmAA ANRS 12109 Study Group. 2009. Tolerance and viral resistance after single-dose

nevirapine with tenofovir and emtricitabine to prevent vertical transmission of

nevirapine resistance mutations in HIV-infected Thai women receiving a single intrapartum dose of NVP followed by a postpartum tail of ZDV/ddI or ZDV/ddI/LPV/r: IMPAACT P1032. Paper presented at: 16th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2009; Montreal, Canada.

Baum, J.M. Tesoriero and R. Savicki. 1998. Abbreviated regimens of zidovudine prophylaxis and perinatal transmission of the human immunodeficiency virus. N.

The European Collaborative Study. 1999. Maternal viral load and vertical transmission of HIV-1: an important factor but not the only one. AIDS 13:1377-1385. Van Dyke, R. and G. Jourdain and D. Shapiro. 2009. A Phase II Study of the incidence of

Wade, N.A. and G.S. Birkhead, B.L. Warren, T.T. Charbonneau, P.T. French, L. Wang, J.B.

WHO & UNAIDS. 2010. Global Report, UNAIDS report on the global AIDS epidemic. WHO & UNAIDS. 1999. HIV in pregnancy. WHO/CHS/RHR/99.15, UNAIDS/99.35E. WHO. 2010. Antiretroviral drugs for treating pregnant women and preventing HIV

http://www.who.int/hiv/pub/mtct/antiretroviral2010/en/index.html

infection in infants: towards universal access 2010

Since the 90s, many developing countries have introduced health strategies aimed at reducing Mother-to-Child transmission rate of HIV. These strategies (PMTCT – Prevention of Mother-to-Child Transmission) are based on (i) adequate counselling for HIV voluntary testing during antenatal care visits, (ii) single dose or a short antiretroviral therapy treatment to the mother and the newly-born baby (WHO, 2001; WHO, 2004; WHO, 2006) and (iii) formula feeding or (iv) exclusive breast-feeding with early weaning (WHO, 2003a; WHO, 2006).

During the last years, thanks to the increase of the PMTCT program coverage, the aim of the program was enlarged to address the needs of all the family members of the HIV-positive women detected by the program. The World Health Organization (WHO) then introduced the Mother-to-Child Transmission Plus (MTCT-plus) program aimed at promoting health, eventually including the use of Highly Active Antiretroviral Treatment (HAART) for the infected mothers, their children, even those whose father is different from the current partner, and the partners themselves (WHO, 2003b).

Aims of the program are, therefore, (i) to reduce HIV vertical transmission rates through specific antiretroviral therapies for the HIV-infected pregnant women, (ii) to involve a larger number of children and partners in the early stage of the disease and (iii) to increase the children survival rates by improving the HIV-positive mothers' life expectancy. (Berer, 1999; Brahmbatt et al, 2006). Psychosocial and nutritional supports and family planning are also integral components of MTCT-plus activities. Programs also focus on health education, including best breastfeeding practices to reduce transmission risk and nutrition.

Effectiveness of the Regular Implementation

undergo the test.

immunological assessment.

**Protocol**

**3. Results** 

Table 1. PMTCT protocols (prophylaxis)

area and centers availability.

a contribution of 3 USD per month was asked (table1).

**AZT** 

(300 mg x 2/day from 28° week of pregnancy)

2006).

of the Mother to Child Transmission Plus (MTCT-Plus) Program in Burkina Faso, West Africa 89

midwives. All women are also asked to invite their partners to undergo the test. Moreover, HIV positive women are invited to test all the children had from previous pregnancies. Should the partner test HIV-infected, possible co-wives are also invited by the partner to

**Screening test:** after informed consent, serological screening is immediately performed by two sequential rapid tests (Determine® and Genie-II®) (Koblavi-Dème et al, 2001) to avoid loss to follow-up and assure adequate post-test counselling. In case of conflicting results, an additional ELISA test is also performed. Polymerase Chain Reaction (PCR) test, available at the Center, is used to detect infection in young children under 18 months (Simporé et al,

**Patients management and PMTCT protocol:** The national guidelines applied by the Center followed the indications provided by WHO during the study period (WHO, 2003c; WHO, 2006) both with regard to PMTCT protocols, and to the indications for therapy and for any

Nevertheless, until 2006, access to treatment has resulted in waiting lists so that even women in need of therapy have made only prophylaxis in pregnancy, starting HAART after delivery. From 2006 onwards, all women in need of therapy started HAART during pregnancy. Prophylaxis, including the breast-milk substitutes, were provided for free, while for HAART

From 1st May 2002 to 30th September 2008, 20,040 deliveries took place at SCMC and 4,028 (20.1%) VCT were carried out in pregnant women, with a seropositivity rate of 20.5%

Only 354 HIV+ pregnant women (42.8%) were enrolled in our MTCT-plus programme while the remaining 472 women were included in other centers' programs according to their living

Catholic religion was declared by 40.4% of the sample (143/354), while the majority of the sample (185/354, 52.3%) declared to be Muslim, as it is the case for the routine attendance of

As to the educational level, 102/354 (28.8%) attended the primary school (including Koranic school), while 35.6% attended the secondary school (126/354). Only 10 women (2.8% of the

(826/4028). These 826 HIV+ pregnant women were enrolled in the PMTCT program.

the Mother-and-Child (MCH) clinic in this predominantly Muslim country.

**During pregnancy During delivery After delivery**

**NVP (**200 mg single dose) + **AZT/3TC (**300mg/150mg)

**Mother**: **AZT/3TC (**300/150mg x 2/day for 1 week)

**Newborn : NVP**  (2mg/kg single dose) + **AZT (**4mg/kg X 2 / day for 1 week )

Another important expectation of the program is to raise awareness and acceptability of HIV testing and early antiretroviral therapy, thus curbing the incidence of overt AIDS.

Since 2003, many MTCT-plus programs have been implemented in HIV endemic developing countries, yielding conflicting results in different settings (Tonwe-Gold et al, 2009).

In Burkina Faso, where a stable HIV-prevalence of 1.8% is recorded (UNAIDS, 2006), the PMTCT program started in 2002 and the MTCT-plus program was introduced later on thanks to the World Bank (TAP-Treatment Acceleration Program) and the Global Fund funding, which increased the availability of antiretroviral drugs.

Aim of our work is to describe the achievements and the constraints faced by the real-life implementation of MTCT-plus program at the St. Camille Medical Center in Ouagadougou, Burkina Faso.

## **2. Methods**

**Study site:** St. Camille Medical Center (SCMC) in Ouagadougou, Burkina Faso, a large mother and child health center, where more than 3000 deliveries take place every year (picture 1). Prevention and care of HIV infection have been carried out since 2002 at the center (WHO 2004). Thanks to a collaborative agreement, physicians from the University of Brescia and from the Spedali Civili General Hospital in Brescia (Italy) have been working in the Center to support local staff and anti-HIV activities.

Fig. 1. St. Camille Medical Center

**Voluntary Counselling and Testing (VCT):** VCT is offered at St. Camille Medical Center to all pregnant women during their prenatal visit (opt-in strategy). Counselling involves an individual pre-test and post-test, conducted during the same morning by specially trained midwives. All women are also asked to invite their partners to undergo the test. Moreover, HIV positive women are invited to test all the children had from previous pregnancies. Should the partner test HIV-infected, possible co-wives are also invited by the partner to undergo the test.

**Screening test:** after informed consent, serological screening is immediately performed by two sequential rapid tests (Determine® and Genie-II®) (Koblavi-Dème et al, 2001) to avoid loss to follow-up and assure adequate post-test counselling. In case of conflicting results, an additional ELISA test is also performed. Polymerase Chain Reaction (PCR) test, available at the Center, is used to detect infection in young children under 18 months (Simporé et al, 2006).

**Patients management and PMTCT protocol:** The national guidelines applied by the Center followed the indications provided by WHO during the study period (WHO, 2003c; WHO, 2006) both with regard to PMTCT protocols, and to the indications for therapy and for any immunological assessment.

Nevertheless, until 2006, access to treatment has resulted in waiting lists so that even women in need of therapy have made only prophylaxis in pregnancy, starting HAART after delivery.

From 2006 onwards, all women in need of therapy started HAART during pregnancy. Prophylaxis, including the breast-milk substitutes, were provided for free, while for HAART a contribution of 3 USD per month was asked (table1).


Table 1. PMTCT protocols (prophylaxis)

## **3. Results**

88 Understanding HIV/AIDS Management and Care – Pandemic Approaches in the 21st Century

Another important expectation of the program is to raise awareness and acceptability of HIV testing and early antiretroviral therapy, thus curbing the incidence of overt AIDS. Since 2003, many MTCT-plus programs have been implemented in HIV endemic developing

In Burkina Faso, where a stable HIV-prevalence of 1.8% is recorded (UNAIDS, 2006), the PMTCT program started in 2002 and the MTCT-plus program was introduced later on thanks to the World Bank (TAP-Treatment Acceleration Program) and the Global Fund

Aim of our work is to describe the achievements and the constraints faced by the real-life implementation of MTCT-plus program at the St. Camille Medical Center in Ouagadougou,

**Study site:** St. Camille Medical Center (SCMC) in Ouagadougou, Burkina Faso, a large mother and child health center, where more than 3000 deliveries take place every year (picture 1). Prevention and care of HIV infection have been carried out since 2002 at the center (WHO 2004). Thanks to a collaborative agreement, physicians from the University of Brescia and from the Spedali Civili General Hospital in Brescia (Italy) have been working in

**Voluntary Counselling and Testing (VCT):** VCT is offered at St. Camille Medical Center to all pregnant women during their prenatal visit (opt-in strategy). Counselling involves an individual pre-test and post-test, conducted during the same morning by specially trained

countries, yielding conflicting results in different settings (Tonwe-Gold et al, 2009).

funding, which increased the availability of antiretroviral drugs.

the Center to support local staff and anti-HIV activities.

Fig. 1. St. Camille Medical Center

Burkina Faso.

**2. Methods** 

From 1st May 2002 to 30th September 2008, 20,040 deliveries took place at SCMC and 4,028 (20.1%) VCT were carried out in pregnant women, with a seropositivity rate of 20.5% (826/4028). These 826 HIV+ pregnant women were enrolled in the PMTCT program.

Only 354 HIV+ pregnant women (42.8%) were enrolled in our MTCT-plus programme while the remaining 472 women were included in other centers' programs according to their living area and centers availability.

Catholic religion was declared by 40.4% of the sample (143/354), while the majority of the sample (185/354, 52.3%) declared to be Muslim, as it is the case for the routine attendance of the Mother-and-Child (MCH) clinic in this predominantly Muslim country.

As to the educational level, 102/354 (28.8%) attended the primary school (including Koranic school), while 35.6% attended the secondary school (126/354). Only 10 women (2.8% of the

Effectiveness of the Regular Implementation

pregnancy after 2006 (Simporé et al, 2007).

Center, 4 are followed by other centers and 5 are lost to follow-up.

SEVERE (< 200 CD4/μl)

**Stage I** 194 (54.8%) 30 55 107 **Stage II** 121 (34.2%) 44 35 41 **Stage III** 36 (10.2%) 22 9 4 **Stage IV** 3 (0.8%) 3 0 0 **TOTAL 354 99 99 152** 

Table 3a. Clinical and immunological staging of HIV-infected pregnant women

SEVERE (< 200 CD4/μl)

**Stage I** 15 (41.7%) 2 6 6 **Stage II** 10 (27.8%) 5 2 3 **Stage III** 11 (30.6%) 7 2 1 **Stage IV** 0 0 0 0 **TOTAL 36 14 10 10** 

Table 3b. Clinical and immunological staging of HIV-infected partners

difference is not statistically significant (p=0.067).

Altogether, the mean CD4+ value in the adult sample is 350.5 CD4+/µl (range 1 - 1769 CD4+/µl), higher in women (361.6 ± σ 253.7) than in men (281.3 ± σ 188), although this

in stage WHO-3 or in stage WHO-4. (table 3a and 3b).

the death of previous children.

**Clinical Stage** 

**Clinical stage** 

§ data not available for 4 patients

**¥** data not available for 2 patients

of the Mother to Child Transmission Plus (MTCT-Plus) Program in Burkina Faso, West Africa 91

Furthermore, a significant proportion of pregnant women (48/354) declared not having other living children due to the negative outcome (abortion) of previous pregnancies or to

HIV testing was carried out in 186/348 (53.4%) children had from previous pregnancies, with a HIV prevalence rate of 10.2% (19/186) and in 231/299 children delivered under the PMTCT program, with a HIV prevalence rate of 4.3% (10/231). In our study, we did not observe any neonatal infection from that sub-set of mother who underwent HAART during

Out of the 29 HIV infected children, 20 are currently followed at the St. Camille Medical

At the time of enrolment most adult people in our sample (354 women and 36 men) were in the early stages of infection: 315/354 (88.9%) women and 25/36 (69.4%) partners were in stages WHO-1 or WHO-2, while only 39/354 (11%) women and 11/36 (30.5%) partners were

**Immunological severity level §** 

INTERMEDIATE (200-349 CD4/μl)

**Immunological severity level ¥** 

INTERMEDIATE (200-349 CD4/μl) MODERATE ( 350 CD4/ μl)

MODERATE ( 350 CD4/μl)

sample) attended higher level schools. A remarkable part of the sample (32.8%, 116/354) declared to be illiterate.

As many as 129/354 women (36.4%) could only speak the local language (Mòoré), including 15 women who declared Koranic school. The remainig 63.6% (225/354) could also speak French. Most women were married (246/354; 69.5%), while 77/354 (21.8%) declared to be unmarried, but with a steady partner. The remaining 18/354 (5.1%) declared to be divorced and 13/354 (3.7%) declared to be widows, 3 of whom married again. (Table 2).


Table 2. Description of the female population

After counseling, 182/344 living partners (52.9%) accepted to undergo the test and 115/182 (63.2%) tested positive. Among those partners who did not accept HIV testing, 82/162 (50.6%) were not informed by the woman, 29/162 (17.9%) were informed but refused to undergo the test, while for the remaining 51/162 (31.5%) information was not available.

Polygamic families in our sample had very low acceptance rate to the test (just 1/46 of the co-wives was tested, 2.1%, with negative result).

Out of the 115 HIV-infected partners, only 36 (31.3%) accepted to be followed at the St. Camille Medical Center, while the remaining 79 did not show up anymore.

The average age of our HIV+ patient cohort (390 in total, 354 women and 36 partners) was 32.3 years old (SD ± 6.3 years old) with a significant difference (p< 0,01) between women (31.5 years old) and their partners (40.8 years old).

At the end of September 2008, we counted 647 living children for the women enrolled in the program, considering all the children had before (348, 53.8%) and those delivered in the current and/or in a later pregnancy (299, 46.2%). As many as 249 dead children were also reported with an overall average of 2.5 children per woman.

The 17.8% (63/354) of our pregnant women was at the first pregnancy at the moment of enrollment.

Furthermore, a significant proportion of pregnant women (48/354) declared not having other living children due to the negative outcome (abortion) of previous pregnancies or to the death of previous children.

HIV testing was carried out in 186/348 (53.4%) children had from previous pregnancies, with a HIV prevalence rate of 10.2% (19/186) and in 231/299 children delivered under the PMTCT program, with a HIV prevalence rate of 4.3% (10/231). In our study, we did not observe any neonatal infection from that sub-set of mother who underwent HAART during pregnancy after 2006 (Simporé et al, 2007).

Out of the 29 HIV infected children, 20 are currently followed at the St. Camille Medical Center, 4 are followed by other centers and 5 are lost to follow-up.

At the time of enrolment most adult people in our sample (354 women and 36 men) were in the early stages of infection: 315/354 (88.9%) women and 25/36 (69.4%) partners were in stages WHO-1 or WHO-2, while only 39/354 (11%) women and 11/36 (30.5%) partners were in stage WHO-3 or in stage WHO-4. (table 3a and 3b).


§ data not available for 4 patients

90 Understanding HIV/AIDS Management and Care – Pandemic Approaches in the 21st Century

sample) attended higher level schools. A remarkable part of the sample (32.8%, 116/354)

As many as 129/354 women (36.4%) could only speak the local language (Mòoré), including 15 women who declared Koranic school. The remainig 63.6% (225/354) could also speak French. Most women were married (246/354; 69.5%), while 77/354 (21.8%) declared to be unmarried, but with a steady partner. The remaining 18/354 (5.1%) declared to be divorced

> 143/354 185/354 26/354

> 225/354 129/354

> 246/354 77/354 18/354 13/354

> 116/354 102/354 126/354 10/354

After counseling, 182/344 living partners (52.9%) accepted to undergo the test and 115/182 (63.2%) tested positive. Among those partners who did not accept HIV testing, 82/162 (50.6%) were not informed by the woman, 29/162 (17.9%) were informed but refused to undergo the test, while for the remaining 51/162 (31.5%) information was not available. Polygamic families in our sample had very low acceptance rate to the test (just 1/46 of the

Out of the 115 HIV-infected partners, only 36 (31.3%) accepted to be followed at the St.

The average age of our HIV+ patient cohort (390 in total, 354 women and 36 partners) was 32.3 years old (SD ± 6.3 years old) with a significant difference (p< 0,01) between women

At the end of September 2008, we counted 647 living children for the women enrolled in the program, considering all the children had before (348, 53.8%) and those delivered in the current and/or in a later pregnancy (299, 46.2%). As many as 249 dead children were also

The 17.8% (63/354) of our pregnant women was at the first pregnancy at the moment of

Camille Medical Center, while the remaining 79 did not show up anymore.

(40.4%) (52.3%) (7,3%)

(63.6%) (36.4%)

(69.5%) (21.8%) (5.1%) (3,7%)

(32.8%) (28.8%) (35.6%) (2.8%)

and 13/354 (3.7%) declared to be widows, 3 of whom married again. (Table 2).

 **Number %**

declared to be illiterate.

**Religion**  - Catholic - Muslim - Others

**Language** 

**Civil Status**  - Married

 - Divorced - Widows

enrollment.

**Educational Level** - Illiterate - Primary School - Secondary school - Higher school level

 - French and local language - Only local language


Table 2. Description of the female population

co-wives was tested, 2.1%, with negative result).

(31.5 years old) and their partners (40.8 years old).

reported with an overall average of 2.5 children per woman.

Table 3a. Clinical and immunological staging of HIV-infected pregnant women


**¥** data not available for 2 patients

Table 3b. Clinical and immunological staging of HIV-infected partners

Altogether, the mean CD4+ value in the adult sample is 350.5 CD4+/µl (range 1 - 1769 CD4+/µl), higher in women (361.6 ± σ 253.7) than in men (281.3 ± σ 188), although this difference is not statistically significant (p=0.067).

Effectiveness of the Regular Implementation

al., 2006).

has taken place.

partners.

(249/896; 27.8%).

of the Mother to Child Transmission Plus (MTCT-Plus) Program in Burkina Faso, West Africa 93

Nevertheless we did not observe any relevant loss in the follow-up, proving that women that accept opt-in VCT are highly motivated and willing to follow the program. In our experience the availability of antiretroviral drugs is not a relevant determinant for the acceptance of VCT. In fact, we did not observe any increase in the VCT acceptance rate linked to the increasing HAART availability over time. This observation suggests that cultural factors (partner's consensus, stigmatisation, illness perception, level of education) still play a very important role. An indirect confirmation comes from the higher educational level of our sample compared to that of the general female population attending the centre, 71% of which is illiterate (UNDP report 2009). Education is one of the most important factors facilitating VCT acceptance, together with obstetric history (Pignatelli et al., 2006; Perez et

The recorded HIV prevalence rate at St. Camille Medical Center is then significantly higher than in the general population of Ouagadougou (about 4%) and than the national statistics (1.8%): this suggests not only a predisposition of the women in our urban environment to

In fact, until the end of 2005, the SCMC in Ouagadougou was the only existing centre in Burkina Faso that implemented the MTCT-plus program. Pregnant seropositive women, followed by other centres, were often reported to SCMC just for the PMTCT before

The choice to implement the MTCT-plus program at the St Camille Medical Center compared to other centers, is due to the possibility to have access to many services that are not available in other centers, such as free formula milk, free laboratory follow up (test for children) and the possibility to have access to a Paediatrics unit and to the only neonatology unit existing in the Country. These facts also explain why many HIV-infected women (472/826; 57.1% of those women tested HIV-infected in our study) chose to deliver at the SCMC and to return for continuous follow-up at the original living area once the delivery

The repeated offer to test the woman's family members in the period before and after childbirth and during the counselling meetings gave good results in our study as already reported. In particular, the involvement of the male partner in the VCT and the couple counselling was a very important element in order to increase the number of people taking

On average, the immune status of the HIV-positive male partners was more compromised than the one of the pregnant females, suggesting the presence of an older infection. This fact, even if not statistically significant, matches with other report in the country (Saleri et al., 2009). The number of HIV-negative male patients is not negligible (67/182, 36.5% of the tested partners). This is in line with data showing that in Burkina Faso about two thirds of HIVinfected couples are sero-discordant (de Walque, 2007). This shows the usefulness of the MTCT-plus protocol as a unique opportunity to promote preventive measure for negative

The average number of living children is low (1.8/woman), especially if compared to the high fertility rate in Burkina Faso (6.2/woman) (CIA, 2009). This is probably due to the high foetal and infant mortality rate in mothers infected at St. Camille Medical Center (Pignatelli et al., 2006). In fact as many as 249 previously dead children were reported in our sample

undergo the test, but a further selection of the population for the PMTCT.

returning to their original center for follow-up.

part in the preventive programs (Katz et al., 2009).

As many as 113/384 (29.4%) patients (99 women and 14 partners) had a CD4+ cell count below 200 cells/µl at recruitment, immediately meeting the 2006 WHO guidelines eligibility criteria to start HAART. As a matter of fact, 95/113 patients started HAART within 6 months while 5/113 (all in clinical stage WHO-1) started HAART at a later time and 13/113 never started treatment because they were lost to follow-up or died soon after recruitment.

## **4. Discussion**

Since the results of the HIVNET-012 (Guay et al, 1999) and other PMTCT clinical trials (Dabis et al., 1999; Shaffer et al., 1999) were made available, the adoption of single-dose NVP at delivery as preventive strategy to reduce mother to child HIV transmission rate has avoided many neonatal infections in Developing Countries, allowing at the same time to detect – and cure - a high number of HIV-infected women.

However, the early emergence of HIV nevirapine-resistant strains urged to identify alternative strategies (Johnson et al, 2005).

To face these limits, WHO guidelines for PMTCT were reviewed in order to avoid the risk of viral resistances (WHO, 2006). Moreover, WHO approved MTCT-plus strategy in 2003, suggesting the adoption of a program of comprehensive care for the HIV-positive woman and for all the members of her family.

These treatments include health care, social and psychological support, reproductive health and family planning services, education and nutritional support. With this initiative, the international community has recognized the centrality of the family's role and the great contribution that women offer to the fight against AIDS (Rabkin et al., 2003).

Our study assesses the effectiveness of the MCTC-plus routine implementation in real life condition in an urban area of a resource-limited Sub-Saharian African country (Burkina Faso) to detect HIV+ family members of HIV-infected pregnant women.

In 2002, the national PMTCT program started in Burkina Faso, in three different pilot sites, including St. Camille Medical Center. The number of PTMCT centers in Burkina Faso has progressively and rapidly expanded to 803 in 2008, with a complete coverage of all Health Districts in the Country (CNLS Data, 2009).

This decentralized approach is in line with the most recent recommendations for the progressive increase of antiretroviral coverage as close as possible to the patients' households (Ferradini et al., 2006).

MTCT-plus program activities are in fact considered as the most important tool to detect HIV-infected people as early as possible.

The effectiveness of MTCT-plus program depends, first of all, on the VCT acceptance rate, the real entry point into the program. Actually, wide variations in the VCT acceptance rate were recorded in different geographic environment especially because of different cultural and organizational factors (Perez et al., 2004; Pignatelli et al., 2006; Tonwe-Gold et al., 2009).

The high number of pregnancies (more than 3,000/year) and of the ante-natal visits did not allowed our staff to provide an individual counselling and obliged us to adopt the opt-in strategy. This may explain the low VCT acceptance rate observed in our centre (20.1%) compared to 80% acceptance rate recorded in other centres in Burkina Faso where the optout strategy is adopted (MSFL, 2006).

As many as 113/384 (29.4%) patients (99 women and 14 partners) had a CD4+ cell count below 200 cells/µl at recruitment, immediately meeting the 2006 WHO guidelines eligibility criteria to start HAART. As a matter of fact, 95/113 patients started HAART within 6 months while 5/113 (all in clinical stage WHO-1) started HAART at a later time and 13/113 never started treatment because they were lost to follow-up or died soon after

Since the results of the HIVNET-012 (Guay et al, 1999) and other PMTCT clinical trials (Dabis et al., 1999; Shaffer et al., 1999) were made available, the adoption of single-dose NVP at delivery as preventive strategy to reduce mother to child HIV transmission rate has avoided many neonatal infections in Developing Countries, allowing at the same time to

However, the early emergence of HIV nevirapine-resistant strains urged to identify

To face these limits, WHO guidelines for PMTCT were reviewed in order to avoid the risk of viral resistances (WHO, 2006). Moreover, WHO approved MTCT-plus strategy in 2003, suggesting the adoption of a program of comprehensive care for the HIV-positive woman

These treatments include health care, social and psychological support, reproductive health and family planning services, education and nutritional support. With this initiative, the international community has recognized the centrality of the family's role and the great

Our study assesses the effectiveness of the MCTC-plus routine implementation in real life condition in an urban area of a resource-limited Sub-Saharian African country (Burkina

In 2002, the national PMTCT program started in Burkina Faso, in three different pilot sites, including St. Camille Medical Center. The number of PTMCT centers in Burkina Faso has progressively and rapidly expanded to 803 in 2008, with a complete coverage of all Health

This decentralized approach is in line with the most recent recommendations for the progressive increase of antiretroviral coverage as close as possible to the patients'

MTCT-plus program activities are in fact considered as the most important tool to detect

The effectiveness of MTCT-plus program depends, first of all, on the VCT acceptance rate, the real entry point into the program. Actually, wide variations in the VCT acceptance rate were recorded in different geographic environment especially because of different cultural and organizational factors (Perez et al., 2004; Pignatelli et al., 2006; Tonwe-Gold et

The high number of pregnancies (more than 3,000/year) and of the ante-natal visits did not allowed our staff to provide an individual counselling and obliged us to adopt the opt-in strategy. This may explain the low VCT acceptance rate observed in our centre (20.1%) compared to 80% acceptance rate recorded in other centres in Burkina Faso where the opt-

contribution that women offer to the fight against AIDS (Rabkin et al., 2003).

Faso) to detect HIV+ family members of HIV-infected pregnant women.

detect – and cure - a high number of HIV-infected women.

alternative strategies (Johnson et al, 2005).

and for all the members of her family.

Districts in the Country (CNLS Data, 2009).

households (Ferradini et al., 2006).

al., 2009).

HIV-infected people as early as possible.

out strategy is adopted (MSFL, 2006).

recruitment.

**4. Discussion** 

Nevertheless we did not observe any relevant loss in the follow-up, proving that women that accept opt-in VCT are highly motivated and willing to follow the program. In our experience the availability of antiretroviral drugs is not a relevant determinant for the acceptance of VCT. In fact, we did not observe any increase in the VCT acceptance rate linked to the increasing HAART availability over time. This observation suggests that cultural factors (partner's consensus, stigmatisation, illness perception, level of education) still play a very important role. An indirect confirmation comes from the higher educational level of our sample compared to that of the general female population attending the centre, 71% of which is illiterate (UNDP report 2009). Education is one of the most important factors facilitating VCT acceptance, together with obstetric history (Pignatelli et al., 2006; Perez et al., 2006).

The recorded HIV prevalence rate at St. Camille Medical Center is then significantly higher than in the general population of Ouagadougou (about 4%) and than the national statistics (1.8%): this suggests not only a predisposition of the women in our urban environment to undergo the test, but a further selection of the population for the PMTCT.

In fact, until the end of 2005, the SCMC in Ouagadougou was the only existing centre in Burkina Faso that implemented the MTCT-plus program. Pregnant seropositive women, followed by other centres, were often reported to SCMC just for the PMTCT before returning to their original center for follow-up.

The choice to implement the MTCT-plus program at the St Camille Medical Center compared to other centers, is due to the possibility to have access to many services that are not available in other centers, such as free formula milk, free laboratory follow up (test for children) and the possibility to have access to a Paediatrics unit and to the only neonatology unit existing in the Country. These facts also explain why many HIV-infected women (472/826; 57.1% of those women tested HIV-infected in our study) chose to deliver at the SCMC and to return for continuous follow-up at the original living area once the delivery has taken place.

