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Background: Low adherence has contributed to disappointing results for trials testing vaginal microbicides for HIV prevention. This study engaged former gel trial participants to understand the reasons behind low adherence and seek suggestions on how to improve products and adherence to microbicides. This analysis examines the impact of participant perceptions of male partners on participant adherence and suggestions on how to address those issues., Methods: Eight focus group discussion workshops were conducted with 46 former microbicide trial participants in South Africa and Tanzania. Participants provided feedback on why women join trials, barriers to using gels and reporting adherence accurately, and how adherence and adherence reporting can be improved., Results: Participants reported that male partners can affect women's ability to use gels. For some, the lubricating effects caused relationship conflicts due to suspicion of male partners about infidelity. Needing to provide sex to partners on demand was a barrier to gel use. Participants suggested a gel formulation which was thicker and less noticeable, and explicit male partner engagement to enhance understanding of the purpose of the gels., Conclusions: The imbalance of power in intimate relationships affects the ability of women to use microbicides as directed. To improve adherence to HIV prevention methods within trials and for successful rollout of proven HIV prevention methods in populations, it is important that the complicated dynamics of sex and relationships be taken into greater consideration and that women receive targeted support to navigate product use and communication within the context of these gender dynamics.

Background: The incidence of human immunodeficiency virus (HIV) infection remains high among women in sub-Saharan Africa. We evaluated the safety and efficacy of extended use of a vaginal ring containing dapivirine for the prevention of HIV infection in 1959 healthy, sexually active women, 18 to 45 years of age, from seven communities in South Africa and Uganda., Methods: In this randomized, double-blind, placebo-controlled, phase 3 trial, we randomly assigned participants in a 2:1 ratio to receive vaginal rings containing either 25 mg of dapivirine or placebo. Participants inserted the rings themselves every 4 weeks for up to 24 months. The primary efficacy end point was the rate of HIV type 1 (HIV-1) seroconversion., Results: A total of 77 participants in the dapivirine group underwent HIV-1 seroconversion during 1888 person-years of follow-up (4.1 seroconversions per 100 person-years), as compared with 56 in the placebo group who underwent HIV-1 seroconversion during 917 person-years of follow-up (6.1 seroconversions per 100 person-years). The incidence of HIV-1 infection was 31% lower in the dapivirine group than in the placebo group (hazard ratio, 0.69; 95% confidence interval [CI], 0.49 to 0.99; P=0.04). There was no significant difference in efficacy of the dapivirine ring among women older than 21 years of age (hazard ratio for infection, 0.63; 95% CI, 0.41 to 0.97) and those 21 years of age or younger (hazard ratio, 0.85; 95% CI, 0.45 to 1.60; P=0.43 for treatment-by-age interaction). Among participants with HIV-1 infection, nonnucleoside reverse-transcriptase inhibitor resistance mutations were detected in 14 of 77 participants in the dapivirine group (18.2%) and in 9 of 56 (16.1%) in the placebo group. Serious adverse events occurred more often in the dapivirine group (in 38 participants [2.9%]) than in the placebo group (in 6 [0.9%]). However, no clear pattern was identified., Conclusions: Among women in sub-Saharan Africa, the dapivirine ring was not associated with any safety concerns and was associated with a rate of acquisition of HIV-1 infection that was lower than the rate with placebo. (Funded by the International Partnership for Microbicides; ClinicalTrials.gov number, NCT01539226 .).

The third variable (V3) loop and the CD4 binding site (CD4bs) of the HIV-1 envelope are frequently targeted by neutralizing antibodies (nAbs) in infected individuals. In chronic infection, HIV-1 escape mutants repopulate the plasma, and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3 and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tier 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A national ART program was launched in Tanzania in October 2004. Due to the existence of multiple HIV-1 subtypes and recombinant viruses co-circulating in Tanzania, it is important to monitor rates of drug resistance. The present study determined the prevalence of HIV-1 drug resistance mutations among ART-naive female bar and hotel workers, a high-risk population for HIV-1 infection in Moshi, Tanzania. A partial HIV-1 pol gene was analyzed by single-genome amplification and sequencing in 45 subjects (622 pol sequences total; median number of sequences per subject, 13; IQR 5-20) in samples collected in 2005. The prevalence of HIV-1 subtypes A1, C, and D, and inter-subtype recombinant viruses, was 36%, 29%, 9% and 27%, respectively. Thirteen different recombination patterns included D/A1/D, C/A1, A1/C/A1, A1/U/A1, C/U/A1, C/A1, U/D/U, D/A1/D, A1/C, A1/C, A2/C/A2, CRF10_CD/C/CRF10_CD and CRF35_AD/A1/CRF35_AD. CRF35_AD was identified in Tanzania for the first time. All recombinant viruses in this study were unique, suggesting ongoing recombination processes among circulating HIV-1 variants. The prevalence of multiple infections in this population was 16% (n = 7). Primary HIV-1 drug resistance mutations to RT inhibitors were identified in three (7%) subjects (K65R plus Y181C; N60D; and V106M). In some subjects, polymorphisms were observed at the RT positions 41, 69, 75, 98, 101, 179, 190, and 215. Secondary mutations associated with NNRTIs were observed at the RT positions 90 (7%) and 138 (6%). In the protease gene, three subjects (7%) had M46I/L mutations. All subjects in this study had HIV-1 subtype-specific natural polymorphisms at positions 36, 69, 89 and 93 that are associated with drug resistance in HIV-1 subtype B. These results suggested that HIV-1 drug resistance mutations and natural polymorphisms existed in this population before the initiation of the national ART program. With increasing use of ARV, these results highlight the importance of drug resistance monitoring in Tanzania.