The repeated offer to test the woman's family members in the period before and after childbirth and during the counselling meetings gave good results in our study as already reported. In particular, the involvement of the male partner in the VCT and the couple counselling was a very important element in order to increase the number of people taking part in the preventive programs (Katz et al., 2009).

On average, the immune status of the HIV-positive male partners was more compromised than the one of the pregnant females, suggesting the presence of an older infection. This fact, even if not statistically significant, matches with other report in the country (Saleri et al., 2009).

The number of HIV-negative male patients is not negligible (67/182, 36.5% of the tested partners). This is in line with data showing that in Burkina Faso about two thirds of HIVinfected couples are sero-discordant (de Walque, 2007). This shows the usefulness of the MTCT-plus protocol as a unique opportunity to promote preventive measure for negative partners.

The average number of living children is low (1.8/woman), especially if compared to the high fertility rate in Burkina Faso (6.2/woman) (CIA, 2009). This is probably due to the high foetal and infant mortality rate in mothers infected at St. Camille Medical Center (Pignatelli et al., 2006). In fact as many as 249 previously dead children were reported in our sample (249/896; 27.8%).

Effectiveness of the Regular Implementation

Arianna Andreoli)

Ouagadougou.

Burkina.

**7. Acknowledgement** 

of the Mother to Child Transmission Plus (MTCT-Plus) Program in Burkina Faso, West Africa 95

**General Hospital** (Dr P. Villani, Dr D. Cattarelli)**; St Camille Medical Center (**Brother Luise, Brother Sébastien Rouamba, Dr Dabogo Sia, Father Marc Zombré, Father François Sedogo, Father Dr Paul Ouedraogo, Dr Ghislaine Sawadogo, Dr Korotimi Sanogo, Dr Jedida Simpore, M.me Agnes Thombiano, M.me Josephine Ouedraogo, M.me Therese Ouba), **National Committee against AIDS and STDs, Burkina Faso** (Dr André Joseph Tiendrebeogo, Dr Marie Joseph Sanou, Dr. Genevieve Onadja, Dr Francine Ouedraogo), **National Institute of Health, Rome, Italy** (Dr Stefano Vella), **Post-Graduate Specialty School in Infectious Diseases, University of Brescia, Italy** (Dr Chiara Baiguera, Dr Daniele Bella, Dr Barbara Cadeo, Dr Chiara Cattaneo, Dr Silvia Lorenzotti, Dr Chiara Pezzoli, Dr Katiela Prestini, Dr Cristina Uccelli, Dr Laura Soavi, Dr Giuseppe Lapadula, Dr. Emanuele Focà, Dr Viviana Bergamaschi, Dr Manuela Rosina, Dr Antonella Ricci), **Post-Graduate Specialty School in Tropical Medicine, University of Brescia, Italy** (Dr Giuseppina De Iaco, Dr Maria Manfrin, Dr Cecilia Pizzocolo, Dr Nuccia Saleri, Dr Susanna Capone, Dr Alessandro Pini, Dr Veronica Del Punta, Dr Sara Bigoni, Dr Silvia Odolini, Dr Lucia Urbinati), **Post-Graduate Specialty School in Pediatrics, University of Brescia, Italy** (Dr Sara Aliprandi, Dr Veronica Bennato, Dr Elisa Bertoni, Dr Sara Boccacci, Dr Federica Bruni, Dr Marco Cattalini, Dr Gemma Cattaneo, Dr Valeria Consolati, Dr Donatella De Martiis, Dr Rosaria Fausti, Dr Monica Filisetti, Dr Chiara Gualeni, Dr Sarah Guarisco, Dr Livia Grazzani, Dr Elisa Marniga, Dr Roberto Marzollo, Dr Moira Pinotti, Dr Chiara Ravelli, Dr Francesca Ricci, Dr Elena Soncini, Dr Mariateresa Sinelli, Dr Elena Spinelli, Dr Laura Morali, Dr Chiara Monfredini), **PhD in Methodologies and Appropriate Technologies in International Co-operation for Development** (Dr Fabio Buelli, Dr Beatrice Autino, Dr Marianna Bettinzoli); **Medicus Mundi Italy** (Mr Massimo Chiappa, Mr Giovanni Zoppi, Mrs Cinzia Ferrante, Mrs Marina Martinetto, Mrs Patrizia Marcotti, Dr Alessia Melzani, Dr

The project was made possible thanks to the agreement between the Spedali Civili General Hospital of Brescia and the Camillian health structures in Burkina Faso and the technical-scientific support assured by the University of Brescia and the University of

Trainees doctors in Tropical Medicine, Infectious Diseases and in Pediatrics at the University of Brescia, acting on the behalf of the NGO Medicus Mundi Italy (grant from the Italian Ministry of Foreign Affairs) work together with the local medical staff from

Many national and international organization participated to the realization of the study: the ESTHER project, the World Bank through TAP (Treatment Acceleration Program), the Global Fund against HIV, Tuberculosis and Malaria, UNICEF, the Italian Ministry of Foreign Affairs, the Italian High Institute of Health, the Lombardy Region, the province of

Special thanks to the strong and indispensable collaboration of the Ministry of Health of Burkina Faso and the Program for the Application and the Decentralization of the Burkinan Health System, the Burkina National Committee against AIDS and Sexually Transmitted Infections, the World Health Organization (WHO), the offices of the World Bank, UNICEF

in Burkina Faso, and the Local Technical Unit of the Italian Embassy in Burkina Faso.

Brescia, the Municipality of Brescia, the CARIPLO Foundation.

The lower fertility rate in HIV+ women, especially in the advanced stages of the illness, could be another possible reason (Le Coeur et al., 2005), and this reinforce the need to link HIV treatment and reproductive health services in the framework of the MTCT-plus initiative.

The screening of the children born from previous pregnancies was probably hampered by the fear of the parents to verify the status of the infection in their children for whom PMTCT protocols was not adopted and by the fear that elder children may reveal the secret in the community.

As expected, HIV infected pregnant women that entered the PMTCT program were almost all asymptomatic. However, CD4+ lymphocyte count is essential in order to identify those HIV-infected women that are eligible for the HAART. The mother to child HIV transmission rate (4.3%) is due to the failure of the nevirapine mono-prophylaxis and to the limited access to the HAART for those who needed it before 2006.

The relative older age of the women in our sample compared to the average of the age of the pregnant women at the SCMC is probably due to the fact that the older women are more free to autonomously accept the VCT proposal and can be more worried about the previous and "unexplainable" loss of a child (Pignatelli et al., 2006).

The decreasing trend recorded in the sero-prevalence rate among the younger pregnant women in Burkina Faso can be due to the campaigns focused on the education to health (UNAIDS/OMS/UNICEF/UE, 2006). This effort needs to be strengthened at every level.

The progressive increase of HAART availability in resource limited Countries underlines the role of MTCT-plus programs as a possible tool capable to identify motivated people in a sufficiently initial stage of the illness in order to benefit from the antiretroviral treatment.

In our study, the following socio-cultural factors have limited the effectiveness of the program: (i) refusal of the male partner to undergo the test (ii) refusal of the parents to test the children from previous pregnancies (iii) refusal of the pregnant woman to inform the partner about her serostatus. These reasons possibly find their explanation in the social stigma that HIV still cause in Western Africa.

## **5. Conclusion**

The MTCT-plus approach might be an important tool to increase the early detection of HIV infected patients in the household of the infected pregnant women, allowing for beneficial early treatment. Furthermore detection of discordant couples offers possibilities to prevent infection. However, its effectiveness in the real-life condition of Western Africa is hampered by cultural factors that act at different levels (VCT uptake, notification to the partner, testing of previous children) and it requires new and innovative approaches in order to expand the adoption of HIV testing in Developing Countries.

The positive impact that HAART has on the lives of those affected may further increase acceptance of VCT and reduce stigma, thus allowing to save ever more people.

## **6. ESTHER-Brescia study group**

**Dpt for Infectious Diseases, Spedali Civili General Hospital** (Dr Lina R. Tomasoni, Dr M. Gulletta, Dr Silvio Caligaris, Dr Eugenia Quiros)**; Dpt of Neonatology, Spedali Civili** 

**General Hospital** (Dr P. Villani, Dr D. Cattarelli)**; St Camille Medical Center (**Brother Luise, Brother Sébastien Rouamba, Dr Dabogo Sia, Father Marc Zombré, Father François Sedogo, Father Dr Paul Ouedraogo, Dr Ghislaine Sawadogo, Dr Korotimi Sanogo, Dr Jedida Simpore, M.me Agnes Thombiano, M.me Josephine Ouedraogo, M.me Therese Ouba), **National Committee against AIDS and STDs, Burkina Faso** (Dr André Joseph Tiendrebeogo, Dr Marie Joseph Sanou, Dr. Genevieve Onadja, Dr Francine Ouedraogo), **National Institute of Health, Rome, Italy** (Dr Stefano Vella), **Post-Graduate Specialty School in Infectious Diseases, University of Brescia, Italy** (Dr Chiara Baiguera, Dr Daniele Bella, Dr Barbara Cadeo, Dr Chiara Cattaneo, Dr Silvia Lorenzotti, Dr Chiara Pezzoli, Dr Katiela Prestini, Dr Cristina Uccelli, Dr Laura Soavi, Dr Giuseppe Lapadula, Dr. Emanuele Focà, Dr Viviana Bergamaschi, Dr Manuela Rosina, Dr Antonella Ricci), **Post-Graduate Specialty School in Tropical Medicine, University of Brescia, Italy** (Dr Giuseppina De Iaco, Dr Maria Manfrin, Dr Cecilia Pizzocolo, Dr Nuccia Saleri, Dr Susanna Capone, Dr Alessandro Pini, Dr Veronica Del Punta, Dr Sara Bigoni, Dr Silvia Odolini, Dr Lucia Urbinati), **Post-Graduate Specialty School in Pediatrics, University of Brescia, Italy** (Dr Sara Aliprandi, Dr Veronica Bennato, Dr Elisa Bertoni, Dr Sara Boccacci, Dr Federica Bruni, Dr Marco Cattalini, Dr Gemma Cattaneo, Dr Valeria Consolati, Dr Donatella De Martiis, Dr Rosaria Fausti, Dr Monica Filisetti, Dr Chiara Gualeni, Dr Sarah Guarisco, Dr Livia Grazzani, Dr Elisa Marniga, Dr Roberto Marzollo, Dr Moira Pinotti, Dr Chiara Ravelli, Dr Francesca Ricci, Dr Elena Soncini, Dr Mariateresa Sinelli, Dr Elena Spinelli, Dr Laura Morali, Dr Chiara Monfredini), **PhD in Methodologies and Appropriate Technologies in International Co-operation for Development** (Dr Fabio Buelli, Dr Beatrice Autino, Dr Marianna Bettinzoli); **Medicus Mundi Italy** (Mr Massimo Chiappa, Mr Giovanni Zoppi, Mrs Cinzia Ferrante, Mrs Marina Martinetto, Mrs Patrizia Marcotti, Dr Alessia Melzani, Dr Arianna Andreoli)

## **7. Acknowledgement**

94 Understanding HIV/AIDS Management and Care – Pandemic Approaches in the 21st Century

The lower fertility rate in HIV+ women, especially in the advanced stages of the illness, could be another possible reason (Le Coeur et al., 2005), and this reinforce the need to link HIV treatment and reproductive health services in the framework of the MTCT-plus

The screening of the children born from previous pregnancies was probably hampered by the fear of the parents to verify the status of the infection in their children for whom PMTCT protocols was not adopted and by the fear that elder children may reveal the secret in the

As expected, HIV infected pregnant women that entered the PMTCT program were almost all asymptomatic. However, CD4+ lymphocyte count is essential in order to identify those HIV-infected women that are eligible for the HAART. The mother to child HIV transmission rate (4.3%) is due to the failure of the nevirapine mono-prophylaxis and to the limited access

The relative older age of the women in our sample compared to the average of the age of the pregnant women at the SCMC is probably due to the fact that the older women are more free to autonomously accept the VCT proposal and can be more worried about the previous

The decreasing trend recorded in the sero-prevalence rate among the younger pregnant women in Burkina Faso can be due to the campaigns focused on the education to health (UNAIDS/OMS/UNICEF/UE, 2006). This effort needs to be strengthened at every level. The progressive increase of HAART availability in resource limited Countries underlines the role of MTCT-plus programs as a possible tool capable to identify motivated people in a sufficiently initial stage of the illness in order to benefit from the antiretroviral

In our study, the following socio-cultural factors have limited the effectiveness of the program: (i) refusal of the male partner to undergo the test (ii) refusal of the parents to test the children from previous pregnancies (iii) refusal of the pregnant woman to inform the partner about her serostatus. These reasons possibly find their explanation in the social

The MTCT-plus approach might be an important tool to increase the early detection of HIV infected patients in the household of the infected pregnant women, allowing for beneficial early treatment. Furthermore detection of discordant couples offers possibilities to prevent infection. However, its effectiveness in the real-life condition of Western Africa is hampered by cultural factors that act at different levels (VCT uptake, notification to the partner, testing of previous children) and it requires new and innovative approaches in order to expand the

The positive impact that HAART has on the lives of those affected may further increase

**Dpt for Infectious Diseases, Spedali Civili General Hospital** (Dr Lina R. Tomasoni, Dr M. Gulletta, Dr Silvio Caligaris, Dr Eugenia Quiros)**; Dpt of Neonatology, Spedali Civili** 

acceptance of VCT and reduce stigma, thus allowing to save ever more people.

to the HAART for those who needed it before 2006.

stigma that HIV still cause in Western Africa.

adoption of HIV testing in Developing Countries.

**6. ESTHER-Brescia study group** 

and "unexplainable" loss of a child (Pignatelli et al., 2006).

initiative.

community.

treatment.

**5. Conclusion** 

The project was made possible thanks to the agreement between the Spedali Civili General Hospital of Brescia and the Camillian health structures in Burkina Faso and the technical-scientific support assured by the University of Brescia and the University of Ouagadougou.

Trainees doctors in Tropical Medicine, Infectious Diseases and in Pediatrics at the University of Brescia, acting on the behalf of the NGO Medicus Mundi Italy (grant from the Italian Ministry of Foreign Affairs) work together with the local medical staff from Burkina.

Many national and international organization participated to the realization of the study: the ESTHER project, the World Bank through TAP (Treatment Acceleration Program), the Global Fund against HIV, Tuberculosis and Malaria, UNICEF, the Italian Ministry of Foreign Affairs, the Italian High Institute of Health, the Lombardy Region, the province of Brescia, the Municipality of Brescia, the CARIPLO Foundation.

Special thanks to the strong and indispensable collaboration of the Ministry of Health of Burkina Faso and the Program for the Application and the Decentralization of the Burkinan Health System, the Burkina National Committee against AIDS and Sexually Transmitted Infections, the World Health Organization (WHO), the offices of the World Bank, UNICEF in Burkina Faso, and the Local Technical Unit of the Italian Embassy in Burkina Faso.

Effectiveness of the Regular Implementation

of the Mother to Child Transmission Plus (MTCT-Plus) Program in Burkina Faso, West Africa 97

Perez F, Orne-Gliemann J, Mukotekwa T, Miller A, Glenshaw M, Mahomva A, Dabis F:

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*district hospital in rural Zimbabwe*. Brit. Med. J., 2004; 329: 1147-1150.

*Ouagadougou (Burkina Faso)*. Trop. Med. Int. Health, 2006; 11:350-357. Rabkin M, El-Sadr WM: *Saving mothers, saving families. The MCTC-plus initiative. WHO perspective and practice in antiretroviral treatment. Case Study* , Geneva, WHO, 2003. Saleri N, Capone S, Pietra V, De Iaco G, Del Punta V, Rizzi M, Levi M, Rouamba S, Somé E,

*in hospitalized HIV-patients in Burkina Faso*. Infection. 2009 Apr;37(2):142-7. Shaffer N, Chuachoowong R, Mock PA, Bhadrakom C, Siriwasin W, Young NL,

*Bangkok,Thailand: a randomised controlled trial*. Lancet. 1999; 353:773-780. Simporé J, Pietra V, Savadogo A, Pignatelli S, Nikiema JB, Nadembega WMC., Yara J,

Simporé J, Pietra V, Pignatelli S, Karou D, Nadembega WM, Ilboudo D, Ceccherini-

Tonwe-Gold B, Ekouevi DK, Bosse CA, Toure S, Koné M, Becquet R, Leroy V, Toro P, Dabis

World Health Organization (WHO): *Prevention of mother-to-child transmission of HIV : selection* 

World Health Organization (WHO*): HIV and Infant Feeding. Framework for priority action*.

World Health Organisation (WHO): *Scaling up antiretroviral therapy in resource-limited settings* 

World Health Organization (WHO): *Saving mothers, saving families : the MTCT-PLUS Initiative. Perspectives and practice in antiretroviral treatment*. Case study. Geneva, 2003c.

*Ouagadougou, Burkina Faso*. J. Med. Virol., 2006; 78:148-152.

*d'Ivoire*. Trop. Med. Int. Health, February 2009; 14:204-212.

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*Burkina Faso*. J. Med. Virol. 2007 Jul;79(7):873-9.

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[http://hdrstats.undp.org/en/indicators/99.html]

*and use of nevirapine*. Technical Notes. Geneva, 2001.

*– Guidelines for a public health approach*. Geneva, 2003b.

United Nations Development Program report 2009,

Geneva, 2003a.

*Prevention of mother to child transmission of HIV: evaluation of a pilot programme in a* 

G, Tall F, Ouiminga A, Carosi G, Castelli F: *Factors predicting uptake of Voluntary Counselling and Testing (VCT) in a real-life setting in a mother-to-child Centre in* 

Simporé J, Patroni A, Carosi G, Castelli F: *Outcome and predictive factors of mortality* 

Chotpitayasunondh T, Chearskul S, Roongpisuthipong A, Chinayon P, Karon J, Mastro TD, Simonds RJ: *Short-course zidovudine for perinatal HIV-1 transmission in* 

Zoungrana N, Bakouan D, Colizzi V, Castelli F, Musumeci S: *Reduction of mother-tochild transmission of HIV (MTCT-HIV) at Saint Camille Medical Center (SCMC) in* 

Silberstein F, Ghilat-Avoid-Belem WN, Bellocchi MC, Saleri N, Sanou MJ, Ouedraogo CM, Nikiema JB, Colizzi V, Perno CF, Castelli F, Musumeci S: *Effective program against mother-to-child transmission of HIV at Saint Camille Medical Centre in* 

F, El Sadr WM, Abrams EJ: *Implementing family-focused HIV care treatment: the first 2 years' experience of the mother-to-child transmission-plus program in Abidjan, Cote* 

#### **8. References**


CIA *World Factbook Demographic Statistics*, 2009.

CNLS Burkina Faso: *Données de la PTME en 2009* ; www.cnls.bf; 2009.


Berer M: *Reducing perinatal HIV transmission in developing countries through antenatal and* 

Brahmbhatt H, Kigozi G, Wabwire-Mangen F, Serwadda D, Lutalo T, Nalugoda F,

Dabis F, Msellati P, Meda N, Welffens-Ekra C, You B, Manigart O, Leroy V, Simonon A,

de Walque D: *Sero-discordant couples in five African countries: implications for prevention strategies*. Population and Development Review, 2007, 33(3):501-523. Ferradini L, Jeannin A, Pinoges L, Izopet J, Odhiambo D, Mankhambo L, Karungi G,

Guay LA, Musoke P, Fleming T, Bagenda D, Allen M, Nakabiito C, Sherman J, Bakaki P,

Katz DA, Kiarie JN, John-Stewart GC, Richardson BA, John FN, Farquhar C: *Male* 

Koblavi-Dème S, Maurice C, Yavo D, Sibailly TS, N'guessan K, Kamelan-Tano Y, Wiktor SZ,

Medecins sans Frontières Luxembourg (MSFL). *Prise en charge des PVVIH/SIDA dans le* 

*Clinic in Abidjan, Ivory Coast*. J. Clin. Microbiol. 2001; 39 (5) : 1808–1812. Le Coeur S, Khlat M, Halembokaka G, Augereau-Vacher C, Batala-M'Pondo G, Baty G,

*Malawi: an effectiveness assessement*. Lancet, 2006; 367: 1335-1369.

*substantially underestimated*. J. Infect. Dis., 2005; 192: 16-23.

*survival.* Bull. World Health Org., 1999; 77: 871-877.

CNLS Burkina Faso: *Données de la PTME en 2009* ; www.cnls.bf; 2009.

*multicentre trial*. Lancet, 1999; 353:781-785.

*delivery care, and breastfeeding: supporting infant survival by supporting mother's* 

Sewankambo N, Kiduggavu M, Wawer M, Gray R: *Mortality in HIV-infected and uninfected children of HIV-infected and uninfected mothers in rural Uganda*. JAIDS, 2006;

Cartoux M, Combe P, Ouangré A, Ramon R, Ky-Zerbo O, Montcho C, Salamon R, Rouzioux C, Van de Perre P, Mandelbrot L: *6-months efficacy, tolerance and acceptability of a short regimen of oral zidovudine to reduce vertical transmission of HIV in breastfed children in Côte d'Ivoire and Burkina Faso: a double blind placebo-controlled* 

Szumilin E, Balandine S, Fedida G, Carrieri MP, Spire B, Ford N, Tassie JM, Guerin PJ, Brasher C*: Scaling up of highly active antiretroviral therapy in a rural distrct of* 

Ducar C, Deseyve M, Emel L, Mirochnick M, Fowler MG, Mofenson L, Miotti P, Dransfield K, Bray D, Mmiro F, Jackson JB: *Intrapartum and neonatal single-dose nevirapine compared with zidovudine for prevention of mother-to-child transmission of HIV-1 in Kampala, Uganda: HIVNET 012 randomised trial*. Lancet 1999; 354: 795-804. Johnson JA, Li JF, Morris L, Martinson N, Gray G, McIntyre J, Heneine W : *Emergence of* 

*drug-resistant HIV-1 after intrapartum administration of single-dose nevirapine is* 

*perspectives on incorporating men into antenatal HIV counselling and testing*. PLoS One.

Roels TH, Chorba T, Nkengasong JN: *Sensitivity and Specificity of Human Immunodeficiency Virus Rapid Serologic Assays and Testing Algorithms in an Antenatal* 

Ronsmans C: *HIV and the magnitude of pregnancy-related mortality in Point Noir,* 

*district sanitaire de Pissy*. Rapport. annuel. 2005. [msfl-ouaga@luxembourg.msf.org]

**8. References** 

41: 504-508.

CIA *World Factbook Demographic Statistics*, 2009.

2009 Nov 2;4(11):e7602.

*Congo*. AIDS, 2005 ; 19 : 69-75.


**7** 

*Canada* 

**Exosomes Decrease** *In Vitro* **Infectivity of HIV-1** 

Since their identification by Johnstone et al (Johnstone et al., 1987), exosomes have gained importance in understanding many biological processes. Exosomes are vesicles expelled by cells into the extracellular milieu. They originate from internal endocytic compartments called multivesicular bodies (MVB) and are released following fusion of MVB with the plasma membrane (Stoorvogel et al., 2002). Numerous cell types, including tumour, foetal, epithelial and haematopoietic cells share the characteristics of releasing exosomes upon activation by cytokines (Abusamra et al., 2005; Ahn and Johnstone, 1993; Altieri, Khan, and Tomasi, 2004; Peche et al., 2006; Segura, Amigorena, and Thery, 2005; Taylor, Akyol, and Gercel-Taylor, 2006; van Niel and Heyman, 2002). Initially associated with the elimination of obsolete proteins during reticulocyte maturation, exosomes are now known to play several roles in intercellular communication (for reviews, (Chaput and Thery, 2010) and (Record et al., 2011)). Based on the presence of various molecules within the vesicle membrane or lumen, it has been proposed that exosomes are particularly involved in regulation of the immune response, for example tolerance induction (Admyre et al., 2006; Frangsmyr et al., 2005; Kapsogeorgou et al., 2005; Karlsson et al., 2001; Kim, Morse, and Choi, 2006; Larregina et al., 2004; Mallegol, van Niel, and Heyman, 2005; Ostman, Taube, and Telemo, 2005; Peche et al., 2003; Peche et al., 2006; Quah and O'Neill, 2005a; Segura, Amigorena, and Thery, 2005; Taylor, Akyol, and Gercel-Taylor, 2006; Van Niel et al., 2003), antigen presentation (Andre et al., 2004; Chaput et al., 2004; Clayton et al., 2003; Kleijmeer et al., 2001; Peche et al., 2003; Raposo et al., 1996; Thery et al., 2002), cancer immunotherapy (Amigorena, 2000; Andre et al., 2001; Mignot et al., 2006; Quah and O'Neill, 2000; Zitvogel et al., 1998), control of receptor expression (Ahn and Johnstone, 1993; Hawari et al., 2004; Levine, 2004), mechanisms involved in cell death (Abusamra et al., 2005; Farsad, 2002; Iero et al., 2008; Lenassi et al., 2010; Zhang et al., 2006) and control of inflammation (Abusamra et al., 2005; Kim et al., 2006; Levine, 2004). Exosomes may also contain functional miRNA (Pegtel et al., 2010) or deliver bioactive lipids (Esser et al., 2010; Subra et al., 2010). Depending on the function and on

**1. Introduction** 

**1.1 Nature and role of exosomes** 

**Preparations: Implication for CD4+T** 

**Lymphocyte Depletion** *In Vivo*

Simard Sébastien and Gilbert Caroline

Subra Caroline, Burelout Chantal, Proulx Sophie,

*Centre de Recherche en Rhumatologie et Immunologie (CRRI),* 

*Département de Médecine, Faculté de Médecine, Université Laval, Québec,* 


## **Exosomes Decrease** *In Vitro* **Infectivity of HIV-1 Preparations: Implication for CD4+T Lymphocyte Depletion** *In Vivo*

Subra Caroline, Burelout Chantal, Proulx Sophie, Simard Sébastien and Gilbert Caroline

*Centre de Recherche en Rhumatologie et Immunologie (CRRI), Département de Médecine, Faculté de Médecine, Université Laval, Québec, Canada* 

## **1. Introduction**

98 Understanding HIV/AIDS Management and Care – Pandemic Approaches in the 21st Century

World Health Organization (WHO): *Antiretroviral drugs and the prevention of mother-to-child* 

Geneva, 2006.

*transmission of HIV infection in resource-constrained settings. Recommendations for use*. 2004 revision Report of a Technical Consultation, Geneva, 5–6 February 2004 . World Health Organization (WHO). *Antiretroviral drugs for treating pregnant women and* 

*preventing HIV infection in infants in resource-limited settings. Towards universal access*.

#### **1.1 Nature and role of exosomes**

Since their identification by Johnstone et al (Johnstone et al., 1987), exosomes have gained importance in understanding many biological processes. Exosomes are vesicles expelled by cells into the extracellular milieu. They originate from internal endocytic compartments called multivesicular bodies (MVB) and are released following fusion of MVB with the plasma membrane (Stoorvogel et al., 2002). Numerous cell types, including tumour, foetal, epithelial and haematopoietic cells share the characteristics of releasing exosomes upon activation by cytokines (Abusamra et al., 2005; Ahn and Johnstone, 1993; Altieri, Khan, and Tomasi, 2004; Peche et al., 2006; Segura, Amigorena, and Thery, 2005; Taylor, Akyol, and Gercel-Taylor, 2006; van Niel and Heyman, 2002). Initially associated with the elimination of obsolete proteins during reticulocyte maturation, exosomes are now known to play several roles in intercellular communication (for reviews, (Chaput and Thery, 2010) and (Record et al., 2011)). Based on the presence of various molecules within the vesicle membrane or lumen, it has been proposed that exosomes are particularly involved in regulation of the immune response, for example tolerance induction (Admyre et al., 2006; Frangsmyr et al., 2005; Kapsogeorgou et al., 2005; Karlsson et al., 2001; Kim, Morse, and Choi, 2006; Larregina et al., 2004; Mallegol, van Niel, and Heyman, 2005; Ostman, Taube, and Telemo, 2005; Peche et al., 2003; Peche et al., 2006; Quah and O'Neill, 2005a; Segura, Amigorena, and Thery, 2005; Taylor, Akyol, and Gercel-Taylor, 2006; Van Niel et al., 2003), antigen presentation (Andre et al., 2004; Chaput et al., 2004; Clayton et al., 2003; Kleijmeer et al., 2001; Peche et al., 2003; Raposo et al., 1996; Thery et al., 2002), cancer immunotherapy (Amigorena, 2000; Andre et al., 2001; Mignot et al., 2006; Quah and O'Neill, 2000; Zitvogel et al., 1998), control of receptor expression (Ahn and Johnstone, 1993; Hawari et al., 2004; Levine, 2004), mechanisms involved in cell death (Abusamra et al., 2005; Farsad, 2002; Iero et al., 2008; Lenassi et al., 2010; Zhang et al., 2006) and control of inflammation (Abusamra et al., 2005; Kim et al., 2006; Levine, 2004). Exosomes may also contain functional miRNA (Pegtel et al., 2010) or deliver bioactive lipids (Esser et al., 2010; Subra et al., 2010). Depending on the function and on

Exosomes Decrease *In Vitro* Infectivity

**1.2 Rapid depletion of CD4TL during primary infection** 

of HIV-1 Preparations: Implication for CD4+T Lymphocyte Depletion *In Vivo* 101

role of the HIV-1 protein Nef (released in association with exosomes) in inducing apoptosis of bystander CD4TL has been published recently (Lenassi et al., 2010). All of these data have led us to examine the involvement of exosomes in CD4TL depletion during HIV-1 infection.

HIV-1-caused disease is characterized by a state of chronic immune activation due to sustained inflammation and immune hyperactivation that persists even under antiretroviral therapy (HAART) (Imami et al., 2001). Several observations from non-pathogenic *simian immunodeficiency virus* (SIV) infection, HIV-1 infected "elite controllers", "elite suppressors" or long-term non-progressors reveal a good correlation between low level of activation of the immune system and absence of clinical signs of AIDS (Bailey et al., 2008; Fontaine et al., 2011; Milush et al., 2007; Shacklett, 2010; Silvestri et al., 2003). In contrast, strongly increased immune activation characterized by dysregulated neutrophil and macrophage functions (Roilides et al., 1990; Torre et al., 2002), polyclonal B cell activation (Aberg et al., 2005), increased T cell turnover (Aberg et al., 2005), increased numbers of T cells with an activated phenotype (Aberg et al., 2005) and increased levels of pro-inflammatory molecules are hallmarks of disease progression in pathogenic infections by primate (HIV/SIV) lentiviruses (Ascher and Sheppard, 1988), (Giorgi et al., 1999; Liu et al., 1997). More significant is that a major rapid loss of mucosal CD4TL occurs in the gut-associated lymphoid tissues quite early in HIV-1 infection (Brenchley, Price, and Douek, 2006; Brenchley et al., 2004; Mehandru et al., 2004). At this stage, both mucosal lymph node destruction (which initiates immune dysfunction) and loss of integrity of the gut epithelium allow microbial products to cross the intestinal barrier. This translocation phenomenon produces high levels of circulating bacterial lipopolysaccharides (Brenchley, Price, and Douek, 2006) and thus contributes to the maintenance of the inflammatory state and systemic immune activation observed in chronic HIV-1-infected patients (Brenchley, Price, and Douek, 2006; Marchetti et al., 2008). These studies all point to early events in HIV-1 infection as decisive determinants of the irreversible damage inflicted on immune cells. It is well established that dendritic cells (DCs) are involved early in HIV-1 transmission (Granelli-Piperno et al., 1998; Manel et al., 2010; Tsunetsugu-Yokota et al., 1997). It is also known that CD4TL, more particularly the Th17 mucosal subset (Cecchinato and Franchini, 2010; Cecchinato et al., 2008; Elhed and Unutmaz, 2010; Favre et al., 2009; Milush et al., 2011; Paiardini, 2010) are dysregulated (Elbim et al., 2009; Hofman et al., 1999; Okada, Takei, and Tashiro, 1997; Okada, Takei, and Tashiro, 1998; Pitrak et al., 1996; Roilides et al., 1993; Roilides et al., 1990; Szelc et al., 1992; Thorsen, Busch-Sorensen, and Sondergaard, 1989) in pathogenic HIV-1/SIV infection

(Brenchley et al., 2008; Elbim et al., 2009; Elbim et al., 2008; Favre et al., 2009).

A rapid decrease thus occurs in the numbers of both infected and uninfected CD4TL within the very first weeks of infection. CD4TL are known to play a pivotal role in orchestrating the immune response as well as in the development, maturation and maintenance of cytotoxic T cells (Matloubian, Concepcion, and Ahmed, 1994; Zajac et al., 1998), the development of a humoral response and B cell antibody class switching (Tsuji et al., 1994), control of the bactericidal activity of macrophages and induction of HIV-1-specific CD4 and CD8 T cell responses. In fact, HIV-1 infection and the specific immune response to it depend largely on CD4TL functionnality and depletion of these cells during primary infection constitutes major interference, perhaps explaining the long-term inability of the host immune response to control the infection. Different mechanisms have been proposed to explain the significant

the activation state of the secreting cells, exosomes thus regulate multiple pathways in neighbouring cells in autocrine, paracrine and juxtacrine fashion.

The mechanism by which molecules are sorted in exosomes involves a recycling process that is influenced by molecular lateral mobility within lipid domains (de Gassart et al., 2003; de Gassart et al., 2004). MHC-II, co-stimulatory molecules, enzymes (Alonso et al., 2007; Baynes et al., 1991) and heat-shock proteins (HSP) (Lancaster and Febbraio, 2005) are among the proteins associated with exosomes (Segura, Amigorena, and Thery, 2005; Segura et al., 2005; Skokos et al., 2001).

Exosomes are similar to retroviruses not only in terms of size but also the molecules they incorporate and their ability to activate immune cells. Exosomes are slightly smaller and more heterogeneous in size (30-100 nm) than HIV-1 particles (100 nm). The most obvious similarity between these two types of particles is the presence of molecules of host origin. For example, incorporation of MHC-I and MHC-II by virions and by exosomes has been described (Cantin, Fortin, and Tremblay, 1996; Cantin, Martin, and Tremblay, 2001; Gansuvd et al., 2003; Raposo et al., 2002; Vincent-Schneider et al., 2002). In addition, several cell-surface molecules such as LFA-1 integrins (CD11a, CD18), co-stimulatory molecules (CD28, CD54) and complement-neutralizing molecules (CD55, CD59) are associated with both particles (Cantin, Methot, and Tremblay, 2005; Nguyen et al., 2003; Thery et al., 2001; Thery et al., 1999). Finally, the buoyant density of exosomes ranges from 1.13 to 1.21 g/l, while that of HIV-1 particles ranges from 1.16 to 1.18 g/l (Thery et al., 2001; Wang et al., 1999). Similar protein and lipid composition as well as buoyant densities render the separation of exosomes from virions quite difficult using standard techniques such as density-gradient centrifugation. These problems prompted us to use an Optiprep™-based velocity gradient method (Cantin et al., 2008), which has allowed us to show clearly that exosomes can be separated completely from viruses, based on detection of exosome marker (acetylcholinesterase) and HIV-1 marker (capsid protein p24).

The relationship between exosome biogenesis and retrovirus assembly has not yet been described in satisfactory detail. Although considerable evidence points to the takeover of the intracellular machinery responsible for MVB biogenesis (located at the cytoplasmic membrane) in the case of HIV-1 budding from CD4+ T lymphocytes (CD4TL), virions are found in endosomes of macrophages and dendritic cells (DCs), suggesting an internal budding process (Booth et al., 2006; Gould, Hildreth, and Booth, 2004; Morita and Sundquist, 2004; Nguyen et al., 2003). Comparative studies of exosomes and HIV-1 particle production pathways (Nguyen et al., 2003) based on observations of similar viral budding (Derse et al., 1987) and uptake by cells (Izquierdo-Useros et al., 2010) indicate that retroviruses evolved by exploiting the exosome release pathway.

The highly varied exosome composition and content suggest crucial roles for these vesicles in intercellular communication (Thery, Zitvogel, and Amigorena, 2002), transport of genetic material (mRNA or microRNA) (Valadi et al., 2007) and exchange of proteins (Andre et al., 2004; Thery, Zitvogel, and Amigorena, 2002), or in inflammation by carrying bioactive lipids (Esser et al.; Subra et al., 2010). Numerous studies indicate more efficient T cell activation by exosomes released from mature (mDCs) than from immature DCs (iDCs) (Admyre et al., 2006; Chaput and Thery, 2010; Segura, Amigorena, and Thery, 2005). In other studies, an inhibitory role for exosomes in the immune response has been described and particularly in the induction of T cell death via either FasL or galactin-9 by tumour-derived exosomes (Abusamra et al., 2005; Alonso et al., 2007; Chaput and Thery, 2010; Klibi et al., 2009; Ren et al.; Xie et al., 2010) (Andreola et al., 2002; Monleon et al., 2001). Compelling evidence for a role of the HIV-1 protein Nef (released in association with exosomes) in inducing apoptosis of bystander CD4TL has been published recently (Lenassi et al., 2010). All of these data have led us to examine the involvement of exosomes in CD4TL depletion during HIV-1 infection.

## **1.2 Rapid depletion of CD4TL during primary infection**

100 Understanding HIV/AIDS Management and Care – Pandemic Approaches in the 21st Century

the activation state of the secreting cells, exosomes thus regulate multiple pathways in

The mechanism by which molecules are sorted in exosomes involves a recycling process that is influenced by molecular lateral mobility within lipid domains (de Gassart et al., 2003; de Gassart et al., 2004). MHC-II, co-stimulatory molecules, enzymes (Alonso et al., 2007; Baynes et al., 1991) and heat-shock proteins (HSP) (Lancaster and Febbraio, 2005) are among the proteins associated with exosomes (Segura, Amigorena, and Thery, 2005; Segura et al., 2005;

Exosomes are similar to retroviruses not only in terms of size but also the molecules they incorporate and their ability to activate immune cells. Exosomes are slightly smaller and more heterogeneous in size (30-100 nm) than HIV-1 particles (100 nm). The most obvious similarity between these two types of particles is the presence of molecules of host origin. For example, incorporation of MHC-I and MHC-II by virions and by exosomes has been described (Cantin, Fortin, and Tremblay, 1996; Cantin, Martin, and Tremblay, 2001; Gansuvd et al., 2003; Raposo et al., 2002; Vincent-Schneider et al., 2002). In addition, several cell-surface molecules such as LFA-1 integrins (CD11a, CD18), co-stimulatory molecules (CD28, CD54) and complement-neutralizing molecules (CD55, CD59) are associated with both particles (Cantin, Methot, and Tremblay, 2005; Nguyen et al., 2003; Thery et al., 2001; Thery et al., 1999). Finally, the buoyant density of exosomes ranges from 1.13 to 1.21 g/l, while that of HIV-1 particles ranges from 1.16 to 1.18 g/l (Thery et al., 2001; Wang et al., 1999). Similar protein and lipid composition as well as buoyant densities render the separation of exosomes from virions quite difficult using standard techniques such as density-gradient centrifugation. These problems prompted us to use an Optiprep™-based velocity gradient method (Cantin et al., 2008), which has allowed us to show clearly that exosomes can be separated completely from viruses, based on detection of exosome marker

The relationship between exosome biogenesis and retrovirus assembly has not yet been described in satisfactory detail. Although considerable evidence points to the takeover of the intracellular machinery responsible for MVB biogenesis (located at the cytoplasmic membrane) in the case of HIV-1 budding from CD4+ T lymphocytes (CD4TL), virions are found in endosomes of macrophages and dendritic cells (DCs), suggesting an internal budding process (Booth et al., 2006; Gould, Hildreth, and Booth, 2004; Morita and Sundquist, 2004; Nguyen et al., 2003). Comparative studies of exosomes and HIV-1 particle production pathways (Nguyen et al., 2003) based on observations of similar viral budding (Derse et al., 1987) and uptake by cells (Izquierdo-Useros et al., 2010) indicate that

The highly varied exosome composition and content suggest crucial roles for these vesicles in intercellular communication (Thery, Zitvogel, and Amigorena, 2002), transport of genetic material (mRNA or microRNA) (Valadi et al., 2007) and exchange of proteins (Andre et al., 2004; Thery, Zitvogel, and Amigorena, 2002), or in inflammation by carrying bioactive lipids (Esser et al.; Subra et al., 2010). Numerous studies indicate more efficient T cell activation by exosomes released from mature (mDCs) than from immature DCs (iDCs) (Admyre et al., 2006; Chaput and Thery, 2010; Segura, Amigorena, and Thery, 2005). In other studies, an inhibitory role for exosomes in the immune response has been described and particularly in the induction of T cell death via either FasL or galactin-9 by tumour-derived exosomes (Abusamra et al., 2005; Alonso et al., 2007; Chaput and Thery, 2010; Klibi et al., 2009; Ren et al.; Xie et al., 2010) (Andreola et al., 2002; Monleon et al., 2001). Compelling evidence for a

neighbouring cells in autocrine, paracrine and juxtacrine fashion.

(acetylcholinesterase) and HIV-1 marker (capsid protein p24).

retroviruses evolved by exploiting the exosome release pathway.

Skokos et al., 2001).

HIV-1-caused disease is characterized by a state of chronic immune activation due to sustained inflammation and immune hyperactivation that persists even under antiretroviral therapy (HAART) (Imami et al., 2001). Several observations from non-pathogenic *simian immunodeficiency virus* (SIV) infection, HIV-1 infected "elite controllers", "elite suppressors" or long-term non-progressors reveal a good correlation between low level of activation of the immune system and absence of clinical signs of AIDS (Bailey et al., 2008; Fontaine et al., 2011; Milush et al., 2007; Shacklett, 2010; Silvestri et al., 2003). In contrast, strongly increased immune activation characterized by dysregulated neutrophil and macrophage functions (Roilides et al., 1990; Torre et al., 2002), polyclonal B cell activation (Aberg et al., 2005), increased T cell turnover (Aberg et al., 2005), increased numbers of T cells with an activated phenotype (Aberg et al., 2005) and increased levels of pro-inflammatory molecules are hallmarks of disease progression in pathogenic infections by primate (HIV/SIV) lentiviruses (Ascher and Sheppard, 1988), (Giorgi et al., 1999; Liu et al., 1997). More significant is that a major rapid loss of mucosal CD4TL occurs in the gut-associated lymphoid tissues quite early in HIV-1 infection (Brenchley, Price, and Douek, 2006; Brenchley et al., 2004; Mehandru et al., 2004). At this stage, both mucosal lymph node destruction (which initiates immune dysfunction) and loss of integrity of the gut epithelium allow microbial products to cross the intestinal barrier. This translocation phenomenon produces high levels of circulating bacterial lipopolysaccharides (Brenchley, Price, and Douek, 2006) and thus contributes to the maintenance of the inflammatory state and systemic immune activation observed in chronic HIV-1-infected patients (Brenchley, Price, and Douek, 2006; Marchetti et al., 2008). These studies all point to early events in HIV-1 infection as decisive determinants of the irreversible damage inflicted on immune cells. It is well established that dendritic cells (DCs) are involved early in HIV-1 transmission (Granelli-Piperno et al., 1998; Manel et al., 2010; Tsunetsugu-Yokota et al., 1997). It is also known that CD4TL, more particularly the Th17 mucosal subset (Cecchinato and Franchini, 2010; Cecchinato et al., 2008; Elhed and Unutmaz, 2010; Favre et al., 2009; Milush et al., 2011; Paiardini, 2010) are dysregulated (Elbim et al., 2009; Hofman et al., 1999; Okada, Takei, and Tashiro, 1997; Okada, Takei, and Tashiro, 1998; Pitrak et al., 1996; Roilides et al., 1993; Roilides et al., 1990; Szelc et al., 1992; Thorsen, Busch-Sorensen, and Sondergaard, 1989) in pathogenic HIV-1/SIV infection (Brenchley et al., 2008; Elbim et al., 2009; Elbim et al., 2008; Favre et al., 2009).

A rapid decrease thus occurs in the numbers of both infected and uninfected CD4TL within the very first weeks of infection. CD4TL are known to play a pivotal role in orchestrating the immune response as well as in the development, maturation and maintenance of cytotoxic T cells (Matloubian, Concepcion, and Ahmed, 1994; Zajac et al., 1998), the development of a humoral response and B cell antibody class switching (Tsuji et al., 1994), control of the bactericidal activity of macrophages and induction of HIV-1-specific CD4 and CD8 T cell responses. In fact, HIV-1 infection and the specific immune response to it depend largely on CD4TL functionnality and depletion of these cells during primary infection constitutes major interference, perhaps explaining the long-term inability of the host immune response to control the infection. Different mechanisms have been proposed to explain the significant

Exosomes Decrease *In Vitro* Infectivity

Fig. 1. Potential Role of exosomes in HIV-1 infection

of HIV-1 Preparations: Implication for CD4+T Lymphocyte Depletion *In Vivo* 103

After their migration to the lymph nodes, iDCs likely transfer HIV-1 to CD4TL with great efficiency and simultaneously release exosomes. The ability of exosomes to activate CD4TL, thereby enhancing HIV-1 replication, or to induce T cell apoptosis directly, could contribute

depletion of CD4TL in the gut-associated lymphatic tissues. Among these, direct infection of CD4TL by the virus (Arnoult et al., 2003), cytotoxic activity of CD8 T cells against infected cells (Sewell et al., 2000) and cytopathic effects on bystander cells or abortive infection (Doitsh et al., 2010) are the most plausible. However, additional factors, including HIV-1 proteins such as Vpr, Tat, Nef, VpU, proteases and gp120 (Varbanov, Espert, and Biard-Piechaczyk, 2006; Wan and Chen, 2010), mechanisms such as activation-induced cell death (AICD) mediated by Fas, TNF and TRAIL/APO2 (Lichtner et al., 2004) or dysregulation of cytokine/chemokine production (Saelens et al., 2004) can contribute to CD4TL death. Moreover, the detection of Nef in exosomes and the known involvement of this viral protein in apoptosis add support to the potential role of exosomes in bystander cell viability (Lenassi et al., 2010). We therefore propose another mechanism involving the release, from HIV-1-loaded iDCs, of exosomes that can induce functional defects in CD4TL and contribute to their elimination.

#### **1.3 The role of dendritic cells in HIV-1 primary infection**

The weakening of the immune system begins soon after the virus enters the body, which it does principally via the mucosal tissues. Following transmission of HIV-1, the virus crosses the mucosal barrier and is met by DCs, which are among the first cells to encounter the virus (Hladik and McElrath, 2008). A major immune system cell type involved in capturing and internalizing HIV-1 is the iDCs, which then migrates principally to the lymph nodes of the gastrointestinal tract, a site of HIV-1 replication during acute infection. Despite the progress that has been made in understanding iDC/HIV-1 interactions as well as virion sequestration and transmission to CD4TL, several fundamental questions surrounding the near total depletion of memory CD4TL observed during the acute infection (Brenchley et al., 2004; Guadalupe et al., 2003; Li et al., 2005; Mattapallil et al., 2005; Mehandru et al., 2004) remain unanswered. It is well known that both cells play a pivotal role in the dissemination of HIV-1, in the establishment of infection and also in anti-HIV-1 immunity.

It is now well established that HIV-1 entry into CD4TL is mediated by cellular chemokine receptors such as CCR5 or CXCR4. However, we, along with others, have found that additional factors, such as the DC-SIGN and DCIR lectins can mediate virus attachment to DCs and its subsequent endocytosis (Cambi et al., 2009; Geijtenbeek et al., 2000; Lambert et al., 2008; Permanyer, Ballana, and Este, 2010). Indeed, several recent studies have shown virions concentrated in late endocytic compartments also called multivesicular bodies (MVBs) or MHC class II compartments in mature DCs (Garcia et al., 2005; Izquierdo-Useros et al., 2009; Kwon et al., 2002), where they are sheltered both from the action of antiviral drugs and the immune response. Mature DCs are capable of stocking viral particles and migrating via the lymphatic network to lymph nodes and thus constitute reservoirs of virions. At this stage, it is thought that mDCs can transmit virions to T cells through two sequential routes: an early route known as trans-infection, via passive transfer through late endosomes, or a later route called cis-infection following productive infection (Turville et al., 2004). Fusion of late endosomes with the DC plasma membrane releases large numbers of virions into intercellular space called the virological synapse, which can infect nearby target cells (Moir, Chun, and Fauci, 2010; Piguet and Steinman, 2007). Endosomes contain the intraluminal vesicles that become exosomes when delivered into this space at the same time as the viral particles contained in HIV-1 loaded DCs. Exosomes and virions thus pass via the late endosome across the cell to be exchanged with other cells (Izquierdo-Useros et al., 2010).

depletion of CD4TL in the gut-associated lymphatic tissues. Among these, direct infection of CD4TL by the virus (Arnoult et al., 2003), cytotoxic activity of CD8 T cells against infected cells (Sewell et al., 2000) and cytopathic effects on bystander cells or abortive infection (Doitsh et al., 2010) are the most plausible. However, additional factors, including HIV-1 proteins such as Vpr, Tat, Nef, VpU, proteases and gp120 (Varbanov, Espert, and Biard-Piechaczyk, 2006; Wan and Chen, 2010), mechanisms such as activation-induced cell death (AICD) mediated by Fas, TNF and TRAIL/APO2 (Lichtner et al., 2004) or dysregulation of cytokine/chemokine production (Saelens et al., 2004) can contribute to CD4TL death. Moreover, the detection of Nef in exosomes and the known involvement of this viral protein in apoptosis add support to the potential role of exosomes in bystander cell viability (Lenassi et al., 2010). We therefore propose another mechanism involving the release, from HIV-1-loaded iDCs, of exosomes that can induce functional defects in CD4TL and contribute

The weakening of the immune system begins soon after the virus enters the body, which it does principally via the mucosal tissues. Following transmission of HIV-1, the virus crosses the mucosal barrier and is met by DCs, which are among the first cells to encounter the virus (Hladik and McElrath, 2008). A major immune system cell type involved in capturing and internalizing HIV-1 is the iDCs, which then migrates principally to the lymph nodes of the gastrointestinal tract, a site of HIV-1 replication during acute infection. Despite the progress that has been made in understanding iDC/HIV-1 interactions as well as virion sequestration and transmission to CD4TL, several fundamental questions surrounding the near total depletion of memory CD4TL observed during the acute infection (Brenchley et al., 2004; Guadalupe et al., 2003; Li et al., 2005; Mattapallil et al., 2005; Mehandru et al., 2004) remain unanswered. It is well known that both cells play a pivotal role in the dissemination of HIV-

It is now well established that HIV-1 entry into CD4TL is mediated by cellular chemokine receptors such as CCR5 or CXCR4. However, we, along with others, have found that additional factors, such as the DC-SIGN and DCIR lectins can mediate virus attachment to DCs and its subsequent endocytosis (Cambi et al., 2009; Geijtenbeek et al., 2000; Lambert et al., 2008; Permanyer, Ballana, and Este, 2010). Indeed, several recent studies have shown virions concentrated in late endocytic compartments also called multivesicular bodies (MVBs) or MHC class II compartments in mature DCs (Garcia et al., 2005; Izquierdo-Useros et al., 2009; Kwon et al., 2002), where they are sheltered both from the action of antiviral drugs and the immune response. Mature DCs are capable of stocking viral particles and migrating via the lymphatic network to lymph nodes and thus constitute reservoirs of virions. At this stage, it is thought that mDCs can transmit virions to T cells through two sequential routes: an early route known as trans-infection, via passive transfer through late endosomes, or a later route called cis-infection following productive infection (Turville et al., 2004). Fusion of late endosomes with the DC plasma membrane releases large numbers of virions into intercellular space called the virological synapse, which can infect nearby target cells (Moir, Chun, and Fauci, 2010; Piguet and Steinman, 2007). Endosomes contain the intraluminal vesicles that become exosomes when delivered into this space at the same time as the viral particles contained in HIV-1 loaded DCs. Exosomes and virions thus pass via the late endosome across the cell to be

to their elimination.

**1.3 The role of dendritic cells in HIV-1 primary infection** 

1, in the establishment of infection and also in anti-HIV-1 immunity.

exchanged with other cells (Izquierdo-Useros et al., 2010).

After their migration to the lymph nodes, iDCs likely transfer HIV-1 to CD4TL with great efficiency and simultaneously release exosomes. The ability of exosomes to activate CD4TL, thereby enhancing HIV-1 replication, or to induce T cell apoptosis directly, could contribute

Fig. 1. Potential Role of exosomes in HIV-1 infection

Exosomes Decrease *In Vitro* Infectivity

Invivogen (San Diego, CA, USA).

days.

**2.2 Virus production and purification** 

**2.3 Separation of exosomes and virions by velocity gradient** 

of HIV-1 Preparations: Implication for CD4+T Lymphocyte Depletion *In Vivo* 105

(Germantown, MD, USA). Experiments were performed with cell preparations that were devoid of contamination (i.e. DC purity > 95%; CD4TL purity > 98%). In all culture media, bovine exosomes from foetal bovine serum (FBS) were eliminated by O/N ultracentrifugation at 100,000*xg*. Complete RPMI 1640 culture medium contains FBS, penicillin G, streptomycin, glutamine from Wisent and primocin and plasmocin from

Virions were produced by transient transfection in human embryonic kidney 293T cells (HEK293T) as previously described (Cantin et al., 1997). Plasmids used include pJR-CSF (R5 tropic), pNLAD8 (R5-tropic), pNL4-3balenv (R5-tropic) and pNL4-3 (X4-tropic). The pNL4- 3balenv vector (provided by R. Pomerantz, Thomas Jefferson University, Philadelphia, PA, USA) was generated by replacing the *env* gene of the T-tropic HIV-1 strain, NL4-3, with that of the macrophage-tropic HIV-1 Bal strain, thus resulting in an infectious molecular clone with R5-tropic properties (Dornadula et al., 1999). Other plasmids were obtained from the AIDS Repository Reagent Program (Germantown, MD, USA). Several viral preparations were obtained from primary cells. Peripheral blood from healthy donors was centrifuged on lymphocyte separation medium (Wisent) to obtain PBMCs. NL4-3Balenv virions were propagated by acute infection of PBMCs (1.5x107 cells/ml) with virus (500 ng/ml) for six

Exosomes, microvesicles and virions contained in cell-free supernatants were passed through a 0.22 µm filter or centrifuged, 10 min at 10,000*xg* to eliminate micro-particles. Filtered virus and/or exosomes from 293T cells or PBMCs were concentrated by ultracentrifugation in an Optima L-90K Beckman Coulter centrifuge (Fullerton, CA) for 45 min at 31,500 rpm (100,000*xg*) in a 70 Ti rotor. The pellet containing virions and microvesicles/exosomes was re-suspended in 500 µl of PBS. HIV-1 viral particles were then centrifuged through a 6-18% Optiprep™ (60% iodixanol) separation gradient from Sigma Aldrich® (Winston Park, CA, USA) as previously described (Dettenhofer and Yu, 1999). The densities of each gradient fraction were below 1.13 g/ml, as shown in Figure 2. The virus preparations were then centrifuged using the Optima L-90K centrifuge for 75 min at 52,000 rpm (250,000*xg*) in a NVT65 rotor. Gradient fractions were collected from the top. Optiprep™ separation medium possesses intrinsic properties such as neutral pH and physiological osmolarity (Dettenhofer and Yu, 1999; Ford, Graham, and Rickwood, 1994; Van Veldhoven, Baumgart, and Mannaerts, 1996) that make it highly suitable for efficient separation of viral entities from cellular debris and microvesicles (Dettenhofer and Yu, 1999; Hermens et al., 1999; Moller-Larsen and Christensen, 1998; Zolotukhin et al., 1999). This technique, identical to the velocity gradient adapted by Dettenhofer (Dettenhofer and Yu, 1999) for HIV-1 separation, is also very efficient for separating microvesicle contaminants from HIV-1 preparations. The results depicted in Figure 3A (black rectangles) express the separation efficiency in terms of quantity of viral p24gag protein, which is concentrated in fraction 15.6. Electron microscopy, performed at the Armand Frappier Institute Microscopy Laboratory using standard protocol (Alain et al., 1987; Hammond et al., 1981), confirmed that fractions 15.6 and 16.8 contain concentrated and homogenous viral particles (1011 per ml). Virion contents were normalized by means of an in-house sensitive double-antibody

to the massive depletion of CD4TL. Indeed, our preliminary observations show that HIV-1 increases exosome release from iDCs. Some studies have shown that exosomes can activate T cells and consequently are involved in the regulation of the immune response (Admyre et al., 2006; Segura et al., 2005; Thery et al., 2002). Furthermore, the Fas-ligand on exosomes can also induce apoptosis of both CD4 and CD8 T cells (Abusamra et al., 2005; Alonso et al., 2007; Alonso et al., 2005; Segura, Amigorena, and Thery, 2005). It should be noted that the capacity of exosomes to regulate the viability of CD4 T cell sub-populations has not yet been fully investigated in the context of HIV-1 infection. In order to answer this important fundamental question, new methods are needed for separating mixtures of exosomes and HIV-1 to purity.