Background: HIV-1 subtype C has emerged as the most prevalent strain of HIV-1 worldwide, leading to speculation that subtype C may be more transmissible than other subtypes. We compared the risk of HIV-1 transmission for subtype C versus non-C subtypes (A, D, G and recombinant forms) among heterosexual African HIV-1 serodiscordant couples., Methods: We conducted a nested case-control analysis using data from two prospective cohort studies of heterosexual HIV-1 serodiscordant couples from six countries in eastern and southern Africa. Cases (N = 121) included incident HIV-1 transmissions that were established as linked within the serodiscordant partnership by viral sequencing; controls (N = 501) were nontransmitting HIV-1-infected partners. Subtype was determined for partial env and gag genes. Multiple logistic regression controlled for age and gender of the HIV-1-nfected partner and self-reported unprotected sex. Plasma and genital HIV-1 RNA concentrations were compared between subtype C and non-C subtypes using generalized estimating equations., Results: HIV-1 subtype C was not associated with increased risk of HIV-1 transmission compared with non-C subtypes: env adjusted odds ratio (adjOR) 1.14 [95% confidence interval (CI) 0.74-1.75, P = 0.6] and gag adjOR 0.98 (95% CI 0.63-1.52, P = 0.9). Plasma and genital HIV-1 RNA levels did not differ significantly for subtype C versus non-C., Conclusion: In a geographically diverse population of heterosexual African HIV-1 serodiscordant couples, subtype C was not associated with greater risk of HIV-1 transmission compared with non-C subtypes, arguing against the hypothesis that subtype C is more transmissible compared with other common subtypes.

Background: Increased understanding of the genetic diversity of HIV-1 is challenging but important in the development of an effective vaccine. We aimed to describe the distribution of HIV-1 subtypes in northern Tanzania among women enrolled in studies preparing for HIV-1 prevention trials (hospitality facility-worker cohorts), and among men and women in an open cohort demographic surveillance system (Kisesa cohort)., Methods: The polymerase encompassing partial reverse transcriptase was sequenced and phylogenetic analysis performed and subtype determined. Questionnaires documented demographic data. We examined factors associated with subtype using multinomial logistic regression, adjusted for study, age, and sex., Results: Among 140 individuals (125 women and 15 men), subtype A1 predominated (54, 39%), followed by C (46, 33%), D (25, 18%) and unique recombinant forms (URFs) (15, 11%). There was weak evidence to suggest different subtype frequencies by study (for example, 18% URFs in the Kisesa cohort versus 5-9% in the hospitality facility-worker cohorts; adjusted relative-risk ratio (aRR) = 2.35 [95% CI 0.59,9.32]; global p = 0.09). Compared to men, women were less likely to have subtype D versus A (aRR = 0.12 [95% CI 0.02,0.76]; global p = 0.05). There was a trend to suggest lower relative risk of subtype D compared to A with older age (aRR = 0.44 [95% CI 0.23,0.85] per 10 years; global p = 0.05)., Conclusions: We observed multiple subtypes, confirming the complex genetic diversity of HIV-1 strains circulating in northern Tanzania, and found some differences between cohorts and by age and sex. This has important implications for vaccine design and development, providing opportunity to determine vaccine efficacy in diverse HIV-1 strains.