HIV-1 is a retrovirus that causes a slow but sustained depletion of CD4TL during the chronic stage of infection, leading to progressive failure of the immune system. The principal immune cell type that captures and internalizes HIV-1 is the iDC. HIV-1-loaded iDCs migrate to the lymph nodes of the gastrointestinal tract, a major site of HIV-1 replication during acute infection. Given that CD4TL play a pivotal role in the orchestration of the immune response, the rapid and sustained disappearance of mucosal CD4TL (within the first 15 days) compromises the development of both the cellular and humoral responses to HIV-1 infection. Exosomes release by DCs or CD4TL can contribute to elimination of CD4TL as well as other cell deregulations characterizing this crucial phase.

### **2. Protocol to study the role of exosomes in CD4TL viability in the context of HIV-1 infection**

#### **2.1 Cell purification**

Experiments were performed using human primary cells, DCs and CD4TL. Cells were isolated from peripheral blood mononuclear cells (PBMCs) obtained from anonymous and healthy volunteer donors. PBMCs were prepared by centrifugation on a lymphocyte separation medium from Wisent Inc. (St Bruno, QC, Canada). CD14+ cells were then isolated using a monocyte positive selection kit according to the manufacturer's instructions (Stemsep Human CD14 positive selection Kit, STEMCell Technologies, Vancouver, BC, Canada) using an AutoMacs (Miltenyl Biotech, Auburn, CA, USA) and a previously established procedure (Bounou et al., 2004; Gilbert et al., 2007a; Gilbert et al., 2007b). CD14+ cells were cultured in six-well plates at a concentration of 1 X 106 cells/ml. To generate iDCs, purified monocytes were cultured in complete culture medium supplemented every two days with granulocyte-macrophage colony-stimulating factor (1,000 U/ml) from Genscript (Cedarlane Laboratories, Burlington, ON, Canada) and IL-4 (200 U/ml) from R&D Systems (Minneapolis, MN, USA) for 6 to 7 days. Expression of CD3 and CD19 was measured to assess contamination with T and B cells respectively. Expression of HLA-DR, CD86, DC-SIGN, CD83 and CD14 was monitored to verify the immature phenotypes of DCs. In the immature state, DCs express a high level of DC-SIGN and low level of CD83, whereas mature DCs express CD83 and high levels of ICAM-1, HLA-DR and CD86.

CD4TL were isolated using a negative selection kit according to the manufacturer's instructions (Stemsep Human CD4 T cell enrichment kit, STEMCell Technologies). In some experiments, these cells were activated with phytohemagglutinin-L (1 µg/ml) to obtain mitogen-stimulated cells and maintained at a density of 2 x 106 cells/ml in RPMI supplemented with IL-2 (30 U/ml) obtained through the AIDS Repository Reagent Program (Germantown, MD, USA). Experiments were performed with cell preparations that were devoid of contamination (i.e. DC purity > 95%; CD4TL purity > 98%). In all culture media, bovine exosomes from foetal bovine serum (FBS) were eliminated by O/N ultracentrifugation at 100,000*xg*. Complete RPMI 1640 culture medium contains FBS, penicillin G, streptomycin, glutamine from Wisent and primocin and plasmocin from Invivogen (San Diego, CA, USA).

## **2.2 Virus production and purification**

104 Understanding HIV/AIDS Management and Care – Pandemic Approaches in the 21st Century

to the massive depletion of CD4TL. Indeed, our preliminary observations show that HIV-1 increases exosome release from iDCs. Some studies have shown that exosomes can activate T cells and consequently are involved in the regulation of the immune response (Admyre et al., 2006; Segura et al., 2005; Thery et al., 2002). Furthermore, the Fas-ligand on exosomes can also induce apoptosis of both CD4 and CD8 T cells (Abusamra et al., 2005; Alonso et al., 2007; Alonso et al., 2005; Segura, Amigorena, and Thery, 2005). It should be noted that the capacity of exosomes to regulate the viability of CD4 T cell sub-populations has not yet been fully investigated in the context of HIV-1 infection. In order to answer this important fundamental question, new methods are needed for separating mixtures of exosomes and

HIV-1 is a retrovirus that causes a slow but sustained depletion of CD4TL during the chronic stage of infection, leading to progressive failure of the immune system. The principal immune cell type that captures and internalizes HIV-1 is the iDC. HIV-1-loaded iDCs migrate to the lymph nodes of the gastrointestinal tract, a major site of HIV-1 replication during acute infection. Given that CD4TL play a pivotal role in the orchestration of the immune response, the rapid and sustained disappearance of mucosal CD4TL (within the first 15 days) compromises the development of both the cellular and humoral responses to HIV-1 infection. Exosomes release by DCs or CD4TL can contribute to elimination of

**2. Protocol to study the role of exosomes in CD4TL viability in the context of** 

Experiments were performed using human primary cells, DCs and CD4TL. Cells were isolated from peripheral blood mononuclear cells (PBMCs) obtained from anonymous and healthy volunteer donors. PBMCs were prepared by centrifugation on a lymphocyte separation medium from Wisent Inc. (St Bruno, QC, Canada). CD14+ cells were then isolated using a monocyte positive selection kit according to the manufacturer's instructions (Stemsep Human CD14 positive selection Kit, STEMCell Technologies, Vancouver, BC, Canada) using an AutoMacs (Miltenyl Biotech, Auburn, CA, USA) and a previously established procedure (Bounou et al., 2004; Gilbert et al., 2007a; Gilbert et al., 2007b). CD14+ cells were cultured in six-well plates at a concentration of 1 X 106 cells/ml. To generate iDCs, purified monocytes were cultured in complete culture medium supplemented every two days with granulocyte-macrophage colony-stimulating factor (1,000 U/ml) from Genscript (Cedarlane Laboratories, Burlington, ON, Canada) and IL-4 (200 U/ml) from R&D Systems (Minneapolis, MN, USA) for 6 to 7 days. Expression of CD3 and CD19 was measured to assess contamination with T and B cells respectively. Expression of HLA-DR, CD86, DC-SIGN, CD83 and CD14 was monitored to verify the immature phenotypes of DCs. In the immature state, DCs express a high level of DC-SIGN and low level of CD83, whereas

CD4TL as well as other cell deregulations characterizing this crucial phase.

mature DCs express CD83 and high levels of ICAM-1, HLA-DR and CD86.

CD4TL were isolated using a negative selection kit according to the manufacturer's instructions (Stemsep Human CD4 T cell enrichment kit, STEMCell Technologies). In some experiments, these cells were activated with phytohemagglutinin-L (1 µg/ml) to obtain mitogen-stimulated cells and maintained at a density of 2 x 106 cells/ml in RPMI supplemented with IL-2 (30 U/ml) obtained through the AIDS Repository Reagent Program

HIV-1 to purity.

**HIV-1 infection 2.1 Cell purification**  Virions were produced by transient transfection in human embryonic kidney 293T cells (HEK293T) as previously described (Cantin et al., 1997). Plasmids used include pJR-CSF (R5 tropic), pNLAD8 (R5-tropic), pNL4-3balenv (R5-tropic) and pNL4-3 (X4-tropic). The pNL4- 3balenv vector (provided by R. Pomerantz, Thomas Jefferson University, Philadelphia, PA, USA) was generated by replacing the *env* gene of the T-tropic HIV-1 strain, NL4-3, with that of the macrophage-tropic HIV-1 Bal strain, thus resulting in an infectious molecular clone with R5-tropic properties (Dornadula et al., 1999). Other plasmids were obtained from the AIDS Repository Reagent Program (Germantown, MD, USA). Several viral preparations were obtained from primary cells. Peripheral blood from healthy donors was centrifuged on lymphocyte separation medium (Wisent) to obtain PBMCs. NL4-3Balenv virions were propagated by acute infection of PBMCs (1.5x107 cells/ml) with virus (500 ng/ml) for six days.

## **2.3 Separation of exosomes and virions by velocity gradient**

Exosomes, microvesicles and virions contained in cell-free supernatants were passed through a 0.22 µm filter or centrifuged, 10 min at 10,000*xg* to eliminate micro-particles. Filtered virus and/or exosomes from 293T cells or PBMCs were concentrated by ultracentrifugation in an Optima L-90K Beckman Coulter centrifuge (Fullerton, CA) for 45 min at 31,500 rpm (100,000*xg*) in a 70 Ti rotor. The pellet containing virions and microvesicles/exosomes was re-suspended in 500 µl of PBS. HIV-1 viral particles were then centrifuged through a 6-18% Optiprep™ (60% iodixanol) separation gradient from Sigma Aldrich® (Winston Park, CA, USA) as previously described (Dettenhofer and Yu, 1999). The densities of each gradient fraction were below 1.13 g/ml, as shown in Figure 2. The virus preparations were then centrifuged using the Optima L-90K centrifuge for 75 min at 52,000 rpm (250,000*xg*) in a NVT65 rotor. Gradient fractions were collected from the top. Optiprep™ separation medium possesses intrinsic properties such as neutral pH and physiological osmolarity (Dettenhofer and Yu, 1999; Ford, Graham, and Rickwood, 1994; Van Veldhoven, Baumgart, and Mannaerts, 1996) that make it highly suitable for efficient separation of viral entities from cellular debris and microvesicles (Dettenhofer and Yu, 1999; Hermens et al., 1999; Moller-Larsen and Christensen, 1998; Zolotukhin et al., 1999). This technique, identical to the velocity gradient adapted by Dettenhofer (Dettenhofer and Yu, 1999) for HIV-1 separation, is also very efficient for separating microvesicle contaminants from HIV-1 preparations. The results depicted in Figure 3A (black rectangles) express the separation efficiency in terms of quantity of viral p24gag protein, which is concentrated in fraction 15.6. Electron microscopy, performed at the Armand Frappier Institute Microscopy Laboratory using standard protocol (Alain et al., 1987; Hammond et al., 1981), confirmed that fractions 15.6 and 16.8 contain concentrated and homogenous viral particles (1011 per ml). Virion contents were normalized by means of an in-house sensitive double-antibody

Exosomes Decrease *In Vitro* Infectivity

**2.4 Rapid purification of exosomes** 

14.4.

**2.5 Analysis of protein contained in each gradient fraction** 

from the HIV-1 preparation increased cell viability.

transmission electron microscope (see Fig. 3A).

**2.7 Virus infection assays** 

of HIV-1 Preparations: Implication for CD4+T Lymphocyte Depletion *In Vivo* 107

A quick alternative procedure for eliminating exosomes prior to contacting the cells with HIV-1 preparation consisted of immuno-depleting AChE-bearing vesicles directly from 100,000*xg* pellet (starting material) as shown in Figure 3B. Briefly, ultracentrifuged cells supernatants were diluted in PBS and incubated with protein A/G beads pre-coated with anti-AChE (AE-1 from ATCC) or with isotypic control antibodies (IgG1). The beads (exosomes) and final supernatants (infectious viruses without exosomes) were kept for viral Titer determination (data not shown) and AChE assay (Fig. 3B). The results show that 95% of the AChE activity was recovered using the anti-AChE beads after the first round of precipitation. Exosomes can be eluted from the beads in two minutes using 50 µl of 0.2M glycine in 0.1M KH2PO4 pH 3. Acidic pH was then neutralized by adding 100 µl of 0.1M K2HPO4 (pH 8.8). It should be noted that this treatment does not affect AChE activity.

Several proteins including LFA-1 are known to be present on both particles and were quantified in exosome/HIV-1 preparations by means of an in-house enzymatic sandwichtype immunoassay. Plates were initially coated with anti-LFA-1 (MEM-25) (50 µg/ml) in carbonate buffer. After three washes with PBS/0.1% Tween 20, the non-specific sites were blocked with PBS/0.1% Tween 20/1% BSA for 1 hr at RT. The plates were washed and 75 µl of each gradient fraction mixed with 0.5% Triton X-100 to cause lysis were added and the plates were then held overnight at 4°C. The plates were then washed three times and biotinylated anti-LFA-1 (TSI/22) (0.5 µg/ml) was added in blocking solution for 1 hr at RT. After three more washings, streptavidin-peroxidase conjugate was added for 30 min at RT. The plates were washed and the detection was performed by the addition of the TMB-S substrate followed by the addition of H3PO4. Absorbance at 450 nm was read to quantify LFA-1 in each fraction. Figure 3C shows that LFA-1 is present on both vesicles (exosomes and fully infectious virions) and also in uncharacterized particles from fractions 13.2 and

Both methods (Optiprep™ gradient and immunodepletion) were used to purify exosomes. These methods were employed to eliminate exosomes from all viral or mock preparations before contact with cells and results depicted in Figure 3D show that depletion of exosomes

**2.6 Electron microscopy procedure and examination of negative-stain specimens**  The protocol described by Hammond et al. and Alain et al was performed with each analytical fraction (Alain et al., 1987; Hammond et al., 1981). Briefly, microvesicle preparation was fixed with an equal volume of 2% paraformaldehyde and 50–200 µL in a micro-ultracentrifuge tube with a formvar-coated electron microscopy grid at the bottom were concentrated for 5 minutes at 120,000*xg* (20 psig) using an Airfuge ultracentrifuge (Beckman, Palo Alto, CA, USA). The grids were dried on bibulous paper and stained for 1 min with a drop of 3% phosphotungstic acid (pH 6.0). The concentration, shape and overall appearance of the microvesicles were examined with a Hitachi 7100 (Hitachi, Japan)

Indicator cell line TZM-bl, which carries a stably integrated luciferase reporter gene under the control of the HIV-1 regulatory element known as the long terminal repeat (Thibault et

sandwich enzyme-linked immunosorbent assay (ELISA) specific for the viral p24gag protein (Bounou, Leclerc, and Tremblay, 2002).

The distribution of fractions containing exosomes along the gradient was evaluated by measuring acetylcholinesterase (AChE) activity, which is a commonly used specific exosome marker (Fig. 3A, open rectangles) (Gastpar et al., 2005; Rieu et al., 2000). This enzyme activity was measured following a procedure previously described (Cantin et al., 2008). Briefly, 30 µl of standard or sample were mixed with PBS pH 8 containing acetylthiocholine and PBS pH 7 containing 5,5-dithio-*bis*-(2-nitrobenzoic acid) to obtain final reagent concentrations of respectively 1.25 mM and 0.1 mM in a volume of 200 µl and held at room temperature. Changes in absorption at 450 nm were monitored for 10 min with a plate reader spectrophotometer (ELX808, BIO-TEK instruments, Winooski, VT, USA). Glycosylphosphatidylinositol-anchored AChE as well as other GPI proteins are localized in the MVB and are part of the exosome membrane (Gastpar et al., 2005; Johnstone et al., 1987). The results illustrated in Figure 3 show that AChE activity is concentrated in fractions 8.4 to 12 and microscopic observations showed that 108 particles/ml are present in these fractions. To confirm the efficiency of this velocity gradient method for separating HIV-1 from exosomes, we also checked for the presence of infectious particles in each gradient fraction (i.e. infectivity on TZM-bl cells (Cantin et al., 2008) or measure of spliced TAT on CD4TL). Infectivity assays using the TZM-bl indicator cell line showed that fractions 14.4 to 16.8 contained fully infectious virus while fractions 8.4 to 12 contained insignificant amounts. These results thus clearly indicate that our procedures can isolate exosomes and HIV-1 particles differentially and independently. We are satisfied that fractions 8.4 to 12 of the Optiprep™ gradients contained exosomes exclusively, while fractions 14.4 to 16.8 contained infectious viruses.

Fig. 2. Density of gradient fractions

The particularity of velocity gradient separation resides in its capacity to isolate particles of similar density. The density range of 6 to 18 % of Optiprep™ is 1.03 to 1.13 g/l.

### **2.4 Rapid purification of exosomes**

106 Understanding HIV/AIDS Management and Care – Pandemic Approaches in the 21st Century

sandwich enzyme-linked immunosorbent assay (ELISA) specific for the viral p24gag protein

The distribution of fractions containing exosomes along the gradient was evaluated by measuring acetylcholinesterase (AChE) activity, which is a commonly used specific exosome marker (Fig. 3A, open rectangles) (Gastpar et al., 2005; Rieu et al., 2000). This enzyme activity was measured following a procedure previously described (Cantin et al., 2008). Briefly, 30 µl of standard or sample were mixed with PBS pH 8 containing acetylthiocholine and PBS pH 7 containing 5,5-dithio-*bis*-(2-nitrobenzoic acid) to obtain final reagent concentrations of respectively 1.25 mM and 0.1 mM in a volume of 200 µl and held at room temperature. Changes in absorption at 450 nm were monitored for 10 min with a plate reader spectrophotometer (ELX808, BIO-TEK instruments, Winooski, VT, USA). Glycosylphosphatidylinositol-anchored AChE as well as other GPI proteins are localized in the MVB and are part of the exosome membrane (Gastpar et al., 2005; Johnstone et al., 1987). The results illustrated in Figure 3 show that AChE activity is concentrated in fractions 8.4 to 12 and microscopic observations showed that 108 particles/ml are present in these fractions. To confirm the efficiency of this velocity gradient method for separating HIV-1 from exosomes, we also checked for the presence of infectious particles in each gradient fraction (i.e. infectivity on TZM-bl cells (Cantin et al., 2008) or measure of spliced TAT on CD4TL). Infectivity assays using the TZM-bl indicator cell line showed that fractions 14.4 to 16.8 contained fully infectious virus while fractions 8.4 to 12 contained insignificant amounts. These results thus clearly indicate that our procedures can isolate exosomes and HIV-1 particles differentially and independently. We are satisfied that fractions 8.4 to 12 of the Optiprep™ gradients contained exosomes exclusively, while fractions 14.4 to 16.8 contained

The particularity of velocity gradient separation resides in its capacity to isolate particles of

similar density. The density range of 6 to 18 % of Optiprep™ is 1.03 to 1.13 g/l.

(Bounou, Leclerc, and Tremblay, 2002).

infectious viruses.

Fig. 2. Density of gradient fractions

A quick alternative procedure for eliminating exosomes prior to contacting the cells with HIV-1 preparation consisted of immuno-depleting AChE-bearing vesicles directly from 100,000*xg* pellet (starting material) as shown in Figure 3B. Briefly, ultracentrifuged cells supernatants were diluted in PBS and incubated with protein A/G beads pre-coated with anti-AChE (AE-1 from ATCC) or with isotypic control antibodies (IgG1). The beads (exosomes) and final supernatants (infectious viruses without exosomes) were kept for viral Titer determination (data not shown) and AChE assay (Fig. 3B). The results show that 95% of the AChE activity was recovered using the anti-AChE beads after the first round of precipitation. Exosomes can be eluted from the beads in two minutes using 50 µl of 0.2M glycine in 0.1M KH2PO4 pH 3. Acidic pH was then neutralized by adding 100 µl of 0.1M K2HPO4 (pH 8.8). It should be noted that this treatment does not affect AChE activity.

## **2.5 Analysis of protein contained in each gradient fraction**

Several proteins including LFA-1 are known to be present on both particles and were quantified in exosome/HIV-1 preparations by means of an in-house enzymatic sandwichtype immunoassay. Plates were initially coated with anti-LFA-1 (MEM-25) (50 µg/ml) in carbonate buffer. After three washes with PBS/0.1% Tween 20, the non-specific sites were blocked with PBS/0.1% Tween 20/1% BSA for 1 hr at RT. The plates were washed and 75 µl of each gradient fraction mixed with 0.5% Triton X-100 to cause lysis were added and the plates were then held overnight at 4°C. The plates were then washed three times and biotinylated anti-LFA-1 (TSI/22) (0.5 µg/ml) was added in blocking solution for 1 hr at RT. After three more washings, streptavidin-peroxidase conjugate was added for 30 min at RT. The plates were washed and the detection was performed by the addition of the TMB-S substrate followed by the addition of H3PO4. Absorbance at 450 nm was read to quantify LFA-1 in each fraction. Figure 3C shows that LFA-1 is present on both vesicles (exosomes and fully infectious virions) and also in uncharacterized particles from fractions 13.2 and 14.4.

Both methods (Optiprep™ gradient and immunodepletion) were used to purify exosomes. These methods were employed to eliminate exosomes from all viral or mock preparations before contact with cells and results depicted in Figure 3D show that depletion of exosomes from the HIV-1 preparation increased cell viability.

## **2.6 Electron microscopy procedure and examination of negative-stain specimens**

The protocol described by Hammond et al. and Alain et al was performed with each analytical fraction (Alain et al., 1987; Hammond et al., 1981). Briefly, microvesicle preparation was fixed with an equal volume of 2% paraformaldehyde and 50–200 µL in a micro-ultracentrifuge tube with a formvar-coated electron microscopy grid at the bottom were concentrated for 5 minutes at 120,000*xg* (20 psig) using an Airfuge ultracentrifuge (Beckman, Palo Alto, CA, USA). The grids were dried on bibulous paper and stained for 1 min with a drop of 3% phosphotungstic acid (pH 6.0). The concentration, shape and overall appearance of the microvesicles were examined with a Hitachi 7100 (Hitachi, Japan) transmission electron microscope (see Fig. 3A).

#### **2.7 Virus infection assays**

Indicator cell line TZM-bl, which carries a stably integrated luciferase reporter gene under the control of the HIV-1 regulatory element known as the long terminal repeat (Thibault et

Exosomes Decrease *In Vitro* Infectivity

Fig. 4. Experimental set-up

**exosomes into the extracellular medium** 

viability.

**3. Results** 

microplate luminometer for 20s/well after a 2-5s delay.

of HIV-1 Preparations: Implication for CD4+T Lymphocyte Depletion *In Vivo* 109

al., 2007; Wei et al., 2002; Zhao et al., 2005), was used to quantify infectious viral particles after exosomes depletion. Each well contained 1.5 x 104 cells plus 100 µl of fraction in a final volume of 200 µl. Plates were incubated for 48 hrs. All experimental points were done in triplicate. Luciferase activity was determined following a modified version of a known protocol (Barbeau et al., 1997; Berube et al., 1996). Briefly, cell-free supernatant (100 µl) withdrawn from each well and mixed with solution containing 25 mM Tris phosphate pH 7.8, 2 mM dithiothreitol, 1% Triton X-100 and 10% glycerol (25 µl) to cause vesicle lysis was held at room temperature for 30 min. A 20-µl aliquot of this mixture was then mixed with 100 µl of luciferase assay buffer (20 mM Tricine, 1.07 mM (MgCO3)4·Mg(OH)2·5 H2O, 2.67 mM MgSO4, 0.1 mM EDTA, 270 µM coenzyme A, 470 µM luciferin, 530 µM ATP and 33.3 mM dithiothreitol) and the luciferase reaction was monitored on a Dynex MLX

This figure illustrates the experimental set-up used to test the impact of exosomes on cell

**3.1 Loading of DCs or CD4TL with HIV-1 induces the release of large amounts of** 

Exosomes contain several molecules that can either eliminate or activate CD4TL (Quah and O'Neill, 2005b; Segura et al., 2005; Thery et al., 2002). Moreover, exosome-like vesicles found in plasma induce apoptosis in a FasL-like manner (Ren et al., 2010). Although exosomes

The upper panel illustrates the purification protocols based on Optiprep™ velocity gradient and on immunocapture. Panel A shows velocity gradient purification of NL4-3balenv virions produced on HEK293T cells by transient transfection. Gradients were overlaid with 100,000*xg* centrifugal pellet. Viral protein p*24gag* (black rectangles) was measured by ELISA. AChE activity (x106 OD/min, open rectangles) was evaluated in each fraction by colorimetry. Each fraction was examined by electron microscopy for particle quality and quantity. Panel B represents the results obtained by immunocapture with AChE. Panel C shows the presence of LFA-1 in each gradient fraction. Finally, panel D shows that depletion of exosomes increased cell viability. These results show that we are able to separate exosomes from HIV-1 particles.

al., 2007; Wei et al., 2002; Zhao et al., 2005), was used to quantify infectious viral particles after exosomes depletion. Each well contained 1.5 x 104 cells plus 100 µl of fraction in a final volume of 200 µl. Plates were incubated for 48 hrs. All experimental points were done in triplicate. Luciferase activity was determined following a modified version of a known protocol (Barbeau et al., 1997; Berube et al., 1996). Briefly, cell-free supernatant (100 µl) withdrawn from each well and mixed with solution containing 25 mM Tris phosphate pH 7.8, 2 mM dithiothreitol, 1% Triton X-100 and 10% glycerol (25 µl) to cause vesicle lysis was held at room temperature for 30 min. A 20-µl aliquot of this mixture was then mixed with 100 µl of luciferase assay buffer (20 mM Tricine, 1.07 mM (MgCO3)4·Mg(OH)2·5 H2O, 2.67 mM MgSO4, 0.1 mM EDTA, 270 µM coenzyme A, 470 µM luciferin, 530 µM ATP and 33.3 mM dithiothreitol) and the luciferase reaction was monitored on a Dynex MLX microplate luminometer for 20s/well after a 2-5s delay.

Fig. 4. Experimental set-up

This figure illustrates the experimental set-up used to test the impact of exosomes on cell viability.

## **3. Results**

108 Understanding HIV/AIDS Management and Care – Pandemic Approaches in the 21st Century

The upper panel illustrates the purification protocols based on Optiprep™ velocity gradient and on immunocapture. Panel A shows velocity gradient purification of NL4-3balenv virions produced on HEK293T cells by transient transfection. Gradients were overlaid with 100,000*xg* centrifugal pellet. Viral protein p*24gag* (black rectangles) was measured by ELISA. AChE activity (x106 OD/min, open rectangles) was evaluated in each fraction by colorimetry. Each fraction was examined by electron microscopy for particle quality and quantity. Panel B represents the results obtained by immunocapture with AChE. Panel C shows the presence of LFA-1 in each gradient fraction. Finally, panel D shows that depletion of exosomes increased cell viability. These results show that we are able to separate exosomes from HIV-1 particles.

Fig. 3. Exosome and virus purification

#### **3.1 Loading of DCs or CD4TL with HIV-1 induces the release of large amounts of exosomes into the extracellular medium**

Exosomes contain several molecules that can either eliminate or activate CD4TL (Quah and O'Neill, 2005b; Segura et al., 2005; Thery et al., 2002). Moreover, exosome-like vesicles found in plasma induce apoptosis in a FasL-like manner (Ren et al., 2010). Although exosomes

Exosomes Decrease *In Vitro* Infectivity

2007b; Turville et al., 2004) as illustrated in Figure 1.

of HIV-1 Preparations: Implication for CD4+T Lymphocyte Depletion *In Vivo* 111

expected, exosomes were concentrated in iodixanol fractions 8.4-12.0% on the Optiprep™ gradient (Fig. 5C, D). Large amounts of exosomes produced by HIV-1-loaded cells accumulated in fractions starting at 9.6% iodixanol (in comparison to the control condition, open bar). The velocity method thus allows efficient separation of exosomes which accumulate in iodixanol fractions 8.4 to 12 % as illustrated in Figure 3. Immature DCs release exosomes and are highly relevant to HIV-1 primary infection since they are involved in the capture of HIV-1 in mucosal tissues and play a crucial role in the subsequent transmission of the virus to CD4TL in the lymph nodes (Gilbert et al., 2007a; Gilbert et al.,

The large increase in exosome release by HIV-1-pulsed cells, combined with the results showing that exosomes from these cells induced apoptosis, is particularly relevant in the context of HIV-1 infection for several reasons. These results could explain in part the severe depletion of mucosal CD4TL, a cell type very susceptible to HIV-1. In addition, these cells play a pivotal role in orchestrating immune response and their decline during the early phase of infection undoubtedly delays the specific response to HIV-1. Furthermore, most laboratory preparations of HIV-1 contain exosomes, which may explain in vitro observations such as cytokine release, apoptosis, atypical gene expression, infectivity and so on. Since it became clear that exosomes play a major role in several aspects of the immune response to HIV-1, we sought to evaluate their impact on HIV-1 p24 production and infectivity. NL4-3balenv produced by transfection of 293T cells was made free of exosomes by immunocapture with anti-AE-1. Activated CD4TL were pre-incubated for 2h at 37°C with either exosome-free or exosome-containing HIV-1 preparation washed and then incubated for up to 5 days. ELISA was used to determine viral protein p24 in culture supernatants. Figure 6A shows that infection in the presence of exosomes is transient and less efficient

Panel A) P24 production was evaluated in the supernatants of CD4TL cultured for up to 5 days after pulsing with NL4-3balenv preparation either free of exosomes (Balenv AE-1) or not (Balenv IgG1). Panel B) TZM-bl cells were incubated with several dilutions of HIV-1 preparation either immunodepleted (Balenv AE-1) or not (Balenv IgG1) and maintained in culture for 48 hrs before lysis. Results are representative of two independent experiments.

Fig. 6. Impact of exosomes on p24 production and HIV-1 infectivity

**3.2 Impact of exosome depletion on CD4TL p24 production and infectivity** 

release by DCs have been studied extensively (Chaput et al., 2006; Izquierdo-Useros et al., 2009; Thery et al., 2001; Thery et al., 1999), the release mechanism and the nature of the exosomes produced by HIV-1-loaded cells (DCs or CD4TL) have not been thoroughly investigated. To begin to answer this question, the experimental set up proposed in Figure 4 and methods presented in Figure 3 were used.

DCs and CD4TL pulsed with NL4-3balenv and washed several times were cultured for respectively 2 or 5 days. Exosomes and virions were isolated initially by differential centrifugation and exosome levels were determined by measuring exosomal AChE activity (Cantin et al., 2008). These results, presented in Figure 5, confirmed higher levels of exosomes secreted by iDCs and CD4TL pulsed with purified HIV-1 (1.4 fold and 1.9 respectively, Fig. 5A, B). Using Optiprep™ velocity gradients to separate exosomes and HIV-1, we processed the pellet obtained following sedimentation centrifugation. As

Fig. 5. Exosomes released by DCs and CD4TL after pulsing with HIV-1 I DCs **(A, C)** or CD4TL **(B, D)** were incubated for 2h with exosome-free NL4-3balenv HIV-1 virus or mock preparation and cultured for an additional 72h. Cell-free supernatants were obtained by centrifugation and exosomes in the pellets were quantified by measuring AChE activity (x106 DO/min). Exosomes were then separated from HIV-1 on an Optiprep gradient and the exosome content (based on AchE activity) of each fraction was determined. Data are representative of five independent donors. These results show that HIV-1 induced exosome release (in fractions 9.6 through 12) by both cell types (mean increases of 1.4-fold for DCs and 1.9-fold for CD4TL, based on at least 5 independent experiments).

release by DCs have been studied extensively (Chaput et al., 2006; Izquierdo-Useros et al., 2009; Thery et al., 2001; Thery et al., 1999), the release mechanism and the nature of the exosomes produced by HIV-1-loaded cells (DCs or CD4TL) have not been thoroughly investigated. To begin to answer this question, the experimental set up proposed in Figure 4

DCs and CD4TL pulsed with NL4-3balenv and washed several times were cultured for respectively 2 or 5 days. Exosomes and virions were isolated initially by differential centrifugation and exosome levels were determined by measuring exosomal AChE activity (Cantin et al., 2008). These results, presented in Figure 5, confirmed higher levels of exosomes secreted by iDCs and CD4TL pulsed with purified HIV-1 (1.4 fold and 1.9 respectively, Fig. 5A, B). Using Optiprep™ velocity gradients to separate exosomes and HIV-1, we processed the pellet obtained following sedimentation centrifugation. As

Fig. 5. Exosomes released by DCs and CD4TL after pulsing with HIV-1

and 1.9-fold for CD4TL, based on at least 5 independent experiments).

I DCs **(A, C)** or CD4TL **(B, D)** were incubated for 2h with exosome-free NL4-3balenv HIV-1 virus or mock preparation and cultured for an additional 72h. Cell-free supernatants were obtained by centrifugation and exosomes in the pellets were quantified by measuring AChE activity (x106 DO/min). Exosomes were then separated from HIV-1 on an Optiprep gradient and the exosome content (based on AchE activity) of each fraction was determined. Data are representative of five independent donors. These results show that HIV-1 induced exosome release (in fractions 9.6 through 12) by both cell types (mean increases of 1.4-fold for DCs

and methods presented in Figure 3 were used.

expected, exosomes were concentrated in iodixanol fractions 8.4-12.0% on the Optiprep™ gradient (Fig. 5C, D). Large amounts of exosomes produced by HIV-1-loaded cells accumulated in fractions starting at 9.6% iodixanol (in comparison to the control condition, open bar). The velocity method thus allows efficient separation of exosomes which accumulate in iodixanol fractions 8.4 to 12 % as illustrated in Figure 3. Immature DCs release exosomes and are highly relevant to HIV-1 primary infection since they are involved in the capture of HIV-1 in mucosal tissues and play a crucial role in the subsequent transmission of the virus to CD4TL in the lymph nodes (Gilbert et al., 2007a; Gilbert et al., 2007b; Turville et al., 2004) as illustrated in Figure 1.

## **3.2 Impact of exosome depletion on CD4TL p24 production and infectivity**

The large increase in exosome release by HIV-1-pulsed cells, combined with the results showing that exosomes from these cells induced apoptosis, is particularly relevant in the context of HIV-1 infection for several reasons. These results could explain in part the severe depletion of mucosal CD4TL, a cell type very susceptible to HIV-1. In addition, these cells play a pivotal role in orchestrating immune response and their decline during the early phase of infection undoubtedly delays the specific response to HIV-1. Furthermore, most laboratory preparations of HIV-1 contain exosomes, which may explain in vitro observations such as cytokine release, apoptosis, atypical gene expression, infectivity and so on. Since it became clear that exosomes play a major role in several aspects of the immune response to HIV-1, we sought to evaluate their impact on HIV-1 p24 production and infectivity. NL4-3balenv produced by transfection of 293T cells was made free of exosomes by immunocapture with anti-AE-1. Activated CD4TL were pre-incubated for 2h at 37°C with either exosome-free or exosome-containing HIV-1 preparation washed and then incubated for up to 5 days. ELISA was used to determine viral protein p24 in culture supernatants. Figure 6A shows that infection in the presence of exosomes is transient and less efficient

Fig. 6. Impact of exosomes on p24 production and HIV-1 infectivity Panel A) P24 production was evaluated in the supernatants of CD4TL cultured for up to 5 days after pulsing with NL4-3balenv preparation either free of exosomes (Balenv AE-1) or not (Balenv IgG1). Panel B) TZM-bl cells were incubated with several dilutions of HIV-1 preparation either immunodepleted (Balenv AE-1) or not (Balenv IgG1) and maintained in culture for 48 hrs before lysis. Results are representative of two independent experiments.

Exosomes Decrease *In Vitro* Infectivity

**5. Acknowledgments** 

Research Program.

**6. References** 

area.

of HIV-1 Preparations: Implication for CD4+T Lymphocyte Depletion *In Vivo* 113

laboratories and lessen discrepancies such as those seen among several studies in recent years as well as facilitate the interpretation of new results to be published in this subject

The authors wish to thank Dr Lahlou Hadji for his technical assistance in editing this manuscript and for constructive comments. We also thank Dr. Stephen Davids for proofreading. We are also grateful to M. Robert Alain for his assistance with the electron microscopy analyses and to Odette Simard for excellent technical contributions. This work was supported by start-up funds to CG from the Centre Hospitalier Universitaire de Québec and an operating grant to CG from the Canadian Institutes of Health Research (CIHR) (MOP-188726). CG is recipient of a level-1 junior researcher award from the Fonds de la Recherche en Santé du Québec and a New Investigator Award from CIHR. C.S. is the recipient of a fellowship award from the Canadian Institutes of Health Research HIV/AIDS

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than in their absence (solid bar). To evaluate virion infectivity, indicator cell line TZM-bl was incubated with several dilutions of HIV-1 preparation either exosome-depleted or not. Panel B of Figure 6 shows that the exosome-depleted preparation is more infectious than the non-depleted preparation. All these results provide additional evidence that exosomes derived from HIV-1-pulsed cells influence cell viability (figure 3D) and indirectly p24 production and infectivity. They suggest that the presence of exosomes in culture supernatants of HIV-1-stimulated cells should be considered in all laboratory experiments with HIV-1.

## **4. Conclusion**

Exosome biogenesis and the HIV-1 virion assembly pathway converge in a common intracellular compartment. Moreover, both types of vesicle can be released during the transinfection process in DCs (Izquierdo-Useros et al., 2009). However, exosome secretion in the context of HIV-1 infection has not been properly investigated, due primarily to lack of effective methods of separating the two types of vesicles. Their separation using antibodies directed against specific membrane antigens is often suboptimal since exosomes and HIV-1 display approximately the same antigen expression pattern in addition to several other surface molecules. This is why flow cytometry, ELISA or bead capture techniques based on specific markers are not sufficiently discriminating for the separation of exosomes, extraneous micro-particles and HIV-1. Alternatively, immunocapture with anti-CD45 (Chertova et al., 2006; Trubey et al., 2003), used to separate only micro-particles derived from leucocyte plasma membranes, does not eliminate exosomes originating from the endosomal membrane and cannot be used to separate exosomes from HIV-1. We have shown that AChE appears essentially excluded from the HIV-1 fraction, since the major portion of its activity is recovered in the early Optiprep™ fractions (8.4 to 12), in which no virus is detected (Cantin et al., 2008). Based on this observation, depletion of 100,000*xg*  pellets with protein-A/G-bound anti-AChE on agarose beads appears to provide excellent means of rapidly purifying virions or capturing exosomes (Cantin et al., 2008). Using these methods, we have observed that HIV-1 contact with DCs or CD4TL enhances extracellular exosome release and that these exosomes can affect the viability of nearby cells such as CD4TL. These results are in agreement with observations concerning the pro-apoptotic role of Nef accessory proteins. Using Optiprep™ gradients, recent work has shown that the viral protein Nef is enclosed in exosomes, conferring to it the capacity to trigger apoptosis of uninfected bystander T cells (Lenassi et al., 2010).

In summary, the results of the present study show that relatively simple methods of purifying both exosomes and HIV-1 contained in the same cell supernatant are now available. Achieving very highly purified exosomes from HIV-1 preparations is a definite advantage in studying the respective roles of both vesicles as well as the links between them. These methods could also provide the opportunity for specific isolation of exosomes secreted by a variety of cell types and could prove useful in experiments that require highly purified exosome preparations to study their roles in various biological processes. Indeed, these purification steps are crucial in studies involving mixtures of exosomes and HIV-1 (or for that matter, other retroviruses) as starting material. We may anticipate that these methods will constitute a significant contribution to the use of exosomes for vaccination or gene therapy. In addition, we strongly believe that an improved and standardized method of exosome purification should lead to more comparable results among different laboratories and lessen discrepancies such as those seen among several studies in recent years as well as facilitate the interpretation of new results to be published in this subject area.

## **5. Acknowledgments**

112 Understanding HIV/AIDS Management and Care – Pandemic Approaches in the 21st Century

than in their absence (solid bar). To evaluate virion infectivity, indicator cell line TZM-bl was incubated with several dilutions of HIV-1 preparation either exosome-depleted or not. Panel B of Figure 6 shows that the exosome-depleted preparation is more infectious than the non-depleted preparation. All these results provide additional evidence that exosomes derived from HIV-1-pulsed cells influence cell viability (figure 3D) and indirectly p24 production and infectivity. They suggest that the presence of exosomes in culture supernatants of HIV-1-stimulated cells should be considered in all laboratory experiments

Exosome biogenesis and the HIV-1 virion assembly pathway converge in a common intracellular compartment. Moreover, both types of vesicle can be released during the transinfection process in DCs (Izquierdo-Useros et al., 2009). However, exosome secretion in the context of HIV-1 infection has not been properly investigated, due primarily to lack of effective methods of separating the two types of vesicles. Their separation using antibodies directed against specific membrane antigens is often suboptimal since exosomes and HIV-1 display approximately the same antigen expression pattern in addition to several other surface molecules. This is why flow cytometry, ELISA or bead capture techniques based on specific markers are not sufficiently discriminating for the separation of exosomes, extraneous micro-particles and HIV-1. Alternatively, immunocapture with anti-CD45 (Chertova et al., 2006; Trubey et al., 2003), used to separate only micro-particles derived from leucocyte plasma membranes, does not eliminate exosomes originating from the endosomal membrane and cannot be used to separate exosomes from HIV-1. We have shown that AChE appears essentially excluded from the HIV-1 fraction, since the major portion of its activity is recovered in the early Optiprep™ fractions (8.4 to 12), in which no virus is detected (Cantin et al., 2008). Based on this observation, depletion of 100,000*xg*  pellets with protein-A/G-bound anti-AChE on agarose beads appears to provide excellent means of rapidly purifying virions or capturing exosomes (Cantin et al., 2008). Using these methods, we have observed that HIV-1 contact with DCs or CD4TL enhances extracellular exosome release and that these exosomes can affect the viability of nearby cells such as CD4TL. These results are in agreement with observations concerning the pro-apoptotic role of Nef accessory proteins. Using Optiprep™ gradients, recent work has shown that the viral protein Nef is enclosed in exosomes, conferring to it the capacity to trigger apoptosis of

In summary, the results of the present study show that relatively simple methods of purifying both exosomes and HIV-1 contained in the same cell supernatant are now available. Achieving very highly purified exosomes from HIV-1 preparations is a definite advantage in studying the respective roles of both vesicles as well as the links between them. These methods could also provide the opportunity for specific isolation of exosomes secreted by a variety of cell types and could prove useful in experiments that require highly purified exosome preparations to study their roles in various biological processes. Indeed, these purification steps are crucial in studies involving mixtures of exosomes and HIV-1 (or for that matter, other retroviruses) as starting material. We may anticipate that these methods will constitute a significant contribution to the use of exosomes for vaccination or gene therapy. In addition, we strongly believe that an improved and standardized method of exosome purification should lead to more comparable results among different

with HIV-1.

**4. Conclusion** 

uninfected bystander T cells (Lenassi et al., 2010).

The authors wish to thank Dr Lahlou Hadji for his technical assistance in editing this manuscript and for constructive comments. We also thank Dr. Stephen Davids for proofreading. We are also grateful to M. Robert Alain for his assistance with the electron microscopy analyses and to Odette Simard for excellent technical contributions. This work was supported by start-up funds to CG from the Centre Hospitalier Universitaire de Québec and an operating grant to CG from the Canadian Institutes of Health Research (CIHR) (MOP-188726). CG is recipient of a level-1 junior researcher award from the Fonds de la Recherche en Santé du Québec and a New Investigator Award from CIHR. C.S. is the recipient of a fellowship award from the Canadian Institutes of Health Research HIV/AIDS Research Program.

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**8** 

**Nikavir in Chemoprevention Regimens** 

Elvira Ivanova1, Nadezhda Shmagel1 and Natalia Vorobeva2

The first cases of HIV infection in the Russian Federation were identified in 1987. Between 1987 and 1996 over 90% of individuals acquired infection via homosexual contacts (Segeda,

Since 1996 there has been a dramatic growth of incidence rate due to the parenteral use of psychoactive drugs (Montgomery, 2000; UNAIDS, 2001; Des Jarlais, 2001). Since then, in the Russian Federation a narcodependent type of epidemic process has formed. It is characterized by high intensity and a rapid growth of the incidence rate (Adabekov &

Due to a large share of women of the active reproductive age among the HIV-infected individuals and the tendency for its further growth as well as the increasing percentage of HIV distribution through sexual transmission (Terentyeva, 2006), there is a current annual growth of pregnancy and delivery rates among women infected with HIV (Terentyeva, 2006). Compared with 2000, in 2010 the absolute number of deliveries has increased 15 times. By 2010 deliveries by HIV-infected women accounted to 0.4% of the total number of

The actual increase in the share of women among the newly identified cases of HIV infection may be regarded as an indirect evidence of activation of heterosexual transmission of HIV. 62% of HIV-positive pregnant women identified in 2010 were infected through sexual contacts. Along with the growth of heterosexual route of infection an associated risk of

The mortality rate among children born from HIV-positive mothers is high. 25% of HIVpositive and 12% of HIV-negative children die before the age of five (Rogers, 1984;

By the end 2010, 12000 individuals with HIV infection were registered in Permsky Krai, over one third of them (35%) being females. Between 1999 and 2010 1634 children were born from HIV-infected mothers. 40 of them died and 73 have got HIV infection. Every 192nd delivery

Thus, a current high intensity of epidemic process of HIV infection has emphasized the necessity for HIV prevention among the newborns. Considering an unprecedented growth of the incidence rate and low birth rate in the Russian Federation this issue is a priority.

is in a woman infected with HIV (in the Russian Federation the ratio is 1 per 250).

**1. Introduction** 

2006; Berrous, 2000).

Rakhmanova, 2006).

Mamaev, 2005; Rakhmanova, 2004).

deliveries in the Russian Federation.

mother-to-child transmission of HIV has increased as well.

**of Vertical HIV Transmission** 

*1Perm Regional AIDS Center* 

*Russian Federation* 

*2Vagner Perm State Academy of Medicine* 


## **Nikavir in Chemoprevention Regimens of Vertical HIV Transmission**

## Elvira Ivanova1, Nadezhda Shmagel1 and Natalia Vorobeva2 *1Perm Regional AIDS Center 2Vagner Perm State Academy of Medicine Russian Federation*

## **1. Introduction**

124 Understanding HIV/AIDS Management and Care – Pandemic Approaches in the 21st Century

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Xiang, J. (2010). Dendritic cells recruit T cell exosomes via exosomal LFA-1 leading to inhibition of CD8+ CTL responses through downregulation of peptide/MHC

and Ahmed, R. (1998). Viral immune evasion due to persistence of activated T cells

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mediated transfer of HIV-1 is compromised by the ability of Leishmania infantum

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The first cases of HIV infection in the Russian Federation were identified in 1987. Between 1987 and 1996 over 90% of individuals acquired infection via homosexual contacts (Segeda, 2006; Berrous, 2000).

Since 1996 there has been a dramatic growth of incidence rate due to the parenteral use of psychoactive drugs (Montgomery, 2000; UNAIDS, 2001; Des Jarlais, 2001). Since then, in the Russian Federation a narcodependent type of epidemic process has formed. It is characterized by high intensity and a rapid growth of the incidence rate (Adabekov & Mamaev, 2005; Rakhmanova, 2004).

Due to a large share of women of the active reproductive age among the HIV-infected individuals and the tendency for its further growth as well as the increasing percentage of HIV distribution through sexual transmission (Terentyeva, 2006), there is a current annual growth of pregnancy and delivery rates among women infected with HIV (Terentyeva, 2006). Compared with 2000, in 2010 the absolute number of deliveries has increased 15 times. By 2010 deliveries by HIV-infected women accounted to 0.4% of the total number of deliveries in the Russian Federation.

The actual increase in the share of women among the newly identified cases of HIV infection may be regarded as an indirect evidence of activation of heterosexual transmission of HIV. 62% of HIV-positive pregnant women identified in 2010 were infected through sexual contacts. Along with the growth of heterosexual route of infection an associated risk of mother-to-child transmission of HIV has increased as well.

The mortality rate among children born from HIV-positive mothers is high. 25% of HIVpositive and 12% of HIV-negative children die before the age of five (Rogers, 1984; Rakhmanova, 2006).

By the end 2010, 12000 individuals with HIV infection were registered in Permsky Krai, over one third of them (35%) being females. Between 1999 and 2010 1634 children were born from HIV-infected mothers. 40 of them died and 73 have got HIV infection. Every 192nd delivery is in a woman infected with HIV (in the Russian Federation the ratio is 1 per 250).

Thus, a current high intensity of epidemic process of HIV infection has emphasized the necessity for HIV prevention among the newborns. Considering an unprecedented growth of the incidence rate and low birth rate in the Russian Federation this issue is a priority.

Nikavir in Chemoprevention Regimens of Vertical HIV Transmission 127

the number of virus antigen expressing cells was 69%. With addition of Nikavir the number of live cells increased to 72% and the number of virus antigen expressing cells dropped to 20%. Similar results were obtained with the addition of zidovudine but compared to Nikavir it proved its considerably higher level of toxicity. The selectivity index of Nikavir was twice

Later, a group of researchers from Canada proved a considerably lower toxicity of Nikavir with lymphoblastoid cell lines compared to zidovudine. Nikavir and zidovudine showed a marked efficiency with experimental HIV-1 infection in cord blood mononuclear cells. However, cytotoxic effect of zidovudine appeared by 33 times higher than that of Nikavir. Selectivity index of Nikavir was by 13.6 times higher than that of zidovudine (Machado,

It is known that in response to each antiretroviral agent drug resistant HIV-1 mutants are developed. It is the result of structural changes of HIV-1 genome due to substitution of one or several nucleic bases. It was found that HIV-1 resistance to Nikavir formed significantly slower than to zidovudine (Selimova, 1999). Resistance to Nikavir develops within a 72 days passivation of the virus (10 infectious cycles) whereas resistance to zidovudine occurs within 26 days (6 infectious cycles). The fact that extensive selection with Nikavir yielded only a single D67N substitution, also associated with resistance to zidovudine, rather than other zidovudine-resistance associated mutations as well, may also be a positive indication

Preclinical toxicological and pharmacokinetic studies of Nikavir were performed at the Institute of Experimental Cardiology of Russian Cardiological Scientific Production Complex. It was established that Nikavir belongs to the category of low-toxic drugs. LD50 (average lethal dose) for mice of BABL/c line given in a single intragastric introduction was equal to 8200- 8830 mg/kg, that for intra-abdominal introduction – 2260-2390 mg/kg. Zidovudine proved to be far more toxic: in intragastric intake its LD50 was 2380-2730 mg/kg and 1320-1660 mg/kg in intra-abdominal introduction. During rat testing Nikavir LD50 in intragastric and intraabdominal introduction were 12200-12950 mg/kg and 2490-2510 mg/kg respectively. No damaging effect was established in the investigation of chronic toxicity in daily (90 days)

Chronic toxicity was studied in dogs following 2-month oral dosing (tablets 0.2 g), 400 mg/kg during the first month and 200 mg/kg body weight during the second. It was found that tablets (400 mg/kg, 20-fold human dose) taken by dogs for a month caused some reduction of appetite and motor activity. Reduction of the doses to 200 mg/kg did not result in toxic effects in chronic experiments. Toxicity was also not observed in pathological experiments after the completion of the chronic testing. It showed that Nikavir was well assumed and did not affect hematological (granulocytopenia and anemia) or biochemical parameters of liver, kidney and pancreas functioning and metabolic reactions

Basic methods (predominant lethality identification and the Ames test) did not show any mutagenic action as well as the DNA-damaging and allergic effects. Experimental studies on pregnant rats demonstrated a sufficiently lower embriotoxic and teratogenic action of

Nikavir is a prodrug: after its per oral introduction to dogs only zidovudine can be identified in their blood. However 40-50 minutes after intragastric introduction to mice of tritium-labeled Nikavir both Nikavir and zidovudine were found in blood plasma. The peak radioactivity level of zidovudine was considerably higher than of Nikavir (Skoblov, 2004).

in regard to the potential of Nikavir to combat HIV disease (Machado, 1999).

intragastric introduction of Nikavir to rats (Khandazhinskaya, 2010).

Nikavir compared with zidovudine (Khandazhinskaya, 2010).

(Khandazhinskaya, 2010).

the number than that of zidovudine (Tarussova, 1990).

1999).

Mother-to-child transmission of HIV occurs during pregnancy, delivery and during the postpartum period while breast-feeding. In 1994 the US Center for Disease Control (CDC) recommended a three-stage chemoprevention with zidovudine (Retrovir) for mothers during pregnancy, delivery and postnatally for children (Barlett & Gallant, 1964; Friis, 2001). Along with the rejection of breast-feeding those measures decreased the risk of infection by 2% (Connor, 1994; European Collaborative Study, 2005; Jasseron, 2008; Townsend, 2008). The earliest recommendations on the vertical chemoprophylaxis of HIV were provided by Rakhmanova in 1997 and later by V.Pokrovsky and O.Yurin (2000-2001). Currently, there are American (CDC, 2008) and European (FACS, 2008) Guidelines. Nevertheless the number of antiretroviral agents available for prevention of vertical HIV transmission is rather small. Moreover, their recognized toxicological manifestations considerably restrict the possibility of HIV chemoprevention. Therefore the search, development and clinical implementation of the new low-toxic anti HIV agents with prolonged action are all of immense importance.

## **2. History of creation of Nikavir**

In the middle of the 1980s academician A. Krayevsky initiated investigations of the anti-HIV activity of a group of nucleoside containing compounds, newly synthesized at the laboratory of the Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences, which including a modified phosphate group in 5'-position. 5'-H-phosphonat 3' azido-3'-desoxythymidine in the form of sodium salt appeared to be a highly active substance with the best cytotoxic properties. It was named phosphazide; its brand name is Nikavir (Fig. 1).

Fig. 1. Formula of Nikavir (phosphazide)*.* 

In 1986 Nikavir was identified as a drug that possesses a high level of anti-HIV efficiency in respect to HIV reproduction in H9, MOLT and MT-4 lymphoblastic cell cultures (Galegov, 1988).

Comparative studies of cytotoxic activity of Nikavir and zidovudine were performed at the Ivanovsky Institute of Virology of the Russian Academy of Medical Sciences. It vividly showed that Nikavir concentrations in the range of 0.25-15 μM considerably decreased the number of viral antigen expressing cells while the number of live cells considerably increased compared to the control sample (infected with the culture virus and not treated with the drug). In the control sample the number of live cells decreased from 97% to 18%,

Mother-to-child transmission of HIV occurs during pregnancy, delivery and during the postpartum period while breast-feeding. In 1994 the US Center for Disease Control (CDC) recommended a three-stage chemoprevention with zidovudine (Retrovir) for mothers during pregnancy, delivery and postnatally for children (Barlett & Gallant, 1964; Friis, 2001). Along with the rejection of breast-feeding those measures decreased the risk of infection by 2% (Connor, 1994; European Collaborative Study, 2005; Jasseron, 2008; Townsend, 2008). The earliest recommendations on the vertical chemoprophylaxis of HIV were provided by Rakhmanova in 1997 and later by V.Pokrovsky and O.Yurin (2000-2001). Currently, there are American (CDC, 2008) and European (FACS, 2008) Guidelines. Nevertheless the number of antiretroviral agents available for prevention of vertical HIV transmission is rather small. Moreover, their recognized toxicological manifestations considerably restrict the possibility of HIV chemoprevention. Therefore the search, development and clinical implementation of the new low-toxic anti HIV agents with prolonged action are all of immense importance.

In the middle of the 1980s academician A. Krayevsky initiated investigations of the anti-HIV activity of a group of nucleoside containing compounds, newly synthesized at the laboratory of the Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences, which including a modified phosphate group in 5'-position. 5'-H-phosphonat 3' azido-3'-desoxythymidine in the form of sodium salt appeared to be a highly active substance with the best cytotoxic properties. It was named phosphazide; its brand name is

<sup>O</sup> <sup>O</sup>

P O

ONa

H

N3

In 1986 Nikavir was identified as a drug that possesses a high level of anti-HIV efficiency in respect to HIV reproduction in H9, MOLT and MT-4 lymphoblastic cell cultures (Galegov,

Comparative studies of cytotoxic activity of Nikavir and zidovudine were performed at the Ivanovsky Institute of Virology of the Russian Academy of Medical Sciences. It vividly showed that Nikavir concentrations in the range of 0.25-15 μM considerably decreased the number of viral antigen expressing cells while the number of live cells considerably increased compared to the control sample (infected with the culture virus and not treated with the drug). In the control sample the number of live cells decreased from 97% to 18%,

O

HN

N

O

CH3

**2. History of creation of Nikavir** 

Fig. 1. Formula of Nikavir (phosphazide)*.* 

Nikavir (Fig. 1).

1988).

the number of virus antigen expressing cells was 69%. With addition of Nikavir the number of live cells increased to 72% and the number of virus antigen expressing cells dropped to 20%. Similar results were obtained with the addition of zidovudine but compared to Nikavir it proved its considerably higher level of toxicity. The selectivity index of Nikavir was twice the number than that of zidovudine (Tarussova, 1990).

Later, a group of researchers from Canada proved a considerably lower toxicity of Nikavir with lymphoblastoid cell lines compared to zidovudine. Nikavir and zidovudine showed a marked efficiency with experimental HIV-1 infection in cord blood mononuclear cells. However, cytotoxic effect of zidovudine appeared by 33 times higher than that of Nikavir. Selectivity index of Nikavir was by 13.6 times higher than that of zidovudine (Machado, 1999).

It is known that in response to each antiretroviral agent drug resistant HIV-1 mutants are developed. It is the result of structural changes of HIV-1 genome due to substitution of one or several nucleic bases. It was found that HIV-1 resistance to Nikavir formed significantly slower than to zidovudine (Selimova, 1999). Resistance to Nikavir develops within a 72 days passivation of the virus (10 infectious cycles) whereas resistance to zidovudine occurs within 26 days (6 infectious cycles). The fact that extensive selection with Nikavir yielded only a single D67N substitution, also associated with resistance to zidovudine, rather than other zidovudine-resistance associated mutations as well, may also be a positive indication in regard to the potential of Nikavir to combat HIV disease (Machado, 1999).