Background: Daily suppressive therapy with valacyclovir reduces risk of sexual transmission of herpes simplex virus type 2 (HSV-2) in HSV-2-serodiscordant heterosexual couples by 48%. Whether suppressive therapy reduces HSV-2 transmission from persons coinfected with HSV-2 and human immunodeficiency virus type 1 (HIV-1) is unknown., Methods: Within a randomized trial of daily acyclovir 400 mg twice daily in African HIV-1 serodiscordant couples, in which the HIV-1-infected partner was HSV-2 seropositive, we identified partnerships in which HIV-1-susceptible partners were HSV-2 seronegative to estimate the effect of acyclovir on risk of HSV-2 transmission., Results: We randomly assigned 911 HSV-2/HIV-1-serodiscordant couples to daily receipt of acyclovir or placebo. We observed 68 HSV-2 seroconversions, 40 and 28 in acyclovir and placebo groups, respectively (HSV-2 incidence, 5.1 cases per 100 person-years; hazard ratio [HR], 1.35 [95% confidence interval, .83-2.20]; P = .22). Among HSV-2-susceptible women, vaginal drying practices (adjusted HR, 44.35; P = .004) and unprotected sex (adjusted HR, 9.91; P = .002) were significant risk factors for HSV-2 acquisition; having more children was protective (adjusted HR, 0.47 per additional child; P = .012). Among HSV-2-susceptible men, only age ≤30 years was associated with increased risk of HSV-2 acquisition (P = .016)., Conclusions: Treatment of African HSV-2/HIV-1-infected persons with daily suppressive acyclovir did not decrease risk of HSV-2 transmission to susceptible partners. More-effective prevention strategies to reduce HSV-2 transmission from HIV-1-infected persons are needed.

The study estimated the prevalence of HIV-1 intra-subtype recombinant variants among female bar and hotel workers in Tanzania. While intra-subtype recombination occurs in HIV-1, it is generally underestimated. HIV-1 env gp120 V1-C5 quasispecies from 45 subjects were generated by single-genome amplification and sequencing (median (IQR) of 38 (28-50) sequences per subject). Recombination analysis was performed using seven methods implemented within the recombination detection program version 3, RDP3. HIV-1 sequences were considered recombinant if recombination signals were detected by at least three methods with p-values of ≤0.05 after Bonferroni correction for multiple comparisons. HIV-1 in 38 (84%) subjects showed evidence for intra-subtype recombination including 22 with HIV-1 subtype A1, 13 with HIV-1 subtype C, and 3 with HIV-1 subtype D. The distribution of intra-patient recombination breakpoints suggested ongoing recombination and showed selective enrichment of recombinant variants in 23 (60%) subjects. The number of subjects with evidence of intra-subtype recombination increased from 29 (69%) to 36 (82%) over one year of follow-up, although the increase did not reach statistical significance. Adjustment for intra-subtype recombination is important for the analysis of multiplicity of HIV infection. This is the first report of high prevalence of intra-subtype recombination in the HIV/AIDS epidemic in Tanzania, a region where multiple HIV-1 subtypes co-circulate. HIV-1 intra-subtype recombination increases viral diversity and presents additional challenges for HIV-1 vaccine design.

Recent data suggest that infection with human immunodeficiency virus type 1 (HIV-1) subtype C results in prolonged high-level viremia (>5 log10 copies/mL) during early infection. We examined the relationship between HIV-1 subtype and plasma viremia among 153 African seroconverters. Mean setpoint viral loads were similar for C and non-C subtypes: 4.36 vs 4.42 log10 copies/mL (P = .61). The proportion of subtype C-infected participants with viral loads >5 log10 copies/mL was not greater than the proportion for those with non-C infection. Our data do not support the hypothesis that higher early viral load accounts for the rapid spread of HIV-1 subtype C in southern Africa.

Plasma HIV-1 RNA set point is an important predictor of HIV-1 disease progression. We hypothesized that inoculum size and HIV-1 exposure prior to HIV-1 transmission may modulate set point. We evaluated predictors of set point among 141 African HIV-1 seroconverters and their HIV-1-infected study partners. We compared characteristics of seroconverters and their HIV-1-infected partners and HIV-1 set point. Data were from a clinical trial of genital HSV-2 suppression with acyclovir to reduce HIV-1 transmission in HIV-1 serodiscordant couples with HIV-1 transmission linkage assigned through virus sequencing. Our analysis includes data from all transmissions including those with transmission linkage to the HIV-1-infected "source partner" and those that were not linked to their HIV-1-infected study partner. In multivariable analysis, higher plasma HIV-1 in source partners was associated with higher seroconverter set point ( + 0.44 log10 copies/ml per log(10) source partner plasma HIV-1, p < 0.001). In addition, bacterial vaginosis (BV) among female source partners near the time of infection was associated with higher set point in their male seroconverters ( + 0.49 log(10), p = 0.04). Source partner characteristics associated with lower set point included male circumcision ( - 0.63 log(10), p = 0.03) and assignment to acyclovir ( - 0.44 log10, p = 0.02). The proportion of variation in set point explained by plasma HIV-1 RNA of the source partner, after controlling for other factors, was 0.06. Source partner plasma HIV-1 level is the most significant predictor of seroconverter set point, possibly reflecting characteristics of the transmitted virus. Acyclovir use, BV among women source partners, and circumcision among male source partners may alter the set point by affecting transmitted virus inoculum in the source partners' genital compartment.

Background: Preexposure prophylaxis with antiretroviral drugs has been effective in the prevention of human immunodeficiency virus (HIV) infection in some trials but not in others., Methods: In this randomized, double-blind, placebo-controlled trial, we assigned 2120 HIV-negative women in Kenya, South Africa, and Tanzania to receive either a combination of tenofovir disoproxil fumarate and emtricitabine (TDF-FTC) or placebo once daily. The primary objective was to assess the effectiveness of TDF-FTC in preventing HIV acquisition and to evaluate safety., Results: HIV infections occurred in 33 women in the TDF-FTC group (incidence rate, 4.7 per 100 person-years) and in 35 in the placebo group (incidence rate, 5.0 per 100 person-years), for an estimated hazard ratio in the TDF-FTC group of 0.94 (95% confidence interval, 0.59 to 1.52; P=0.81). The proportions of women with nausea, vomiting, or elevated alanine aminotransferase levels were significantly higher in the TDF-FTC group (P=0.04, P<0.001, and P=0.03, respectively). Rates of drug discontinuation because of hepatic or renal abnormalities were higher in the TDF-FTC group (4.7%) than in the placebo group (3.0%, P=0.051). Less than 40% of the HIV-uninfected women in the TDF-FTC group had evidence of recent pill use at visits that were matched to the HIV-infection window for women with seroconversion. The study was stopped early, on April 18, 2011, because of lack of efficacy., Conclusions: Prophylaxis with TDF-FTC did not significantly reduce the rate of HIV infection and was associated with increased rates of side effects, as compared with placebo. Despite substantial counseling efforts, drug adherence appeared to be low. (Supported by the U.S. Agency for International Development and others; FEM-PrEP ClinicalTrials.gov number, NCT00625404.).

Plasma from a small subset of subjects chronically infected with HIV-1 shows remarkable magnitude and breadth of neutralizing activity. From one of these individuals (CH0219), we isolated two broadly neutralizing antibodies (bnAbs), CH01 and VRC-CH31, from two clonal lineages of memory B cells with distinct specificities (variable loop 1 and 2 [V1V2] conformational specificity and CD4-binding site specificity, respectively) that recapitulate 95% of CH0219 serum neutralization breadth. These data provide proof of concept for an HIV-1 vaccine that aims to elicit bnAbs of multiple specificities.

Background: Bacterial vaginosis (BV), a disruption of the normal vaginal flora, has been associated with a 60% increased risk of HIV-1 acquisition in women and higher concentration of HIV-1 RNA in the genital tract of HIV-1-infected women. However, whether BV, which is present in up to half of African HIV-1-infected women, is associated with an increase in HIV-1 transmission to male partners has not been assessed in previous studies., Methods and Findings: We assessed the association between BV on female-to-male HIV-1 transmission risk in a prospective study of 2,236 HIV-1-seropositive women and their HIV-1 uninfected male partners from seven African countries from a randomized placebo-controlled trial that enrolled heterosexual African adults who were seropositive for both HIV-1 and herpes simplex virus (HSV)-2, and their HIV-1-seronegative partners. Participants were followed for up to 24 months; every three months, vaginal swabs were obtained from female partners for Gram stain and male partners were tested for HIV-1. BV and normal vaginal flora were defined as a Nugent score of 7-10 and 0-3, respectively. To reduce misclassification, HIV-1 sequence analysis of viruses from seroconverters and their partners was performed to determine linkage of HIV-1 transmissions. Overall, 50 incident HIV-1 infections occurred in men in which the HIV-1-infected female partner had an evaluable vaginal Gram stain. HIV-1 incidence in men whose HIV-1-infected female partners had BV was 2.91 versus 0.76 per 100 person-years in men whose female partners had normal vaginal flora (hazard ratio 3.62, 95% CI 1.74-7.52). After controlling for sociodemographic factors, sexual behavior, male circumcision, sexually transmitted infections, pregnancy, and plasma HIV-1 RNA levels in female partners, BV was associated with a greater than 3-fold increased risk of female-to-male HIV-1 transmission (adjusted hazard ratio 3.17, 95% CI 1.37-7.33)., Conclusions: This study identified an association between BV and increased risk of HIV-1 transmission to male partners. Several limitations may affect the generalizability of our results including: all participants underwent couples HIV counseling and testing and enrolled in an HIV-1 prevention trial, and index participants had a baseline CD4 count ≥ 250 cells/mm³ and were HSV-2 seropositive. Given the high prevalence of BV and the association of BV with increased risk of both female HIV-1 acquisition and transmission found in our study, if this association proves to be causal, BV could be responsible for a substantial proportion of new HIV-1 infections in Africa. Normalization of vaginal flora in HIV-1-infected women could mitigate female-to-male HIV-1 transmission., Trial Registration: ClinicalTrials.com NCT00194519.