Preclinical toxicological and pharmacokinetic studies of Nikavir were performed at the Institute of Experimental Cardiology of Russian Cardiological Scientific Production Complex. It was established that Nikavir belongs to the category of low-toxic drugs. LD50 (average lethal dose) for mice of BABL/c line given in a single intragastric introduction was equal to 8200- 8830 mg/kg, that for intra-abdominal introduction – 2260-2390 mg/kg. Zidovudine proved to be far more toxic: in intragastric intake its LD50 was 2380-2730 mg/kg and 1320-1660 mg/kg in intra-abdominal introduction. During rat testing Nikavir LD50 in intragastric and intraabdominal introduction were 12200-12950 mg/kg and 2490-2510 mg/kg respectively. No damaging effect was established in the investigation of chronic toxicity in daily (90 days) intragastric introduction of Nikavir to rats (Khandazhinskaya, 2010).

Chronic toxicity was studied in dogs following 2-month oral dosing (tablets 0.2 g), 400 mg/kg during the first month and 200 mg/kg body weight during the second. It was found that tablets (400 mg/kg, 20-fold human dose) taken by dogs for a month caused some reduction of appetite and motor activity. Reduction of the doses to 200 mg/kg did not result in toxic effects in chronic experiments. Toxicity was also not observed in pathological experiments after the completion of the chronic testing. It showed that Nikavir was well assumed and did not affect hematological (granulocytopenia and anemia) or biochemical parameters of liver, kidney and pancreas functioning and metabolic reactions (Khandazhinskaya, 2010).

Basic methods (predominant lethality identification and the Ames test) did not show any mutagenic action as well as the DNA-damaging and allergic effects. Experimental studies on pregnant rats demonstrated a sufficiently lower embriotoxic and teratogenic action of Nikavir compared with zidovudine (Khandazhinskaya, 2010).

Nikavir is a prodrug: after its per oral introduction to dogs only zidovudine can be identified in their blood. However 40-50 minutes after intragastric introduction to mice of tritium-labeled Nikavir both Nikavir and zidovudine were found in blood plasma. The peak radioactivity level of zidovudine was considerably higher than of Nikavir (Skoblov, 2004).

Nikavir in Chemoprevention Regimens of Vertical HIV Transmission 129

44.7% of patients received Nikavir as monotherapy and 55.3% as a component of highly active antiretroviral therapy (HAART). No cases of Nikavir-associated untoward events were observed and there were no cases of discontinued therapy. Mild nausea was noted in 6.4% of cases and 2.1% had grade 1 toxic anemia. Besides, 36-48 weeks following substitution of zidovudine by Nikavir a considerable growth of CD4 lymphocytes by 70-100

Since 1999 Nikavir was approved for clinical application in the chemotherapy of HIVinfected patients. Currently it is manufactured in the tablet form of 200 mg N 20 and is used

In terms of anemia, significantly fewer Nikavir-associated hemopoetic impairments make it a more perspective drug for therapeutic application for HIV-infected pregnant women than zidovudine. Because of this, evidence-based findings obtained in the comparative studies of embriotoxic and teratogenic properties of zidovudine and Nikavir performed in 2005 at the laboratory of drug toxicology of Institute of Experimental Cardiology under the guidance of

Tests were performed among 80 pregnant rats of Wistar line divided into 4 equal groups. Group 1 included animals for control, group 2 received zidovudine (dose of 100mg/kg of body weight once a day), group 3 received Nikavir (dose of 100mg/kg of body weight once a day). As the period of zidovudine half-excretion is less than that of Nikavir group 4 received zidovudine in the total daily dosage of 100 mg/kg in 2 doses (50 mg/kg at 9 a.m. and 5 p.m.). The tested doses corresponded to a 12.5 multiple of the maximum daily doses of 600

Intragastric introduction of zidovudine and Nikavir to pregnant rats in the tested doses of 100 mg/kg provided the significant (p<0.05) evidence of the body mass retardation in pregnant rats compared with the controls on the 3rd week, while the less marked body mass retardation was observed in Nikavir administration and a two-dose introduction of

No statistically significant difference was established in such parameters evaluated for embriotoxicity of zidovudine and Nikavir as the duration of pregnancy, numbers of alive fetuses, implantation places, yellow bodies, fetal body mass, cranioclaudal size in the pregnant rats receiving preparations in the dose of 100 mg/kg and the controls. Likewise data of preimplantation and postimplantation death in experimental groups did not

Controls Zidovudine

Table 1. Body mass dynamics in pregnant rats (% ratio of baseline parameters) in

100 mg/kg

1st week 1101.6 1071.6 1071.1 1091.4 2nd week 1211.7 1161.8 1201.4 1191.3 3rd week 1353.0 1212.4 1302.6 1241.7

Animal groups

Zidovudine 50+50 mg/kg

Nikavir 100 mg/kg

**3. Comparative studies of embriotoxic and teratogenic properties of** 

cells/mm3 was found (Yurin, 2000).

**zidovudine and Nikavir** 

zidovudine (Table 1).

Observation periods

for treatment of HIV and AIDS in Russian Federation.

professor E. Arzamastsev are of a considerably interest.

mg/individual or 8 mg/kg approved for pregnant women with HIV.

significantly differ from those in the controls as well (Table 2).

intragastric introduction of zidovudine and Nikavir.

The pharmacokinetic studies established that Nikavir is characterized by a smoother pharmacokinetic curve compared to zidovudine (Galegov, 2004; Khandazhinskaya, 2010). The half-life time of Nikavir from blood plasma surpasses zidovudine by 4 times and this allows to recommend fewer daily intakes.

Thus, due to its high anti-HIV efficiency *in vitro*, low cytotoxicity, favorable pharmacokinetic indices and low toxicity in laboratory animals Nikavir was recommended for clinical evaluation studies.

The first phase of clinical investigation (safety and tolerance) was conducted in 1997. It showed that Nikavir was well-tolerated by all patients. There were no main side-effects observed in zidovudine administration (anemia, neutropenia) as well as less frequent untoward gastrointestinal manifestations, headache and insomnia (Yurin, 1998). It should be noted that not a single case of anemia known to the major side-effect of zidovudine was observed.

Further clinical trial (therapeutic efficiency) was conducted as a multicenter clinical trial under coordination of the Russian Federal AIDS Center, Moscow. The local participating centers were Republican Clinical Infection Hospital in Izhora settlement (St. Pertersburg), Regional AIDS Centers in Tver, Nizhny Novgorod and Tyumen (Yurin, 2001).

At the first stage Nikavir was used as a monotherapy. The trial included 103 patients (75 males and 28 females, average age 26 years). According to HIV-infection classification (USA, CDC, 1987) 69.9% were diagnosed A2 stage, 23.3% had A1 stage and 6.8% had B1 and B2 stages. The therapy course lasted 12 weeks. Patients received daily doses ranging from 400 mg to 1200 mg Nikavir. Its therapeutic efficiency was assessed by such clinical criteria as disease progression or its absence; immunological criteria included changes CD4+ Tlymphocytes count per 1 mm3 of blood; virological criteria included changes of HIV RNA levels per 1 ml of plasma. CD4 lymphocytes were counted with flow cytometry method using Fac Scan apparatus (Becton Dickenson, USA) and monoclonal antibodies (Becton Dickenson, USA). HIV RNA levels were measured with PCR method (Amplicor Roche HIV-1 Monitor, Hoffmann-La Roch, Switzerland).

During treatment no cases of HIV progress were noted. The pretreatment baseline mean CD4 lymphocytes count was 350 cells/mm3. After 4 weeks of treatment the mean CD4 lymphocyte index increased by 20 cells and by 80 cells following 12 weeks (p<0.05). A reliable decrease of HIV RNA levels was observed starting with the second week of therapy (-0.53 lg copies/ml, p<0.05), which remained unchanged after 4 and 12 weeks of therapy (- 0.53 and -0.44 lg copies/ml respectively).

The most frequent negative Nikavir-related effects were mild nausea and malaise registered in 30% of patients receiving maximal daily dosage of 1200 mg. No essential changes in hematological indices were revealed. For 1% of patients therapy was temporary stopped due to a moderate granulocytopenia. In 5.8% of cases Nikavir therapy was initiated in spite of grade 1-2 toxicity thrombocytopenia. In all cases the on-going therapy was associated with notable increase of thrombocyte count and disappearing signs of toxicity. During the trial no worth considering changes in biochemical blood values were registered.

The conducted trial demonstrated good efficiency and tolerability of Nikavir monotherapy and allowed to recommend a regimen of 400 mg twice daily (Yurin, 2001).

The next stage was aimed at investigation of possible outcomes following change of zidovudine to Nikavir regimens due to the development of untoward events of grade 2-4 toxicity. In 47 patients zidovudine was substituted by Nikavir because of nausea and vomiting (40.4% of cases), anemia (46.8%) and granulocytopenia (12.8% of cases) whereas

The pharmacokinetic studies established that Nikavir is characterized by a smoother pharmacokinetic curve compared to zidovudine (Galegov, 2004; Khandazhinskaya, 2010). The half-life time of Nikavir from blood plasma surpasses zidovudine by 4 times and this

Thus, due to its high anti-HIV efficiency *in vitro*, low cytotoxicity, favorable pharmacokinetic indices and low toxicity in laboratory animals Nikavir was recommended for clinical

The first phase of clinical investigation (safety and tolerance) was conducted in 1997. It showed that Nikavir was well-tolerated by all patients. There were no main side-effects observed in zidovudine administration (anemia, neutropenia) as well as less frequent untoward gastrointestinal manifestations, headache and insomnia (Yurin, 1998). It should be noted that not a single case of anemia known to the major side-effect of zidovudine was

Further clinical trial (therapeutic efficiency) was conducted as a multicenter clinical trial under coordination of the Russian Federal AIDS Center, Moscow. The local participating centers were Republican Clinical Infection Hospital in Izhora settlement (St. Pertersburg),

At the first stage Nikavir was used as a monotherapy. The trial included 103 patients (75 males and 28 females, average age 26 years). According to HIV-infection classification (USA, CDC, 1987) 69.9% were diagnosed A2 stage, 23.3% had A1 stage and 6.8% had B1 and B2 stages. The therapy course lasted 12 weeks. Patients received daily doses ranging from 400 mg to 1200 mg Nikavir. Its therapeutic efficiency was assessed by such clinical criteria as disease progression or its absence; immunological criteria included changes CD4+ Tlymphocytes count per 1 mm3 of blood; virological criteria included changes of HIV RNA levels per 1 ml of plasma. CD4 lymphocytes were counted with flow cytometry method using Fac Scan apparatus (Becton Dickenson, USA) and monoclonal antibodies (Becton Dickenson, USA). HIV RNA levels were measured with PCR method (Amplicor Roche HIV-

During treatment no cases of HIV progress were noted. The pretreatment baseline mean CD4 lymphocytes count was 350 cells/mm3. After 4 weeks of treatment the mean CD4 lymphocyte index increased by 20 cells and by 80 cells following 12 weeks (p<0.05). A reliable decrease of HIV RNA levels was observed starting with the second week of therapy (-0.53 lg copies/ml, p<0.05), which remained unchanged after 4 and 12 weeks of therapy (-

The most frequent negative Nikavir-related effects were mild nausea and malaise registered in 30% of patients receiving maximal daily dosage of 1200 mg. No essential changes in hematological indices were revealed. For 1% of patients therapy was temporary stopped due to a moderate granulocytopenia. In 5.8% of cases Nikavir therapy was initiated in spite of grade 1-2 toxicity thrombocytopenia. In all cases the on-going therapy was associated with notable increase of thrombocyte count and disappearing signs of toxicity. During the

The conducted trial demonstrated good efficiency and tolerability of Nikavir monotherapy

The next stage was aimed at investigation of possible outcomes following change of zidovudine to Nikavir regimens due to the development of untoward events of grade 2-4 toxicity. In 47 patients zidovudine was substituted by Nikavir because of nausea and vomiting (40.4% of cases), anemia (46.8%) and granulocytopenia (12.8% of cases) whereas

trial no worth considering changes in biochemical blood values were registered.

and allowed to recommend a regimen of 400 mg twice daily (Yurin, 2001).

Regional AIDS Centers in Tver, Nizhny Novgorod and Tyumen (Yurin, 2001).

allows to recommend fewer daily intakes.

1 Monitor, Hoffmann-La Roch, Switzerland).

0.53 and -0.44 lg copies/ml respectively).

evaluation studies.

observed.

44.7% of patients received Nikavir as monotherapy and 55.3% as a component of highly active antiretroviral therapy (HAART). No cases of Nikavir-associated untoward events were observed and there were no cases of discontinued therapy. Mild nausea was noted in 6.4% of cases and 2.1% had grade 1 toxic anemia. Besides, 36-48 weeks following substitution of zidovudine by Nikavir a considerable growth of CD4 lymphocytes by 70-100 cells/mm3 was found (Yurin, 2000).

Since 1999 Nikavir was approved for clinical application in the chemotherapy of HIVinfected patients. Currently it is manufactured in the tablet form of 200 mg N 20 and is used for treatment of HIV and AIDS in Russian Federation.

## **3. Comparative studies of embriotoxic and teratogenic properties of zidovudine and Nikavir**

In terms of anemia, significantly fewer Nikavir-associated hemopoetic impairments make it a more perspective drug for therapeutic application for HIV-infected pregnant women than zidovudine. Because of this, evidence-based findings obtained in the comparative studies of embriotoxic and teratogenic properties of zidovudine and Nikavir performed in 2005 at the laboratory of drug toxicology of Institute of Experimental Cardiology under the guidance of professor E. Arzamastsev are of a considerably interest.

Tests were performed among 80 pregnant rats of Wistar line divided into 4 equal groups. Group 1 included animals for control, group 2 received zidovudine (dose of 100mg/kg of body weight once a day), group 3 received Nikavir (dose of 100mg/kg of body weight once a day). As the period of zidovudine half-excretion is less than that of Nikavir group 4 received zidovudine in the total daily dosage of 100 mg/kg in 2 doses (50 mg/kg at 9 a.m. and 5 p.m.).

The tested doses corresponded to a 12.5 multiple of the maximum daily doses of 600 mg/individual or 8 mg/kg approved for pregnant women with HIV.

Intragastric introduction of zidovudine and Nikavir to pregnant rats in the tested doses of 100 mg/kg provided the significant (p<0.05) evidence of the body mass retardation in pregnant rats compared with the controls on the 3rd week, while the less marked body mass retardation was observed in Nikavir administration and a two-dose introduction of zidovudine (Table 1).

No statistically significant difference was established in such parameters evaluated for embriotoxicity of zidovudine and Nikavir as the duration of pregnancy, numbers of alive fetuses, implantation places, yellow bodies, fetal body mass, cranioclaudal size in the pregnant rats receiving preparations in the dose of 100 mg/kg and the controls. Likewise data of preimplantation and postimplantation death in experimental groups did not significantly differ from those in the controls as well (Table 2).


Table 1. Body mass dynamics in pregnant rats (% ratio of baseline parameters) in intragastric introduction of zidovudine and Nikavir.

Nikavir in Chemoprevention Regimens of Vertical HIV Transmission 131

It was established that an intragastric once-daily dose of 100 mg/kg and a 50+50 mg/kg dose of zidovudine as well as Nikavir daily dose of 100 mg/kg given within 1-19 days of

Body mass dynamics and offspring postnatal mortality indices in the experimental zidovudine and Nikavir groups treated perinatally did not significantly differ compared

Controls Zidovudine

Number of born offspring 8.81.4 8.10.9 8.51.2 8.31.5 Postnatal mortality, % 3.6 4.2 3.9 3.8

Table 4. Postnatal development in the group receiving 100 mg/kg of zidovudine and

maturation, etc.) were within the normal term limits for this animal species.

at 9 a.m. and 5 p.m. partly moderates its negative effect on the body mass gain.

and retardation of embryonic skeletal ossification were observed.

(50+50 mg/kg) and a once-daily intragastric 100 mg/kg dose of Nikavir.

Other parameters of the offspring development observed (hair covering, incisor eruption, opening of eyes, helix detachment, vagina opening, testicle descending, time of reflex

In conclusion it should be noted that daily intragastric administration of zidovudine and Nikavir in a once-daily dose of 100 mg/kg (a 12.5 multiple of the maximum daily doses for pregnant women) to pregnant rats from within 1-19 days of gestation was found to retard their body mass gain in the third trimester. A twice-daily dose of zidovudine (50+50 mg/kg)

A once-daily dose intragastric introduction of zidovudine and Nikavir as well as a twicedaily dose zidovudine (50+50 mg/kg) do not influence such embryotoxicity criteria as the duration of pregnancy, number of yellow bodies, number of alive fetuses, number of implantation places, embryo body mass, cranioclaudal size, as well as preimplantation and

A single-dose intragastric 100 mg/kg zidovudine and Nikavir given within 1-19 days of gestation did not cause any malformations and developmental defects in the offspring. However, in the introduction of theses preparations in a daily intragastric dose of 100 mg/kg within 1-19 days of gestation both the decrease in the number of ossification centers

It was due to the effect of zidovudine and Nikavir that there were no ossification centers in the 2nd and 4th metacarpal bones, the 3rd and the 4th metatarsal bones, sublingual and pubic bones of the embryos. The noted changes were more marked in the embryos of the experimental group which received a once-daily dose of 100 mg/kg zidovudine in the perinatal period. Adverse effects reduced in a twice-daily dose introduction of zidovudine

During the observation period no further adverse influence of the tested agents on the following offspring development was noted. It was not accompanied by any term deviations

Nikavir doses in the prenatal period (1-19 days of gestation).

At birth 7.60.2 7.30.6 7.40.5 7.30.7 7th day of life 20.31.3 18.31.8 20.11.1 19.11.6 14th day of life 37.33.5 35.51.6 36.41.5 35.61.7 2nd day of life 43.83.1 47.72.0 45.91.8 46.01.5 28th day of life 56.32.5 58.12.6 56.72.5 57.52.1

100 mg/kg

Animal groups

Zidovudine 50+50 mg/kg

Nikavir 100 mg/kg

gestation did not cause any change in the number of the offspring born.

with the controls (Table 4).

Body mass, g

Parameters

postimplantation death rate.


Table 2. Embriotoxicity indices of zidovudine and Nikavir administered in the intragastric dose of 100 mg/kg introduced to rats within 1-19 days of gestation.

Microscopy and microanatomical examination (standard Wilson-Dyiban dissection) of fetuses perinatally exposed to zidovudine and Nikavir in tested doses did not reveal any malformations or defects of the visceral development. The incidence rate of malformations in the experimental groups did not significantly differ from the controls.

Development of the skeletal system in rat fetuses treated perinatally with zidovudine and Nikavir in the tested dose of 100 mg/kg was studied. The analysis of the alizarin stained total samples from the experimental groups showed the reduction in the number of ossification centers in the 2nd and 4th metacarpal bones, the 3rd and the 4th metatarsal bones, sublingual and pubic bones. These changes were more evident in the zidovudine group on a daily dose of 100 mg/kg. (Table 3).


Table 3. Fetal skeletal development on the 20th day of prenatal development.

Duration of pregnancy, days 22.90.4 22.40.5 22.50.4 22.70.4 Number of fetuses per 1 rat 8.00.9 7.61.1 7.61.1 8.11.2 Number of implantation places per rat 8.20.6 8.02.1 7.91.0 8.41.4 Number of yellow bodies per rat 8.70.7 8.60.7 8.40.8 8.90.8 Preimplantation death, % 5.8 7.0 5.9 5.6 Postimplantation death, % 2.4 5.0 3.8 3.6 Craniocaudal fetal size, cm 2.70.2 2.50.1 2.50,2 2.60.1 Fetal body mass, g 3.10.1 3.10.2 3.30,1 3.20.1 Table 2. Embriotoxicity indices of zidovudine and Nikavir administered in the intragastric

Microscopy and microanatomical examination (standard Wilson-Dyiban dissection) of fetuses perinatally exposed to zidovudine and Nikavir in tested doses did not reveal any malformations or defects of the visceral development. The incidence rate of malformations

Development of the skeletal system in rat fetuses treated perinatally with zidovudine and Nikavir in the tested dose of 100 mg/kg was studied. The analysis of the alizarin stained total samples from the experimental groups showed the reduction in the number of ossification centers in the 2nd and 4th metacarpal bones, the 3rd and the 4th metatarsal bones, sublingual and pubic bones. These changes were more evident in the zidovudine group on a

Controls Zidovudine

brood 2.6 2.7 2.4 2.5 Sublingual bone, % 2.4 6.6 4.3 4.8

2nd metacarpal bone 13.5 20.9 15.1 14.2 3rd metacarpal bone 3.6 4.2 4.1 3.2 4th metacarpal bone 4.8 8.9 5.3 3.3

2nd metatarsal bones 7.0 12.0 12.1 11.2 3rd metatarsal bones 8.3 8.0 7.8 7.0 4th metatarsal bones 7.0 14.2 8.6 8.1

ischiadic 0 0 0 0 iliac 0 0 0 0 pubic 4.0 6.6 5.8 5.2

Table 3. Fetal skeletal development on the 20th day of prenatal development.

100 mg/kg

dose of 100 mg/kg introduced to rats within 1-19 days of gestation.

in the experimental groups did not significantly differ from the controls.

Controls Zidovudine

100 mg/kg

Animal groups

Animal groups

Zidovudine 50+50 mg/kg

Nikavir 100 mg/kg

Zidovudine 50+50 mg/kg

Nikavir 100 mg/kg

Parameters

daily dose of 100 mg/kg. (Table 3).

Absence of ossification centers in fetal skeletons

Sternum, absolute number per

Forefeet, %

Hind limbs, %

Bones of the trunk, %

It was established that an intragastric once-daily dose of 100 mg/kg and a 50+50 mg/kg dose of zidovudine as well as Nikavir daily dose of 100 mg/kg given within 1-19 days of gestation did not cause any change in the number of the offspring born.

Body mass dynamics and offspring postnatal mortality indices in the experimental zidovudine and Nikavir groups treated perinatally did not significantly differ compared with the controls (Table 4).


Table 4. Postnatal development in the group receiving 100 mg/kg of zidovudine and Nikavir doses in the prenatal period (1-19 days of gestation).

Other parameters of the offspring development observed (hair covering, incisor eruption, opening of eyes, helix detachment, vagina opening, testicle descending, time of reflex maturation, etc.) were within the normal term limits for this animal species.

In conclusion it should be noted that daily intragastric administration of zidovudine and Nikavir in a once-daily dose of 100 mg/kg (a 12.5 multiple of the maximum daily doses for pregnant women) to pregnant rats from within 1-19 days of gestation was found to retard their body mass gain in the third trimester. A twice-daily dose of zidovudine (50+50 mg/kg) at 9 a.m. and 5 p.m. partly moderates its negative effect on the body mass gain.

A once-daily dose intragastric introduction of zidovudine and Nikavir as well as a twicedaily dose zidovudine (50+50 mg/kg) do not influence such embryotoxicity criteria as the duration of pregnancy, number of yellow bodies, number of alive fetuses, number of implantation places, embryo body mass, cranioclaudal size, as well as preimplantation and postimplantation death rate.

A single-dose intragastric 100 mg/kg zidovudine and Nikavir given within 1-19 days of gestation did not cause any malformations and developmental defects in the offspring. However, in the introduction of theses preparations in a daily intragastric dose of 100 mg/kg within 1-19 days of gestation both the decrease in the number of ossification centers and retardation of embryonic skeletal ossification were observed.

It was due to the effect of zidovudine and Nikavir that there were no ossification centers in the 2nd and 4th metacarpal bones, the 3rd and the 4th metatarsal bones, sublingual and pubic bones of the embryos. The noted changes were more marked in the embryos of the experimental group which received a once-daily dose of 100 mg/kg zidovudine in the perinatal period. Adverse effects reduced in a twice-daily dose introduction of zidovudine (50+50 mg/kg) and a once-daily intragastric 100 mg/kg dose of Nikavir.

During the observation period no further adverse influence of the tested agents on the following offspring development was noted. It was not accompanied by any term deviations

Nikavir in Chemoprevention Regimens of Vertical HIV Transmission 133

cases of moderate abdominal pain which did not require its cessation. No deviations in

The end of Nikavir therapy was followed by elevation of the mean CD4 lymphocyte counts. Of note, before delivery 50% of women were referred to a higher immunological category.

There was a marked reduction of viral load noted in the process of therapy. In 4 weeks of treatment the level of viral load reduced below the level of detection in 60% of women in

Thus Nikavir appears to be a highly efficient agent for treatment of HIV-infected pregnant women as its effect has been confirmed by the obtained clinical and immunological evidence. A clinical Nikavir trial at St. Petersburg Regional AIDS Center involved 30 pregnant women aged 20-35 years (mean age 26 years) at 14-34 weeks of gestation and infection term from 1 to 6 years. 36.6% of examined women presented with a history of drug addiction and 50%

In accordance with the baseline viral load 23 patients were administered Nikavir as a

In a monotherapy schedule Nikavir was given in a dosage of 200 mg in 3 doses. In combined dual therapy Nikavir was supplemented with Epivir in a daily dosage of 150 mg in 2 doses. In accordance with the Russian Federation standards at the onset of labour the women were given intravenous zidovudine. Newborns received an extended therapy with

The pretreatment viral load in the majority of women (67%) did not exceed 10000 copies/ml. By the 4th week the total of the group demonstrated a significant reduction of viral load indices (p<0.05). At 28 weeks of gestation the total number of patients with undetectable optimal level of viral load was 33%. At 36 weeks the reduction tendency was stable. In the following postpartum period HIV RNA levels did not exceed the baseline indices (Table 5).

> 400-1000 copies/ml

Pretreatment, patients 3 (10%) 1 (3%) 19 (67%) 3 (10%) 3 (10%)

therapy, patients 9 (37%) 3 (13%) 9 (37%) 2 (9%) 0

patients 8 (35%) 2 (9%) 11 (47%) 2 (9%) 0

patients 7 (37%) 4 (21%) 8 (42%) 0 0

patients 2 (11%) 0 13 (68%) 4 (21%) 0

patients 3 (16%) 2 (11%) 10 (52%) 4 (21%) 0

Table 5. The intra-treatment dynamics of viral load in pregnant women.

patients 2 (10%) 4 (20%) 13 (65%) 0 1 (5%)

Viral load levels

1000-10000 copies/ml 10000-50000 copies/ml

>50000 copies/ml

Such elevation tendency persisted until the end of the investigation.

No cases of perinatal HIV transmission were registered.

monotherapy and 7 patients received dual therapy.

zidovudine in syrup (Retrovir) for 6 weeks following the delivery.

<400 copies/ml

both groups and in 90% before the delivery but it was less than 1000 copies/ml.

chronic hepatitis C. A total of patients did not receive anti-retroviral preparations.

laboratory findings were noted.

Observation time

After 4 weeks of

28 weeks of gestation,

36 weeks of gestation,

1 month postpartum,

3 months postpartum,

6 months postpartum,

and was within the normal time limits natural for the normal physiological development of this animal species.

Thus, studies on animal models have provided reliable evidence that compared to zidovudine Nikavir possesses a less damaging impact on fetal development and thus may be a more preferable choice for ART in HIV-infected pregnant women.

## **4. Experience of Nikavir use in various regiments of vertical HIV transmission chemoprophylaxis**

The first experience of Nikavir use in different regiments of vertical HIV chemoprophylaxis was obtained by the staff workers of the Russian Federal AIDS Center (Detkova, 2003).

Three groups of 96 HIV-infected pregnant women were observed. Groups 1 and 2 received Nikavir in the dose of 200 mg/kg 3 times a day after 14 gestation weeks (within 16 to 36 weeks of gestation, the average time 25-27 weeks of gestation). Group 1 of 27 women were administered intravenous zidovudine in labour. Newborns were given oral zidovudine syrup (Retrovir) in the dose of 2 mg/kg body mass every 6 hours for 6 weeks. Group 2 (17 women) were given a single dose of 200 mg Viramun at the onset of labour. Newborns were given Viramun suspension in the dose of 2 mg/kg body mass once daily for 3 days. Group 3 included 52 women who did not receive chemoprevention. The only preventive measure was exclusion of breastfeeding.

A total of the women under observation delivered alive babied (27 newborns in group 1, 17 newborns in group 2 and 52 newborns in group 3). Body weight parameters of newborns given chemoprevention slightly surpassed those in group 3 though the difference was not significant (3022223 and 2731558 g respectively, p=0.196). The children were followed-up during 72 weeks. By 72 weeks 34.6% of group 3 were diagnosed HIV-infection (stable positive serological evidence HIV DNA in PCR). Children in group 1 were born healthy. Only one newborn in group 2 was diagnosed HIV which was possibly due to the continuous drug addiction of his mother and her inappropriate following the Nikavir regimen during pregnancy (adherence to preventive therapy was <60%).