This study analyzed the distribution and prevalence of HIV-1 subtypes, multiplicity of HIV-1 infection, and frequency of inter-subtype recombination among HIV-1-infected female bar and hotel workers in Moshi, Kilimanjaro Region, Tanzania, from 2004 to 2007. The HIV-1 viral sequences spanning the V1-C5 region of HIV-1 env gp120 were analyzed from 50 subjects by single genome amplification and sequencing (SGA/S) technique. A total of 1740 sequences were amplified and sequenced from the HIV-1 proviral DNA template. The median env sequences analyzed per subject per two time points was 38 (IQR 28-50) over one year of HIV infection. In a subset of 14 subjects, a total of 239 sequences were obtained from HIV-1 RNA template at the baseline visit. The most prevalent HIV-1 subtypes were A1 (56%) and C (30%), while HIV-1 subtype D and inter-subtype recombinant viruses were found in 6% and 8% of subjects respectively. Transmission of multiple HIV-1 variants was evident in 27% of the subjects infected with pure HIV-1 subtypes A1, C, or D. The HIV-1 inter-subtype recombinants were found in 8% including HIV-1 C/A, D/A, and complex mosaic recombinants. Multiple viral variants were found in two subjects infected with inter-subtype recombinants. One subject harbored quasispecies of both pure HIV-1 A1 and C/A recombinant. The other subject was infected with two complex mosaic inter-subtype recombinant variants belonging to subtype D. HIV-1 multiple infections and ongoing recombination contribute significantly to the genetic diversity of circulating HIV-1 in Tanzania and have important implications for vaccine design and the development of therapeutic strategies.

Variable regions 1 and 2 (V1/V2) of human immunodeficiency virus-1 (HIV-1) gp120 envelope glycoprotein are critical for viral evasion of antibody neutralization, and are themselves protected by extraordinary sequence diversity and N-linked glycosylation. Human antibodies such as PG9 nonetheless engage V1/V2 and neutralize 80% of HIV-1 isolates. Here we report the structure of V1/V2 in complex with PG9. V1/V2 forms a four-stranded β-sheet domain, in which sequence diversity and glycosylation are largely segregated to strand-connecting loops. PG9 recognition involves electrostatic, sequence-independent and glycan interactions: the latter account for over half the interactive surface but are of sufficiently weak affinity to avoid autoreactivity. The structures of V1/V2-directed antibodies CH04 and PGT145 indicate that they share a common mode of glycan penetration by extended anionic loops. In addition to structurally defining V1/V2, the results thus identify a paradigm of antibody recognition for highly glycosylated antigens, which-with PG9-involves a site of vulnerability comprising just two glycans and a strand.

V2/V3 conformational epitope antibodies that broadly neutralize HIV-1 (PG9 and PG16) have been recently described. Since an elicitation of previously known broadly neutralizing antibodies has proven elusive, the induction of antibodies with such specificity is an important goal for HIV-1 vaccine development. A critical question is which immunogens and vaccine formulations might be used to trigger and drive the development of memory B cell precursors with V2/V3 conformational epitope specificity. In this paper we identified a clonal lineage of four V2/V3 conformational epitope broadly neutralizing antibodies (CH01 to CH04) from an African HIV-1-infected broad neutralizer and inferred their common reverted unmutated ancestor (RUA) antibodies. While conformational epitope antibodies rarely bind recombinant Env monomers, a screen of 32 recombinant envelopes for binding to the CH01 to CH04 antibodies showed monoclonal antibody (MAb) binding to the E.A244 gp120 Env and to chronic Env AE.CM243; MAbs CH01 and CH02 also bound to transmitted/founder Env B.9021. CH01 to CH04 neutralized 38% to 49% of a panel of 91 HIV-1 tier 2 pseudoviruses, while the RUAs neutralized only 16% of HIV-1 isolates. Although the reverted unmutated ancestors showed restricted neutralizing activity, they retained the ability to bind to the E.A244 gp120 HIV-1 envelope with an affinity predicted to trigger B cell development. Thus, E.A244, B.9021, and AE.CM243 Envs are three potential immunogen candidates for studies aimed at defining strategies to induce V2/V3 conformational epitope-specific antibodies.

Antibody VRC01 is a human immunoglobulin that neutralizes about 90% of HIV-1 isolates. To understand how such broadly neutralizing antibodies develop, we used x-ray crystallography and 454 pyrosequencing to characterize additional VRC01-like antibodies from HIV-1-infected individuals. Crystal structures revealed a convergent mode of binding for diverse antibodies to the same CD4-binding-site epitope. A functional genomics analysis of expressed heavy and light chains revealed common pathways of antibody-heavy chain maturation, confined to the IGHV1-2*02 lineage, involving dozens of somatic changes, and capable of pairing with different light chains. Broadly neutralizing HIV-1 immunity associated with VRC01-like antibodies thus involves the evolution of antibodies to a highly affinity-matured state required to recognize an invariant viral structure, with lineages defined from thousands of sequences providing a genetic roadmap of their development.

Background: Innovative prevention strategies for HIV-1 transmission are urgently needed. PRO2000 vaginal gel was efficacious against HIV-1 transmission in studies in macaques; we aimed to assess efficacy and safety of 2% and 0·5% PRO2000 gels against vaginal HIV-1 transmission in women in sub-Saharan Africa., Methods: Microbicides Development Programme 301 was a phase 3, randomised, double-blind, parallel-group trial, undertaken at 13 clinics in South Africa, Tanzania, Uganda, and Zambia. We randomly assigned sexually active women, aged 18 years or older (≥16 years in Tanzania and Uganda) without HIV-1 infection in a 1:1:1 ratio to 2% PRO2000, 0·5% PRO2000, or placebo gel groups for 52 weeks (up to 104 weeks in Uganda). Randomisation was done by computerised random number generator. Investigators and participants were masked to group assignment. The primary efficacy outcome was incidence of HIV-1 infection before week 52, which was censored for pregnancy and excluded participants without HIV-1 follow-up data or with HIV-1 infection at enrolment. HIV-1 status was established by rapid tests or ELISA at screening at 12 weeks, 24 weeks, 40 weeks, and 52 weeks, and confirmed in a central reference laboratory. The primary safety endpoint was an adverse event of grade 3 or worse. Use of 2% PRO2000 gel was discontinued on Feb 14, 2008, on the recommendation of the Independent Data Monitoring Committee because of low probability of benefit. This trial is registered at http://isrctn.org, number ISRCTN 64716212., Findings: We enrolled 9385 of 15 818 women screened. 2591 (95%) of 2734 participants enrolled to the 2% PRO2000 group, 3156 (95%) of 3326 in the 0·5% PRO2000 group, and 3112 (94%) of 3325 in the placebo group were included in the primary efficacy analysis. Mean reported gel use at last sex act was 89% (95% CI 86-91). HIV-1 incidence was much the same between groups at study end (incidence per 100 woman-years was 4·5 [95% CI 3·8-5·4] for 0·5% PRO2000 vs 4·3 [3·6-5·2] for placebo, hazard ratio 1·05 [0·82-1·34], p=0·71), and at discontinuation (4·7 [3·8-5·8] for 2% PRO2000 gel, 3·9 [3·0-4·9] for 0·5% PRO2000 gel, and 3·9 [3·1-5·0] for placebo gel). Incidence of the primary safety endpoint at study end was 4·6 per 100 woman-years (95% CI 3·9-5·4) in the 0·5% PRO2000 group and 3·9 (3·2-4·6) in the placebo group; and was 4·5 (3·7-5·5) in the 2% PRO2000 group at discontinuation., Interpretation: Although safe, 0·5% PRO2000 and 2% PRO2000 are not efficacious against vaginal HIV-1 transmission and are not indicated for this use., Funding: UK Department for International Development, UK Medical Research Council, European and Developing Countries Clinical Trials Partnership, International Partnership for Microbicides, and Endo Pharmaceuticals Solutions., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Previous papers in this supplement have reviewed the evidence of the effectiveness of alternative HIV prevention methods from randomized controlled trials and other studies. This paper draws together the main conclusions from these reviews. A conceptual framework is presented that maps the proximal and distal determinants of sexual HIV transmission and helps to identify the stages in the causal pathway at which each intervention approach acts. The advances, gaps and challenges emerging from the reviews of individual intervention methods are summarized and cross-cutting themes identified. Approximately 90% of HIV prevention trials have found no effect on HIV incidence and we explore the alternative explanations for the large number of 'flat' trials. We conclude that there is no single explanation for these flat results, which may be due to interventions that are ineffective or inappropriately targeted or implemented, or to factors related to the design or conduct of trials. We examine the lessons from these flat results and provide recommendations on what should be done differently in future trials. HIV prevention remains of critical importance in an era of expanded delivery of antiretroviral therapy. In future HIV prevention research, it is important that resources are used as efficiently as possible to provide rigorous evidence of the effectiveness of a wider array of complementary prevention tools.