Application of Nikavir in pregnancy showed its good tolerability. The major therapyassociated side-effect was a mild gastric syndrome found in 25% of women. Application of Nikavir aimed at prevention during pregnancy was not found to affect either the pregnancy course in HIV-infected pregnant women or maturation and vital capacity of newborns. No significant association between application of Nikavir as intrapartum prevention and both the pregnancy course in HIV-infected pregnant women and maturation and vital capacity of newborns was established.

A further clinical trial of efficiency and safety of chemoprevention with Nikavir in pregnant HIV-infected women was carried out at the Republican Clinical Infection Hospital (Izhora settlement, St. Petersburg) (unpublished data) as well as at Regional AIDS Centers in St. Petersburg (Zakharova, 2008) and Perm (Ivanova, 2010).

The clinical trial conducted at the Republican Clinical Infection Hospital in 2005-2006 involved 20 pregnant women aged 20-31 at 26-28 weeks of gestation with normal laboratory values.

Group 1 (10 women) was given 200 mg Nikavir 3 times daily. Their viral load <3000 copies/ml. Group 2 (10 women) was given 200 mg Nikavir 3 times daily + Epivir in conventional dosage. Their baseline viral load was 3000-30000 copies/ml.

Assessment of therapy was based on registration of clinical and laboratory indicators of HIV progress.

Nikavir therapy demonstrated good tolerance (100% of patients have finished research). No severe adverse events were observed. There were only 2 associated with therapy registered

and was within the normal time limits natural for the normal physiological development of

Thus, studies on animal models have provided reliable evidence that compared to zidovudine Nikavir possesses a less damaging impact on fetal development and thus may

**4. Experience of Nikavir use in various regiments of vertical HIV transmission** 

The first experience of Nikavir use in different regiments of vertical HIV chemoprophylaxis was obtained by the staff workers of the Russian Federal AIDS Center (Detkova, 2003). Three groups of 96 HIV-infected pregnant women were observed. Groups 1 and 2 received Nikavir in the dose of 200 mg/kg 3 times a day after 14 gestation weeks (within 16 to 36 weeks of gestation, the average time 25-27 weeks of gestation). Group 1 of 27 women were administered intravenous zidovudine in labour. Newborns were given oral zidovudine syrup (Retrovir) in the dose of 2 mg/kg body mass every 6 hours for 6 weeks. Group 2 (17 women) were given a single dose of 200 mg Viramun at the onset of labour. Newborns were given Viramun suspension in the dose of 2 mg/kg body mass once daily for 3 days. Group 3 included 52 women who did not receive chemoprevention. The only preventive measure

A total of the women under observation delivered alive babied (27 newborns in group 1, 17 newborns in group 2 and 52 newborns in group 3). Body weight parameters of newborns given chemoprevention slightly surpassed those in group 3 though the difference was not significant (3022223 and 2731558 g respectively, p=0.196). The children were followed-up during 72 weeks. By 72 weeks 34.6% of group 3 were diagnosed HIV-infection (stable positive serological evidence HIV DNA in PCR). Children in group 1 were born healthy. Only one newborn in group 2 was diagnosed HIV which was possibly due to the continuous drug addiction of his mother and her inappropriate following the Nikavir regimen during

Application of Nikavir in pregnancy showed its good tolerability. The major therapyassociated side-effect was a mild gastric syndrome found in 25% of women. Application of Nikavir aimed at prevention during pregnancy was not found to affect either the pregnancy course in HIV-infected pregnant women or maturation and vital capacity of newborns. No significant association between application of Nikavir as intrapartum prevention and both the pregnancy course in HIV-infected pregnant women and maturation and vital capacity of

A further clinical trial of efficiency and safety of chemoprevention with Nikavir in pregnant HIV-infected women was carried out at the Republican Clinical Infection Hospital (Izhora settlement, St. Petersburg) (unpublished data) as well as at Regional AIDS Centers in St.

The clinical trial conducted at the Republican Clinical Infection Hospital in 2005-2006 involved 20 pregnant women aged 20-31 at 26-28 weeks of gestation with normal laboratory values. Group 1 (10 women) was given 200 mg Nikavir 3 times daily. Their viral load <3000 copies/ml. Group 2 (10 women) was given 200 mg Nikavir 3 times daily + Epivir in

Assessment of therapy was based on registration of clinical and laboratory indicators of HIV

Nikavir therapy demonstrated good tolerance (100% of patients have finished research). No severe adverse events were observed. There were only 2 associated with therapy registered

be a more preferable choice for ART in HIV-infected pregnant women.

this animal species.

**chemoprophylaxis** 

was exclusion of breastfeeding.

newborns was established.

progress.

pregnancy (adherence to preventive therapy was <60%).

Petersburg (Zakharova, 2008) and Perm (Ivanova, 2010).

conventional dosage. Their baseline viral load was 3000-30000 copies/ml.

cases of moderate abdominal pain which did not require its cessation. No deviations in laboratory findings were noted.

The end of Nikavir therapy was followed by elevation of the mean CD4 lymphocyte counts. Of note, before delivery 50% of women were referred to a higher immunological category. Such elevation tendency persisted until the end of the investigation.

There was a marked reduction of viral load noted in the process of therapy. In 4 weeks of treatment the level of viral load reduced below the level of detection in 60% of women in both groups and in 90% before the delivery but it was less than 1000 copies/ml.

No cases of perinatal HIV transmission were registered.

Thus Nikavir appears to be a highly efficient agent for treatment of HIV-infected pregnant women as its effect has been confirmed by the obtained clinical and immunological evidence.

A clinical Nikavir trial at St. Petersburg Regional AIDS Center involved 30 pregnant women aged 20-35 years (mean age 26 years) at 14-34 weeks of gestation and infection term from 1 to 6 years. 36.6% of examined women presented with a history of drug addiction and 50% chronic hepatitis C. A total of patients did not receive anti-retroviral preparations.

In accordance with the baseline viral load 23 patients were administered Nikavir as a monotherapy and 7 patients received dual therapy.

In a monotherapy schedule Nikavir was given in a dosage of 200 mg in 3 doses. In combined dual therapy Nikavir was supplemented with Epivir in a daily dosage of 150 mg in 2 doses. In accordance with the Russian Federation standards at the onset of labour the women were given intravenous zidovudine. Newborns received an extended therapy with zidovudine in syrup (Retrovir) for 6 weeks following the delivery.

The pretreatment viral load in the majority of women (67%) did not exceed 10000 copies/ml. By the 4th week the total of the group demonstrated a significant reduction of viral load indices (p<0.05). At 28 weeks of gestation the total number of patients with undetectable optimal level of viral load was 33%. At 36 weeks the reduction tendency was stable. In the following postpartum period HIV RNA levels did not exceed the baseline indices (Table 5).


Table 5. The intra-treatment dynamics of viral load in pregnant women.

Nikavir in Chemoprevention Regimens of Vertical HIV Transmission 135

45% women in group 1 and 44% women in group 2 had transvaginal delivery. Pre-term delivery was registered in 2 women – 1 from each group at 32 and 34 weeks of gestation

Prior to chemoprophylaxis a total of group 1 women were clinically diagnosed the stage A1 HIV infection. Half of group 2 women were diagnosed the stage B1 HIV infection associated with oral candidiasis and grade 1 anemia. On clinical examination of both groups performed 1.5 months after delivery no progression of HIV was revealed. The structure of associated diseases included viral hepatitis C in 45% and 60% of women from group 1 and group 2

Prior chemoprophylaxis the baseline viral load in group 1 women ranged from 500 to 382000 copies/ml (mean 8280); 4 weeks after the start of chemoprevention it dropped by 6 times to 886; it was undetectable at 36 weeks of gestation (<500 copies/ml). The pretreatment baseline mean CD4 lymphocytes count was 478 cells/mm3 and 545 cells/mm3

The baseline viral load in group 2 women was >200 000 copies/ml, 4 weeks after the start of chemoprevention it dropped by about 300 times. Prepartum viral load was undetectable (<500 copies/ml) and 1.5 months after delivery with discontinuation of treatment it increased to over 20000 copies/ml. The pretreatment baseline mean CD4 lymphocytes count was twice lower compared with group 1; after 4 weeks of HAART and before delivery it increased and insignificantly lowered 1.5 month postpartum when treatment was discontinued. Therefore, following the discontinuation of HAART in group 2 viral load

<500 <500 <sup>886</sup>

gestation

545

1,5 months postpartum 420

440

460

480

500

CD4 cells/mm3

520

540

560

respectively. There were no cases of intrapartum complications and breast-feeding.

respectively. Chlamydial infection was identified in 5% of women in group 1.

parameters increased while CD4 lymphocytes count decreased.

<sup>471</sup> <sup>470</sup> <sup>478</sup>

Pretritment Post 4 weeks At 36 weeks of

**Evaluation time**

Fig. 2. Parameters of viral load and CD4 lymphocytes at various terms of examination of

Viral load CD4 lymphocyte count

8280

before delivery (Fig. 2).

women administered Nikavir+Epivir.

VL copies/ml

Patients with viral load over 10000 copies/ml received the dual therapy (Nikavir+Epivir). Already by the 4th week of therapy the viral load in half of the patients reduced to the undetectable level and remained at that level till 36 weeks of gestation. The same was true about the patient who was transferred from the monotherapy group due to the increase of her viral load above 10000 copies/ml.

Starting with the 4th week of treatment an overall significant increase of the mean values of the percentile CD4 lymphocytes indices irrespective of the drug intake regimen was noted. After discontinuation of treatment CD4 lymphocytes indices returned to the baseline.

The mean Hb values did not exceed the norms before initiation of therapy (117 g/l). It was noted that mean Hb values decreased during chemoprophylaxis. Hb decrease lower 100 g/l was improved with administration of iron-containing preparations. By 36 weeks of gestation mean Hb values were not different from baseline (Table 6).


Table 6. Hb level in Nikavir treatment of pregnant women.

The controllable biochemical blood serum values did not correlate with the therapeutic regimen and did not deviate from the normal during the whole observation time.

Proper adherence to therapy was associated with good tolerance of the applied regimens.

In the majority of patients the labour course and the delivery methods did not differ from those in the average population.

Viral load monitoring findings in children provided by the attending pediatricians confirmed the absence of HIV-1 virus in 100% of newborns at three examinations during a 6 months period.

Thus, the obtained findings allow considering Nikavir one of the most perspective agents for the practice of perinatal prophylaxis of vertical transmission of HIV-1 virus. However, a continuous monitoring of Hb levels and viral load for the prompt correction of the switch regimen from monotherapy to combination (dual) therapy as well as an additional administration of iron-containing preparations is necessary.

The clinical trial conducted at Perm Regional AIDS Center involved 38 HIV infected and their 38 newborns. Group 1 (20 women, aged 18-30) was given Nikavir+Epivir therapy. Group 2 (18 women aged 19-32) was given one the HAART regimens (Nikavir+Epivir+Viramun or Kaletra). The total of patients on chemoprevention schedule did not take the agents previously. The therapy was started at 23-32 weeks of gestation depending on the time of their first visit.

During the first hour of labor a dose of 2 mg/kg/h of zidovudine was given intravenously followed by 1 mg/kg/h until the end of the labor. Starting with the 8th hour of life the newborns were given of zidovudine in syrup (Retrovir) in an oral dose of 2 mg/kg every 6 hours during 6 weeks.

Patients with viral load over 10000 copies/ml received the dual therapy (Nikavir+Epivir). Already by the 4th week of therapy the viral load in half of the patients reduced to the undetectable level and remained at that level till 36 weeks of gestation. The same was true about the patient who was transferred from the monotherapy group due to the increase of

Starting with the 4th week of treatment an overall significant increase of the mean values of the percentile CD4 lymphocytes indices irrespective of the drug intake regimen was noted. After discontinuation of treatment CD4 lymphocytes indices returned to the baseline. The mean Hb values did not exceed the norms before initiation of therapy (117 g/l). It was noted that mean Hb values decreased during chemoprophylaxis. Hb decrease lower 100 g/l was improved with administration of iron-containing preparations. By 36 weeks of gestation

Pretreatment Post 4 weeks

Mean value, g/liter 116.5±9.2 106.3±8.5 107.9±7.3 114.9±8.1 Minimal value, g/liter 89 91 86 96 Maximal value, g/liter 150 130 128 140 < 100 g/liter, patients 3 (10%) 12 (40%) 6 (20%) 2 (7%) >100 g/liter, patients 27 (90%) 18 (60%) 24 (80%) 26 (93%)

The controllable biochemical blood serum values did not correlate with the therapeutic

Viral load monitoring findings in children provided by the attending pediatricians confirmed the absence of HIV-1 virus in 100% of newborns at three examinations during a 6

Thus, the obtained findings allow considering Nikavir one of the most perspective agents for the practice of perinatal prophylaxis of vertical transmission of HIV-1 virus. However, a continuous monitoring of Hb levels and viral load for the prompt correction of the switch regimen from monotherapy to combination (dual) therapy as well as an additional

The clinical trial conducted at Perm Regional AIDS Center involved 38 HIV infected and their 38 newborns. Group 1 (20 women, aged 18-30) was given Nikavir+Epivir therapy. Group 2 (18 women aged 19-32) was given one the HAART regimens (Nikavir+Epivir+Viramun or Kaletra). The total of patients on chemoprevention schedule did not take the agents previously. The therapy was started at 23-32 weeks of gestation

During the first hour of labor a dose of 2 mg/kg/h of zidovudine was given intravenously followed by 1 mg/kg/h until the end of the labor. Starting with the 8th hour of life the newborns were given of zidovudine in syrup (Retrovir) in an oral dose of 2 mg/kg every 6

Proper adherence to therapy was associated with good tolerance of the applied regimens. In the majority of patients the labour course and the delivery methods did not differ from

regimen and did not deviate from the normal during the whole observation time.

Observation time

28 week of gestation

36 week of gestation

of treatment

her viral load above 10000 copies/ml.

Hemoglobin

those in the average population.

depending on the time of their first visit.

hours during 6 weeks.

months period.

mean Hb values were not different from baseline (Table 6).

Table 6. Hb level in Nikavir treatment of pregnant women.

administration of iron-containing preparations is necessary.

45% women in group 1 and 44% women in group 2 had transvaginal delivery. Pre-term delivery was registered in 2 women – 1 from each group at 32 and 34 weeks of gestation respectively. There were no cases of intrapartum complications and breast-feeding.

Prior to chemoprophylaxis a total of group 1 women were clinically diagnosed the stage A1 HIV infection. Half of group 2 women were diagnosed the stage B1 HIV infection associated with oral candidiasis and grade 1 anemia. On clinical examination of both groups performed 1.5 months after delivery no progression of HIV was revealed. The structure of associated diseases included viral hepatitis C in 45% and 60% of women from group 1 and group 2 respectively. Chlamydial infection was identified in 5% of women in group 1.

Prior chemoprophylaxis the baseline viral load in group 1 women ranged from 500 to 382000 copies/ml (mean 8280); 4 weeks after the start of chemoprevention it dropped by 6 times to 886; it was undetectable at 36 weeks of gestation (<500 copies/ml). The pretreatment baseline mean CD4 lymphocytes count was 478 cells/mm3 and 545 cells/mm3 before delivery (Fig. 2).

The baseline viral load in group 2 women was >200 000 copies/ml, 4 weeks after the start of chemoprevention it dropped by about 300 times. Prepartum viral load was undetectable (<500 copies/ml) and 1.5 months after delivery with discontinuation of treatment it increased to over 20000 copies/ml. The pretreatment baseline mean CD4 lymphocytes count was twice lower compared with group 1; after 4 weeks of HAART and before delivery it increased and insignificantly lowered 1.5 month postpartum when treatment was discontinued. Therefore, following the discontinuation of HAART in group 2 viral load parameters increased while CD4 lymphocytes count decreased.

Fig. 2. Parameters of viral load and CD4 lymphocytes at various terms of examination of women administered Nikavir+Epivir.

Nikavir in Chemoprevention Regimens of Vertical HIV Transmission 137

With the aim of the analysis of the adherence to chemoprevention two groups of patients with the past history of intravenous psycho-active agents were suggested a self-completed questionnaire. The group 1 included 31 women who had not been administered chemoprevention therapy due to the early gestation term. The group 2 contained 23 women

The answers to the question on the time of registration their pregnancy at the women consultation center were as follows: 45.2% in the group 1 and 30.4% in the group 2 were registered at the term before 12 weeks of gestation. Thus, the majority of the respondents delayed their visit to their gynecologist for registration of their pregnancy for later than 12 weeks of gestation (54.8% and 69.4% respectively).Their attendance of gynecological checkups was self-assessed as neither regular nor frequent by 22.6% of respondents in group 1 and 17.4% in group 2. 77.4% attended gynecologist at the women consultation center but

It is worth noting the fact that before starting chemoprophylaxis almost 30% of woman did not visit gynecologist regularly and 9% missed such visits during the antiviral

A considerable part of pregnant women (80.6% and 73.9% respectively) strictly followed the administrations of their doctors while the others neglected the professional advice. Thus, 3% of women in group 1 and 6% in group 2 have taken responsibility to decide themselves which of the doctor's recommendations they are to follow. And 16.2% noted that prior chemoprophylaxis they underwent only those examinations which considered being necessary. However, with the beginning of antiretroviral therapy women become more

The majority of women in both groups consider the ultimate goal of chemotherapy to be the

45.3% of women did not express apprehension of chemoprevention and revealed an adequately positive attitude to it. The investigation analyzed persistent detrimental habits in pregnant HIV infected women which they could not abandon even being pregnant. About half of them smoked (45.1% and 43.5% respectively) and 3.2% women of group 1 took alcohol. 2 patients of group 1 (6.4%) gave a negative answer to the question about the influence of irregular and incorrect intake of antiretroviral preparations on the therapeutic

A considerable number of pregnant women strictly followed recommendations of their doctors (80.6% and 73.9% respectively) while others neglect certain administrations on diet

In summary, investigation of adherence to perinatal chemoprevention in HIV infected pregnant women demonstrated high level of motivation aimed at birth of a healthy child. However, along with following the therapeutic regimen their specialist check-up visiting was neither regular nor timely. The majority of women kept to their harmful habits (smocking) and did not follow recommendations on their diet which was possibly due to their low social status. Consequently, every third pregnant woman before administration of chemoprevention and every second woman during chemoprevention did not attend their obstetricians and gynecologists regularly. In this connection one should note the necessity of organization of School of Adherence to Chemoprevention which can provide social-

**5.2 Adherence to HAART for perinatal prevention in HIV infected pregnant women**  An important factor of the prevention of the perinatal transmission of HIV virus is the

formation of an adherence to persistent intake of antiretroviral preparations.

undergoing HAART. The age in both groups was 18-25 years.

only 64.5% attended the HIV/AIDS Prevention Center.

responsible and underwent all the administered examinations.

birth of a healthy child (83.6% and 95.6% respectively).

therapy.

effect.

(34.7% and 54.6% respectively).

The total of children born to HIV-infected mothers was referred to the category of risk with diagnosed perinatal HIV infection contact and was examined for the presence of HIV-1 DNA. The obtained results were negative.

The agents proved an appropriate tolerance in both therapeutic regimens. No significant side effects and adverse events associated with the tested agents were noted. The parameters of vital capacity were in compliance with the normal natural course of pregnancy. The haemogram analysis was performed to predict the possible side effects of chemoprevention. In this connection it was found that at the time of conception both red blood cell counts and white blood cell counts were normal. In both groups the Hb level was insignificantly decreased: 101 g/l in group 1 and 106 g/l in 17% of group 2 respectively. 4 weeks after the start of therapy and at 36 weeks of gestation no changes in the parameters of peripheral blood were noted (p>0.05). The total of women received the preventive therapy of anemia including the diet and iron-containing preparations in conventional doses. By 36 weeks of gestation a tendency of thrombocyte count elevation was observed – 297.9 and 271.8 g/l respectively. At different terms the findings of the functional liver tests (ALT, AST, bilirubin) were within the normal limits in both groups.

Thus, absence of HIV-1 infection in children born to HIV-infected pregnant women testifies high efficiency of both chemoprevention regimens with Nikavir both in combination with Epivir and in HAART. An evident positive outcome of this therapy is confirmed by the significant decrease of viral load to undetectable level of viral RNA during therapy starting with the 4th week of gestation up to delivery. Simultaneous elevation of CD4 lymphocytes is undoubtedly an evidence of beneficial effect of both regimens of chemoprevention on the immune status of HIV-infected pregnant women. Excellent adherence to chemoprevention therapy (100%) was associated with good tolerance of the employed agents. The safety of Nikavir application both in combination with Epivir and in HAART schedule was proved by the absence of toxic effect on biochemical blood values at various gestation terms. An insignificant elevation of thrombocyte count by 36 weeks of gestation in both groups may be regarded as a physiological factor preparing the organism of a woman to delivery.

The obtained results allow to regard Nikavir to be one of the most potent perspective agents used in the schedules of chemoprevention of vertical transmission of HIV-1infection.

## **5. Comparative characteristics of methods of perinatal chemoprophylaxis with Nikavir**

### **5.1 Actuality**

Currently, the choice of available antiretroviral agents for chemoprevention of perinatal infection is not extensive. The standard schedules of HAART are widely used. A number of various prevention patterns based on the expert opinion, theoretical research and evidence of preclinical animal studies has been suggested. However, substantiation of choice of methods of chemoprevention of perinatal HIV infection, efficiency and safety of different preparations and their side-effect estimation have not been sufficiently investigated.

Thus, method of chemoprophylaxis of vertical transmission of HIV-1 with Nikavir+Epivir in HAART schedule which is known to produce less side-effects compared to the analogue schedules with Combivir is becoming actual.

The present investigation was performed in June 2007 – October 2008 in the setting of Perm Regional AIDS Center and is based on the analysis of epidemiology data as well as the evidence from clinical and laboratory studies.

The total of children born to HIV-infected mothers was referred to the category of risk with diagnosed perinatal HIV infection contact and was examined for the presence of HIV-1

The agents proved an appropriate tolerance in both therapeutic regimens. No significant side effects and adverse events associated with the tested agents were noted. The parameters of vital capacity were in compliance with the normal natural course of pregnancy. The haemogram analysis was performed to predict the possible side effects of chemoprevention. In this connection it was found that at the time of conception both red blood cell counts and white blood cell counts were normal. In both groups the Hb level was insignificantly decreased: 101 g/l in group 1 and 106 g/l in 17% of group 2 respectively. 4 weeks after the start of therapy and at 36 weeks of gestation no changes in the parameters of peripheral blood were noted (p>0.05). The total of women received the preventive therapy of anemia including the diet and iron-containing preparations in conventional doses. By 36 weeks of gestation a tendency of thrombocyte count elevation was observed – 297.9 and 271.8 g/l respectively. At different terms the findings of the functional liver tests (ALT, AST,

Thus, absence of HIV-1 infection in children born to HIV-infected pregnant women testifies high efficiency of both chemoprevention regimens with Nikavir both in combination with Epivir and in HAART. An evident positive outcome of this therapy is confirmed by the significant decrease of viral load to undetectable level of viral RNA during therapy starting with the 4th week of gestation up to delivery. Simultaneous elevation of CD4 lymphocytes is undoubtedly an evidence of beneficial effect of both regimens of chemoprevention on the immune status of HIV-infected pregnant women. Excellent adherence to chemoprevention therapy (100%) was associated with good tolerance of the employed agents. The safety of Nikavir application both in combination with Epivir and in HAART schedule was proved by the absence of toxic effect on biochemical blood values at various gestation terms. An insignificant elevation of thrombocyte count by 36 weeks of gestation in both groups may be

regarded as a physiological factor preparing the organism of a woman to delivery.

used in the schedules of chemoprevention of vertical transmission of HIV-1infection.

preparations and their side-effect estimation have not been sufficiently investigated.

**5. Comparative characteristics of methods of perinatal chemoprophylaxis** 

The obtained results allow to regard Nikavir to be one of the most potent perspective agents

Currently, the choice of available antiretroviral agents for chemoprevention of perinatal infection is not extensive. The standard schedules of HAART are widely used. A number of various prevention patterns based on the expert opinion, theoretical research and evidence of preclinical animal studies has been suggested. However, substantiation of choice of methods of chemoprevention of perinatal HIV infection, efficiency and safety of different

Thus, method of chemoprophylaxis of vertical transmission of HIV-1 with Nikavir+Epivir in HAART schedule which is known to produce less side-effects compared to the analogue

The present investigation was performed in June 2007 – October 2008 in the setting of Perm Regional AIDS Center and is based on the analysis of epidemiology data as well as the

DNA. The obtained results were negative.

bilirubin) were within the normal limits in both groups.

**with Nikavir 5.1 Actuality** 

schedules with Combivir is becoming actual.

evidence from clinical and laboratory studies.

#### **5.2 Adherence to HAART for perinatal prevention in HIV infected pregnant women**

An important factor of the prevention of the perinatal transmission of HIV virus is the formation of an adherence to persistent intake of antiretroviral preparations.

With the aim of the analysis of the adherence to chemoprevention two groups of patients with the past history of intravenous psycho-active agents were suggested a self-completed questionnaire. The group 1 included 31 women who had not been administered chemoprevention therapy due to the early gestation term. The group 2 contained 23 women undergoing HAART. The age in both groups was 18-25 years.

The answers to the question on the time of registration their pregnancy at the women consultation center were as follows: 45.2% in the group 1 and 30.4% in the group 2 were registered at the term before 12 weeks of gestation. Thus, the majority of the respondents delayed their visit to their gynecologist for registration of their pregnancy for later than 12 weeks of gestation (54.8% and 69.4% respectively).Their attendance of gynecological checkups was self-assessed as neither regular nor frequent by 22.6% of respondents in group 1 and 17.4% in group 2. 77.4% attended gynecologist at the women consultation center but only 64.5% attended the HIV/AIDS Prevention Center.

It is worth noting the fact that before starting chemoprophylaxis almost 30% of woman did not visit gynecologist regularly and 9% missed such visits during the antiviral therapy.

A considerable part of pregnant women (80.6% and 73.9% respectively) strictly followed the administrations of their doctors while the others neglected the professional advice. Thus, 3% of women in group 1 and 6% in group 2 have taken responsibility to decide themselves which of the doctor's recommendations they are to follow. And 16.2% noted that prior chemoprophylaxis they underwent only those examinations which considered being necessary. However, with the beginning of antiretroviral therapy women become more responsible and underwent all the administered examinations.

The majority of women in both groups consider the ultimate goal of chemotherapy to be the birth of a healthy child (83.6% and 95.6% respectively).

45.3% of women did not express apprehension of chemoprevention and revealed an adequately positive attitude to it. The investigation analyzed persistent detrimental habits in pregnant HIV infected women which they could not abandon even being pregnant. About half of them smoked (45.1% and 43.5% respectively) and 3.2% women of group 1 took alcohol. 2 patients of group 1 (6.4%) gave a negative answer to the question about the influence of irregular and incorrect intake of antiretroviral preparations on the therapeutic effect.

A considerable number of pregnant women strictly followed recommendations of their doctors (80.6% and 73.9% respectively) while others neglect certain administrations on diet (34.7% and 54.6% respectively).