Objective: Male circumcision reduces female-to-male HIV-1 transmission risk by approximately 60%. Data assessing the effect of circumcision on male-to-female HIV-1 transmission are conflicting, with one observational study among HIV-1-serodiscordant couples showing reduced transmission but a randomized trial suggesting no short-term benefit of circumcision., Design/methods: Data collected as part of a prospective study among African HIV-1-serodiscordant couples were analyzed for the relationship between circumcision status of HIV-1-seropositive men and risk of HIV-1 acquisition among their female partners. Circumcision status was determined by physical examination. Cox proportional hazards analysis was used., Results: A total of 1096 HIV-1-serodiscordant couples in which the male partner was HIV-1-infected were followed for a median of 18 months; 374 (34%) male partners were circumcised. Sixty-four female partners seroconverted to HIV-1 (incidence 3.8 per 100 person-years). Circumcision of the male partner was associated with a nonstatistically significant approximately 40% lower risk of HIV-1 acquisition by the female partner (hazard ratio 0.62, 95% confidence interval 0.35-1.10, P = 0.10). The magnitude of this effect was similar when restricted to the subset of HIV-1 transmission events confirmed by viral sequencing to have occurred within the partnership (n = 50, hazard ratio 0.57, P = 0.11), after adjustment for male partner plasma HIV-1 concentrations (hazard ratio 0.60, P = 0.13), and when excluding follow-up time for male partners who initiated antiretroviral therapy (hazard ratio 0.53, P = 0.07)., Conclusion: Among HIV-1-serodiscordant couples in which the HIV-1-seropositive partner was male, we observed no increased risk and potentially decreased risk from circumcision on male-to-female transmission of HIV-1.

Background: Most people infected with HIV-1 are dually infected with herpes simplex virus type 2. Daily suppression of this herpes virus reduces plasma HIV-1 concentrations, but whether it delays HIV-1 disease progression is unknown. We investigated the effect of acyclovir on HIV-1 progression., Methods: In a trial with 14 sites in southern Africa and east Africa, 3381 heterosexual people who were dually infected with herpes simplex virus type 2 and HIV-1 were randomly assigned in a 1:1 ratio to acyclovir 400 mg orally twice daily or placebo, and were followed up for up to 24 months. Eligible participants had CD4 cell counts of 250 cells per mL or higher and were not taking antiretroviral therapy. We used block randomisation, and patients and investigators were masked to treatment allocation. Effect of acyclovir on HIV-1 disease progression was defined by a primary composite endpoint of first occurrence of CD4 cell counts of fewer than 200 cells per microL, antiretroviral therapy initiation, or non-trauma related death. As an exploratory analysis, we assessed the endpoint of CD4 falling to <350 cells per microL. Analysis was by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT00194519., Findings: At enrollment, the median CD4 cell count was 462 cells per microL and median HIV-1 plasma RNA was 4.1 log(10) copies per microL. Acyclovir reduced risk of HIV-1 disease progression by 16%; 284 participants assigned acyclovir versus 324 assigned placebo reached the primary endpoint (hazard ratio [HR] 0.84, 95% CI 0.71-0.98, p=0.03). In those with CD4 counts >or=350 cells per microL, aciclovir delayed risk of CD4 cell counts falling to <350 cells per microL by 19% (0.81, 0.71-0.93, p=0.002), Interpretation: The role of suppression of herpes simplex virus type 2 in reduction of HIV-1 disease progression before initiation of antiretroviral therapy warrants consideration., Funding: Bill & Melinda Gates Foundation., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Background: The Partners HSV-2/HIV-1 Transmission Study (Partners Study) is a phase III, placebo-controlled trial of daily acyclovir for genital herpes (HSV-2) suppression among HIV-1/HSV-2 co-infected persons to reduce HIV-1 transmission to their HIV-1 susceptible partners, which requires recruitment of HIV-1 serodiscordant heterosexual couples. We describe the baseline characteristics of this cohort., Methods: HIV-1 serodiscordant heterosexual couples, in which the HIV-1 infected partner was HSV-2 seropositive, had a CD4 count >or=250 cells/mcL and was not on antiretroviral therapy, were enrolled at 14 sites in East and Southern Africa. Demographic, behavioral, clinical and laboratory characteristics were assessed., Results: Of the 3408 HIV-1 serodiscordant couples enrolled, 67% of the HIV-1 infected partners were women. Couples had cohabitated for a median of 5 years (range 2-9) with 28% reporting unprotected sex in the month prior to enrollment. Among HIV-1 susceptible participants, 86% of women and 59% of men were HSV-2 seropositive. Other laboratory-diagnosed sexually transmitted infections were uncommon (<5%), except for Trichomonas vaginalis in 14% of HIV-1 infected women. Median baseline CD4 count for HIV-1 infected participants was 462cells/mcL and median HIV-1 plasma RNA was 4.2 log(10) copies/mL. After adjusting for age and African region, correlates of HIV-1 RNA level included male gender (+0.24 log(10) copies/mL; p<0.001) and CD4 count (-0.25 and -0.55 log(10) copies/mL for CD4 350-499 and >500 relative to <350, respectively, p<0.001)., Conclusions: The Partners Study successfully enrolled a cohort of 3408 heterosexual HIV-1 serodiscordant couples in Africa at high risk for HIV-1 transmission. Follow-up of this cohort will evaluate the efficacy of acyclovir for HSV-2 suppression in preventing HIV-1 transmission and provide insights into biological and behavioral factors determining heterosexual HIV-1 transmission., Trial Registration: ClinicalTrials.gov NCT00194519.