In summary, investigation of adherence to perinatal chemoprevention in HIV infected pregnant women demonstrated high level of motivation aimed at birth of a healthy child. However, along with following the therapeutic regimen their specialist check-up visiting was neither regular nor timely. The majority of women kept to their harmful habits (smocking) and did not follow recommendations on their diet which was possibly due to their low social status. Consequently, every third pregnant woman before administration of chemoprevention and every second woman during chemoprevention did not attend their obstetricians and gynecologists regularly. In this connection one should note the necessity of organization of School of Adherence to Chemoprevention which can provide social-

Nikavir in Chemoprevention Regimens of Vertical HIV Transmission 139

month after delivery. At the same terms the patients were examined by different specialists to register HIV associated diseases and adverse effects of therapy. Women were examined by infectionists, gynecologists, obstetricians, immunologists, etc. and the newborns by neonatologists, infectionists, pediatricians and other specialists according to indications. The HIV diagnosis in women was based on enzyme immunoassay (EIA) detecting HIV antibodies ("Jenscreen Ulra HIV Ag/At") and immunoblot analysis (IMB) ("Blot-HIV") for HIV-1 virus specific proteins antibodies. HIV diagnosis in newborns was excluded on the basis of EIA and IMB monitoring during the period of observation starting at birth and

Laboratory examination included leucocyte and thrombocyte counts performed with MEK-7222 hemoanalyzer and standard urinalyses. Biochemical blood values were identified with Conelab, 20 automated analyzer supplied with ion selection section for evaluation of the functional state of the liver and kidneys. The studied parameters were compared with the

The associated diseases of HIV infected pregnant women were revealed with serological IMB tests for HBsAg, hepatitis C virus, herpes simplex, cytomegalovirus, toxoplasmosis,

Instrumental methods included ECG, ultrasonic examination abdominal and pelvic organs

Cellular immunity in pregnant HIV infected women was assessed by the absolute and percentage levels of CD4 lymphocyte subpopulation with monoclonal antibodies ("Beston Diskinzon" USA) at "FACS Caliber" cytofluorimeter by flow cytometer method. The obtained findings were compared with the norms established by the Russian Federal AIDS

Molecule-biological diagnosis in pregnant HIV infected women receiving chemoprevention was based on the detection of HIV-1 RNA plasma levels with polymerase chain reaction (PCR) and "Amplisensу HIV-monitor FRT" test-systems ("Interlabservice") before antiretroviral therapy, 4 weeks after it was started, 4 weeks before the supposed delivery

For the early diagnosis of HIV in newborns detection of HIV-1 DNA plasma levels with PCR and "Amplisense DNA HIV-96" test-systems ("Interlabservice") was carried out. They were

Adherence to antiretroviral perinatal prevention regimen in pregnant HIV infected women was studied with the questionnaire method assessing their social profile, clinical and

From 28 week of gestation until the delivery they received Nikavir administered in the dosage of 600 mg for 3 intakes daily. Combivir and Kaletra were given in conventional doses. During the first hour of labor 2 mg/kg/h of zidovudine were given intravenously

Starting with their eighth hour of life the newborn babies were given a 6 weeks course of

The overall data of pregnant HIV infected women receiving the targeted agents in therapeutic doses have been statistically assessed. Descriptive and frequency ratio analyses of the total adverse events revealed within the investigation period have been performed.

laboratory examinations, intake of preparations and attitude to chemoprevention.

zidovudine in syrup (Retrovir) orally in the dose of 2 mg/kg every 6 hours.

thereafter at the age of 1.5 and 3 months.

standards established for Perm.

chlamydia and Wassermann test.

Center (Pokrovsky, 2001).

term and 1.5 month after delivery.

performed two tests at the age of 1.5 and 3 months.

followed by 1 mg/kg/h within the labor.

**5.6 Statistical analysis** 

if indicated.

psychological counseling aimed at formation of positive motivation to doctor's recommendations, following the daily regimen and regular intake of antiretroviral preparations as well as refuse of detrimental habits which is of a particular importance for pregnant drug-addicts.

## **5.3 Purpose**


## **5.4 Materials**

The start of antiretroviral prevention was determined by the time of the first appointment with a gynecologist for pregnancy diagnosis. Chemoprevention of vertical HIV mother-tochild virus transmission with various agents was performed in 36 women with A1 (62%) and B2 (38%) HIV stage (USA, CDC, 1987) at 23-32 gestation weeks as well as their 36 newborns. B2 stage manifested with moderate symptoms of oral mucosa candidiasis. 65% of observation group were diagnosed anemia mild to moderate degrees. No intrapartum complications occurred. There were no cases of breast-feeding.

The group 1 included 18 pregnant HIV infected women aged 19-32 (mean age 24 years) receiving Nikavir+Epivir+Kaletra chemoprevention. The group 2 included 18 women aged 19-28 (mean age 23.5 years) receiving Combivir+Kaletra chemoprevention. From the 28th week of gestation until the delivery they received Nikavir administered in the dosage of 600 vg for 3 intakes daily. Combivir and Kaletra were given in standard schedule.

During the first hour of labor 2 mg/kg/h of zidovudine were given intravenously followed by 1 mg/kg/h until the end of the labor.

Starting with the 8th hour of life the newborn babies were given a 6 weeks course of zidovudine in syrup (Retrovir) in the dosage of 2 mg/kg every 6 hours.

Epidemiological analysis of the routes of infection revealed the leading share of sexual HIV-1 transmission: 83% of pregnant women in Nikavir chemoprevention schedule group 1 and 89% in group 2. Parenteral route was revealed in 17% and 11% of cases respectively. Thus, there was a mixed type of epidemic process, the sexual transmission rate 5-8 times surpassing the parenteral one (cases of intravenous drugs).

#### **5.5 Methods**

The evaluation of the results of the investigation was based on analysis of the clinical, epidemiological and laboratory monitoring of HIV infection course in pregnant women and their newborns.

Manifestations of HIV epidemic process were studied according to the following criteria:


Clinical assessment included evaluation of HIV manifestations in pregnant women before chemoprevention, 4 weeks after its start, before delivery at 36 weeks of gestation and 1.5

psychological counseling aimed at formation of positive motivation to doctor's recommendations, following the daily regimen and regular intake of antiretroviral preparations as well as refuse of detrimental habits which is of a particular importance for



The start of antiretroviral prevention was determined by the time of the first appointment with a gynecologist for pregnancy diagnosis. Chemoprevention of vertical HIV mother-tochild virus transmission with various agents was performed in 36 women with A1 (62%) and B2 (38%) HIV stage (USA, CDC, 1987) at 23-32 gestation weeks as well as their 36 newborns. B2 stage manifested with moderate symptoms of oral mucosa candidiasis. 65% of observation group were diagnosed anemia mild to moderate degrees. No intrapartum

The group 1 included 18 pregnant HIV infected women aged 19-32 (mean age 24 years) receiving Nikavir+Epivir+Kaletra chemoprevention. The group 2 included 18 women aged 19-28 (mean age 23.5 years) receiving Combivir+Kaletra chemoprevention. From the 28th week of gestation until the delivery they received Nikavir administered in the dosage of 600

During the first hour of labor 2 mg/kg/h of zidovudine were given intravenously followed

Starting with the 8th hour of life the newborn babies were given a 6 weeks course of

Epidemiological analysis of the routes of infection revealed the leading share of sexual HIV-1 transmission: 83% of pregnant women in Nikavir chemoprevention schedule group 1 and 89% in group 2. Parenteral route was revealed in 17% and 11% of cases respectively. Thus, there was a mixed type of epidemic process, the sexual transmission rate 5-8 times

The evaluation of the results of the investigation was based on analysis of the clinical, epidemiological and laboratory monitoring of HIV infection course in pregnant women and


Clinical assessment included evaluation of HIV manifestations in pregnant women before chemoprevention, 4 weeks after its start, before delivery at 36 weeks of gestation and 1.5

Manifestations of HIV epidemic process were studied according to the following criteria:

PCR reaction for DNA HIV-1 at the age of 1.5 months and 3 months;

vg for 3 intakes daily. Combivir and Kaletra were given in standard schedule.

zidovudine in syrup (Retrovir) in the dosage of 2 mg/kg every 6 hours.


methods, chemoprevention schedules, cases of breast feeding).

surpassing the parenteral one (cases of intravenous drugs).

complications occurred. There were no cases of breast-feeding.

by 1 mg/kg/h until the end of the labor.

pregnant drug-addicts.

**5.3 Purpose** 

**5.4 Materials** 

**5.5 Methods** 

their newborns.

month after delivery. At the same terms the patients were examined by different specialists to register HIV associated diseases and adverse effects of therapy. Women were examined by infectionists, gynecologists, obstetricians, immunologists, etc. and the newborns by neonatologists, infectionists, pediatricians and other specialists according to indications.

The HIV diagnosis in women was based on enzyme immunoassay (EIA) detecting HIV antibodies ("Jenscreen Ulra HIV Ag/At") and immunoblot analysis (IMB) ("Blot-HIV") for HIV-1 virus specific proteins antibodies. HIV diagnosis in newborns was excluded on the basis of EIA and IMB monitoring during the period of observation starting at birth and thereafter at the age of 1.5 and 3 months.

Laboratory examination included leucocyte and thrombocyte counts performed with MEK-7222 hemoanalyzer and standard urinalyses. Biochemical blood values were identified with Conelab, 20 automated analyzer supplied with ion selection section for evaluation of the functional state of the liver and kidneys. The studied parameters were compared with the standards established for Perm.

The associated diseases of HIV infected pregnant women were revealed with serological IMB tests for HBsAg, hepatitis C virus, herpes simplex, cytomegalovirus, toxoplasmosis, chlamydia and Wassermann test.

Instrumental methods included ECG, ultrasonic examination abdominal and pelvic organs if indicated.

Cellular immunity in pregnant HIV infected women was assessed by the absolute and percentage levels of CD4 lymphocyte subpopulation with monoclonal antibodies ("Beston Diskinzon" USA) at "FACS Caliber" cytofluorimeter by flow cytometer method. The obtained findings were compared with the norms established by the Russian Federal AIDS Center (Pokrovsky, 2001).

Molecule-biological diagnosis in pregnant HIV infected women receiving chemoprevention was based on the detection of HIV-1 RNA plasma levels with polymerase chain reaction (PCR) and "Amplisensу HIV-monitor FRT" test-systems ("Interlabservice") before antiretroviral therapy, 4 weeks after it was started, 4 weeks before the supposed delivery term and 1.5 month after delivery.

For the early diagnosis of HIV in newborns detection of HIV-1 DNA plasma levels with PCR and "Amplisense DNA HIV-96" test-systems ("Interlabservice") was carried out. They were performed two tests at the age of 1.5 and 3 months.

Adherence to antiretroviral perinatal prevention regimen in pregnant HIV infected women was studied with the questionnaire method assessing their social profile, clinical and laboratory examinations, intake of preparations and attitude to chemoprevention.

From 28 week of gestation until the delivery they received Nikavir administered in the dosage of 600 mg for 3 intakes daily. Combivir and Kaletra were given in conventional doses. During the first hour of labor 2 mg/kg/h of zidovudine were given intravenously followed by 1 mg/kg/h within the labor.

Starting with their eighth hour of life the newborn babies were given a 6 weeks course of zidovudine in syrup (Retrovir) orally in the dose of 2 mg/kg every 6 hours.

#### **5.6 Statistical analysis**

The overall data of pregnant HIV infected women receiving the targeted agents in therapeutic doses have been statistically assessed. Descriptive and frequency ratio analyses of the total adverse events revealed within the investigation period have been performed.

Nikavir in Chemoprevention Regimens of Vertical HIV Transmission 141

In both groups no thrombopenia was noted before initiation, during and after

During pregnancy the leucocyte formula values were within the normal limits in women of

Thus, 4 weeks after the start of therapy and at 36 weeks of gestation there was no significant decrease in the peripheral blood parameters. The total of women underwent anemia preventive treatment with diet and preparations of iron in the standard daily dosing

In both groups no statistically significant difference in functional liver test values (ALT,

Assessment of chemoprevention efficiency t was based on HIV-1 RNA viral load level and the CD4 lymphocyte count before antiretroviral therapy, 4 weeks after its start, at 36 weeks

At the beginning of treatment CD4 lymphocytes values were 1.5 times lower in group 1 patients (259 and 376 cells/mm3 consequently). With the concurrent chemoprevention there was an almost double increase in group 1 prepartum CD4 lymphocytes levels (by 1.93 times) and that by 1.3 times in group 2 compared to the baseline values. After interruption of therapy in group 1 (1.5 month after deliver) CD4 lymphocytes count decreased to 321

3.9

3.6

1.5 months postpartum

**5.7.2 Immunological and virological evaluation of chemoprevention schedules** 

3.5

HAART

**Examination terms**

CBV+LPV/r Nikavir+3TC+LPV/r

Pre HAART Post 4 weeks of

Fig. 4. Erythrocyte values in pregnant HIV-infected women.

3.6

3.5

At 36 weeks of gestation

3.7

AST, bilirubin) at different terms of pregnancy was found.

of gestation and 1.5 month after delivery.

3.9

3.5

chemoprevention (Fig. 5).

both groups (Fig. 6).

schedule.

**efficiency** 

cells/mm3 (Fig. 7).

3,2 3,3 3,4 3,5 3,6 3,7 3,8 3,9 4 4,1

**Erythrocyte count** 

In considering antiretroviral efficiency the data of the number (index) of women with RNA HIV-1 levels lower than 500 cells/ml blood serum (test-system sensitivity rate) were analyzed.

The number of DNA HIV-1 negative children aged 1.5 and 3 months has been assumed to be the paramount index of antiretroviral efficiency. Safety was assessed using the mean indices of clinical and laboratory control.

## **5.7 Results**

## **5.7.1 Clinical examination; evaluation of side effects**

Tolerance of therapeutic schedules proved to be satisfactory. No marked therapy-related side effects and adverse events were revealed. Vital indices corresponded to physiological course of pregnancy.

No signs of HIV progress were noted on the clinical evaluation of women in both groups performed 1.5 month after delivery.

Parameters of side effects of chemoprevention were analyzed within the on-going clinical observation considering hemoglobin levels, erythrocyte, thrombocyte and leucocyte count values at the established terms.

Pregnancy in group 1 women was associated with the concurrent anemia grade 1-2 (mean Hb 101 g/l) while women in group 2 had normal red blood values (mean Hb 114 g/l). Following a 4 week intake of preparations hemoglobin level decreased to 109 g/l (Fig. 3).

CBV+LPV/r Nikavir+3TC+LPV/r

Erythrocyte count level parameters prior chemoprevention was 3.5x1012/l and 3.9x1012/l in group 1 and 2 respectively. After 4 weeks of therapy and at 36 weeks of gestation a certain decrease of this parameter in group 2 women was noted (Fig. 4).

In considering antiretroviral efficiency the data of the number (index) of women with RNA HIV-1 levels lower than 500 cells/ml blood serum (test-system sensitivity rate) were

The number of DNA HIV-1 negative children aged 1.5 and 3 months has been assumed to be the paramount index of antiretroviral efficiency. Safety was assessed using the mean

Tolerance of therapeutic schedules proved to be satisfactory. No marked therapy-related side effects and adverse events were revealed. Vital indices corresponded to physiological

No signs of HIV progress were noted on the clinical evaluation of women in both groups

Parameters of side effects of chemoprevention were analyzed within the on-going clinical observation considering hemoglobin levels, erythrocyte, thrombocyte and leucocyte count

Pregnancy in group 1 women was associated with the concurrent anemia grade 1-2 (mean Hb 101 g/l) while women in group 2 had normal red blood values (mean Hb 114 g/l). Following a 4 week intake of preparations hemoglobin level decreased to 109 g/l (Fig. 3).

109

103

HAART

**Examination terms**

Erythrocyte count level parameters prior chemoprevention was 3.5x1012/l and 3.9x1012/l in group 1 and 2 respectively. After 4 weeks of therapy and at 36 weeks of gestation a certain

CBV+LPV/r Nikavir+3TC+LPV/r

111

109

At 36 weeks of gestation

120

111

1.5 months postpartum

analyzed.

**5.7 Results** 

course of pregnancy.

indices of clinical and laboratory control.

performed 1.5 month after delivery.

114

101

Pre HAART Post 4 weeks of

Fig. 3. Dynamics of Hb levels in pregnant HIV-infected women.

decrease of this parameter in group 2 women was noted (Fig. 4).

90

95

100

105

110

**Hb levels**

115

120

125

values at the established terms.

**5.7.1 Clinical examination; evaluation of side effects** 

In both groups no thrombopenia was noted before initiation, during and after chemoprevention (Fig. 5).

During pregnancy the leucocyte formula values were within the normal limits in women of both groups (Fig. 6).

Thus, 4 weeks after the start of therapy and at 36 weeks of gestation there was no significant decrease in the peripheral blood parameters. The total of women underwent anemia preventive treatment with diet and preparations of iron in the standard daily dosing schedule.

In both groups no statistically significant difference in functional liver test values (ALT, AST, bilirubin) at different terms of pregnancy was found.

#### **5.7.2 Immunological and virological evaluation of chemoprevention schedules efficiency**

Assessment of chemoprevention efficiency t was based on HIV-1 RNA viral load level and the CD4 lymphocyte count before antiretroviral therapy, 4 weeks after its start, at 36 weeks of gestation and 1.5 month after delivery.

At the beginning of treatment CD4 lymphocytes values were 1.5 times lower in group 1 patients (259 and 376 cells/mm3 consequently). With the concurrent chemoprevention there was an almost double increase in group 1 prepartum CD4 lymphocytes levels (by 1.93 times) and that by 1.3 times in group 2 compared to the baseline values. After interruption of therapy in group 1 (1.5 month after deliver) CD4 lymphocytes count decreased to 321 cells/mm3 (Fig. 7).

Fig. 4. Erythrocyte values in pregnant HIV-infected women.

Nikavir in Chemoprevention Regimens of Vertical HIV Transmission 143

504

At 36 weeks of gestation

304

HAART

**Examination terms**

CBV+LPV/r Nikavir+3TC+LPV/r

<500 <sup>1034</sup>

**Examinaition terms**

CBV+LPV/r Nikavir+3TC+LPV/r

868 <500

At 36 weeks of gestation

372

376

259

Pre HAART Post 4 weeks of

Fig. 7. CD4 lymphocytes profile in pregnant women.

253226

Pre HAART Post 4 weeks of

HAART

93153

Fig. 8. Viral load profile in pregnant women.

0

50000

100000

150000

**HIV-1 RNA viral load**

200000

250000

300000

0

100

200

300

**CD4 lymphocytes count** 

400

500

600

321

1.5 months postpartum

27472

1.5 months postpartum

66965

501 484

Fig. 5. Thrombocyte count values in pregnant women.

Fig. 6. Leucocyte values in pregnant HIV-infected women.

202

HAART

7.6

HAART

**Examination terms**

CBV+LPV/r Nikavir+3TC+LPV/r

**Examination terms**

7.7 7.7

CBV+LPV/r Nikavir+3TC+LPV/r

224

226

209

Pre HAART Post 4 weeks of

Fig. 5. Thrombocyte count values in pregnant women.

Pre HAART Post 4 weeks of

Fig. 6. Leucocyte values in pregnant HIV-infected women.

6.7

8.5

0

0 1 2

**Leucocyte valeus** 

50

100

150

**Thrombocyte count valeus** 

200

250

300

243

1.5 months postpartum

5.5

1.5 months postpartum

5.8

238

226

At 36 weeks of gestation

7.3

At 36 weeks of gestation

271

Fig. 7. CD4 lymphocytes profile in pregnant women.

Fig. 8. Viral load profile in pregnant women.

Nikavir in Chemoprevention Regimens of Vertical HIV Transmission 145

Low toxicity and good tolerance of Nikavir open up new perspectives for its therapeutic application in HIV patients suffering from chronic liver diseases (Kvartchenko, 2006). Finally, some recent studies have reported on the successful application of Nikavir in the treatment of coinfections: HIV+ hepatitis C (Gankina, 2010), HIV + tuberculosis (Panteleyev, 2010). Treatment of such categories of patients is an extremely challenging task of paramount importance. At present several on-going intensive studies continue to

We would like to express our gratitude to the head doctor of Perm Regional AIDS Center, merited doctor of the Russian Federation K. Khafizov for providing an opportunity to

We would also thank pediatricians, obstetricians, gynecologists and all our colleagues who

We express our gratitude to the head of the diagnostic laboratory of Perm Center for

Also we are grateful to Alexander Kononov (the General Director of Joint-Stock Company «Association AZT») for the presented materials on stories of creation of a preparation of Nikavir and results of comparative studying embryotoxicity and teratogenicity Nikavir and

Detkova N. (2003). Chemoprevention of mother-to-child HIV transmission. *Authorized* 

Detkova N., Sokolova E., Bobkova M., et al. (2003). Nevirapin (viramun) – an agent for

Galegov G. (2004). Nikavir (phosphazide) as an antiretroviral agent: antiHIV efficiency,

Galegov G., Korneeva M., Nosyik D., et al. (1988).Azidothymidine and some of its analogues

Gankina N., Kravchenko A., Kuimova U., et al. (2010).Nucleoside inhibitors in ART schedules of HIV and chronic hepatitis C. *Infectious Diseases*, N 1, p. 14-18. Ivanova E., et al. (2006). Clinical and immunological features of HIV infection in pregnant

Ivanova E., Vorobeva N., Krasnopyerova N., et al. (2007). Prevention of HIV infection in

Ivanova E., Shapiro A., Martsenyuk M., et al. (2007). Adherence to chemoprophylaxis in

medical personnel. Scientific Session, Perm, p. 110-112.

prevention of mother-to-child transmission of HIV. *Epidemiology and Infectious* 

toxicology, pharmacology and certain perspectives of its clinical application.

women. The 7th Russia Congress of Infectionists on New technologies in diagnosis and prevention of infectious diseases, Abstract Book, Nizhny Novgorod, p. 115. Ivanova E., Vorobeva N. (2009). Nikavir in perinatal chemoprophylaxis. The IV (XIII)

International Scientific Conference Oncology – the 21st century, Hoschimin, Viet-

pregnant HIV-infected women. AIDS, *Cancer and Public Health Russia Journal*, v. 11,

*reference work of dissertation of candidate of medical sciences*, Moscow.

as inhibitors of HIV production. *Molecular Biology*, v. 2, p. 802-806.

investigate potentials of Nikavir in the treatment of coinfections.

Eradication and Prevention of AIDS and Infectious Diseases S. Zverev.

**7. Acknowledgements** 

contributed to our research.

zidovudine.

**8. References** 

conduct our research on the basis of his center.

*Diseases*, N 6, p. 55-57.

Nam, p. 88-91.

N1, p. 65-67.

*Atibiotics and Chemotherapy*, v. 49, N7, p. 3-8.

The baseline HIV-1 RNA viral load in group 1 was 253226 copies/ml of blood; 4 weeks after the start of chemoprevention it dropped by 293 times (3 lg10); it was undetectable before delivery while 1.5 month after interruption it increased to 27472 copies. At the beginning of chemoprevention HIV-1 RNA viral load in group 2 ranged between 8010 and 1 930000 copies/ml (mean 93153), it dropped to undetectable level before delivery (<500 copies/ml) and remained unchanged till delivery (Fig. 8).

## **5.7.3 Assessment of HIVchemoprophylaxis efficiency in newborns**

Efficiency assessment of chemoprevention of mother-to-child transmission of HIV infection in both groups was based on exclusion of HIV in newborns with perinatal HIV contact aged 1.5 and 3 months. All children of HIV-infected mothers were assigned to the risk category with the diagnosis of perinatal HIV contact and were examined for the presence of HIV-1 DNA using PCR at the above mentioned terms. There were no positive results.

According to the current regulations children of HIV-infected mothers are to be followed-up till the age of 18 months. At present they are under the on-going observation.

## **5.8 Summary**

Negative HIV-1 test in 100% of 3 months children born from HIV mothers is a reliable proof of a high efficiency of applied HAART schedules aimed at perinatal chemoprevention.

Efficiency of HAART schedules in vertical HIV transmission both with Nikavir+Epivir+Kaletra and combivir+Kaletra was confirmed by a significant stable decrease of viral load from the 4th week of gestation until delivery.

Increase of CD4 lymphocyte parameters in the time of chemoprevention is the evidence of the positive effect of such therapy schedules on the immune status of pregnant HIV-infected women.

A considerably high degree of adherence to chemoprevention was associated with good tolerability of the applied schedules.

Safety of HAART schedules was proved by the absence of toxic outcomes in biochemical indices in pregnant HIV-infected women at different terms of pregnancy.

However, application of Combivir+Kaletra schedule revealed the tendency to the decrease of red blood parameters (hemoglobin and erythrocytes) and thrombocytes at the 4th week of therapy and before delivery.

In HAART schedule with Nikavir+Epivir+Kaletra no decrease of hemoglobin, erythrocytes and thrombocytes counts at the fourth week of therapy and before delivery was revealed.

## **6. Conclusion**

Despite the rapid development of chemoprophylaxis of HIV infection for as many as almost 30 years the range of ART preparations used currently is limited. The most widely applied agent is zidovudine though its major recognized side effect is still hemotoxicity (in the first place anemia). As in this respect Nikavir appears to be a considerably more advantageous component of chemoprophylaxis of HIV vertical transmission comparing to zidovudine its application seems to be extremely beneficial. The above mentioned positive data on successful replacement of zidovudine in cases of its intolerance (namely, anemia cases) with Nikavir makes it the preparation of choice. Besides, there is a reported evidence from the previous comparative Nikavir - zidovudine studies of a better tolerance of Nikavir in prevention of parenteral transmission of HIV (Ivanova, 2007).

Low toxicity and good tolerance of Nikavir open up new perspectives for its therapeutic application in HIV patients suffering from chronic liver diseases (Kvartchenko, 2006).

Finally, some recent studies have reported on the successful application of Nikavir in the treatment of coinfections: HIV+ hepatitis C (Gankina, 2010), HIV + tuberculosis (Panteleyev, 2010). Treatment of such categories of patients is an extremely challenging task of paramount importance. At present several on-going intensive studies continue to investigate potentials of Nikavir in the treatment of coinfections.

## **7. Acknowledgements**

144 Understanding HIV/AIDS Management and Care – Pandemic Approaches in the 21st Century

The baseline HIV-1 RNA viral load in group 1 was 253226 copies/ml of blood; 4 weeks after the start of chemoprevention it dropped by 293 times (3 lg10); it was undetectable before delivery while 1.5 month after interruption it increased to 27472 copies. At the beginning of chemoprevention HIV-1 RNA viral load in group 2 ranged between 8010 and 1 930000 copies/ml (mean 93153), it dropped to undetectable level before delivery (<500 copies/ml)

Efficiency assessment of chemoprevention of mother-to-child transmission of HIV infection in both groups was based on exclusion of HIV in newborns with perinatal HIV contact aged 1.5 and 3 months. All children of HIV-infected mothers were assigned to the risk category with the diagnosis of perinatal HIV contact and were examined for the presence of HIV-1

According to the current regulations children of HIV-infected mothers are to be followed-up

Negative HIV-1 test in 100% of 3 months children born from HIV mothers is a reliable proof of a high efficiency of applied HAART schedules aimed at perinatal chemoprevention. Efficiency of HAART schedules in vertical HIV transmission both with Nikavir+Epivir+Kaletra and combivir+Kaletra was confirmed by a significant stable

Increase of CD4 lymphocyte parameters in the time of chemoprevention is the evidence of the positive effect of such therapy schedules on the immune status of pregnant HIV-infected

A considerably high degree of adherence to chemoprevention was associated with good

Safety of HAART schedules was proved by the absence of toxic outcomes in biochemical

However, application of Combivir+Kaletra schedule revealed the tendency to the decrease of red blood parameters (hemoglobin and erythrocytes) and thrombocytes at the 4th week of

In HAART schedule with Nikavir+Epivir+Kaletra no decrease of hemoglobin, erythrocytes and thrombocytes counts at the fourth week of therapy and before delivery was revealed.

Despite the rapid development of chemoprophylaxis of HIV infection for as many as almost 30 years the range of ART preparations used currently is limited. The most widely applied agent is zidovudine though its major recognized side effect is still hemotoxicity (in the first place anemia). As in this respect Nikavir appears to be a considerably more advantageous component of chemoprophylaxis of HIV vertical transmission comparing to zidovudine its application seems to be extremely beneficial. The above mentioned positive data on successful replacement of zidovudine in cases of its intolerance (namely, anemia cases) with Nikavir makes it the preparation of choice. Besides, there is a reported evidence from the previous comparative Nikavir - zidovudine studies of a better tolerance of Nikavir in

and remained unchanged till delivery (Fig. 8).

**5.8 Summary** 

women.

tolerability of the applied schedules.

therapy and before delivery.

**6. Conclusion** 

**5.7.3 Assessment of HIVchemoprophylaxis efficiency in newborns** 

DNA using PCR at the above mentioned terms. There were no positive results.

till the age of 18 months. At present they are under the on-going observation.

decrease of viral load from the 4th week of gestation until delivery.

indices in pregnant HIV-infected women at different terms of pregnancy.

prevention of parenteral transmission of HIV (Ivanova, 2007).

We would like to express our gratitude to the head doctor of Perm Regional AIDS Center, merited doctor of the Russian Federation K. Khafizov for providing an opportunity to conduct our research on the basis of his center.

We would also thank pediatricians, obstetricians, gynecologists and all our colleagues who contributed to our research.

We express our gratitude to the head of the diagnostic laboratory of Perm Center for Eradication and Prevention of AIDS and Infectious Diseases S. Zverev.

Also we are grateful to Alexander Kononov (the General Director of Joint-Stock Company «Association AZT») for the presented materials on stories of creation of a preparation of Nikavir and results of comparative studying embryotoxicity and teratogenicity Nikavir and zidovudine.

## **8. References**


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**Part 3** 

**Understanding Immunological** 

**Aspects of HIV in an Infected Person** 

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