Objective: To examine the relationship between multiple dimensions of socioeconomic status (SES) and HIV seroprevalence in Tanzania., Methods: Using a large nationally representative sample of 7515 sexually active adults drawn from the 2003-04 Tanzania HIV/AIDS Indicator Survey, we analysed the relationship between multiple SES measures and HIV seroprevalence using weighted logistic regression models., Results: In adjusted models, individuals in the highest quintile of standard of living had increased odds ratio (OR) of being HIV-positive (male: OR 2.38, 95% CI 1.17-4.82; female: OR 3.74, 95% CI 2.16-6.49). Occupational status was differentially associated with HIV in men and women; women in professional jobs had higher OR of being HIV-positive (OR 1.54, 95% CI 1.02-2.38), whereas unemployed men had higher risk of being HIV-positive (OR 3.49, 95% CI 1.43-8.58). No marked association was found between increasing education and HIV seroprevalence for men (P = 0.83) and women (P = 0.87)., Conclusion: Contrary to the prevailing perception that low SES individuals tend to be more vulnerable to HIV-infection, we found a positive association between standard of living and HIV-infection. Strategies aimed at reducing HIV-infection needs to be cognizant of the complex social heterogeneity in the patterns of HIV-infection.

Objective: To examine the extent to which the regional and neighborhood distribution of HIV in Tanzania is caused by the differential distribution of individual correlates and risk factors., Methods: Nationally representative, cross-sectional data on 12,522 women and men aged 15-49 years from the 2003-2004 Tanzanian AIDS Indicator Survey. Three-level multilevel binary logistic regression models were specified to estimate the relative contribution of regions and neighborhoods to the variation in HIV seroprevalence., Results: Spatial distribution of individual correlates (and risk factors) of HIV do not explain the neighborhood and regional variation in HIV seroprevalence. Neighborhoods and regions accounted for approximately 14 and 6% of the total variation in HIV. HIV prevalence ranged from 1.8% (Kigoma) to 6.7% (Iringa) even after adjusting for the compositional make-up of these regions. An inverse association was observed between log odds of being HIV positive and neighborhood poverty [odds ratio (OR) 0.24, 95% confidence interval (CI) 0.09-0.61] and regional poverty (OR 0.97, 95% CI 0.95-0.99)., Conclusion: Our study provides evidence for independent contextual variations in HIV, above and beyond that which can be ascribed to geographical variations in individual-level correlates and risk factors. We emphasize the need to adopt both a group-based and a place-based approach, as opposed to the dominant high-risk group approach, for understanding the epidemiology of HIV as well as for developing HIV intervention activities.

Background: We examined the role of herpes simplex virus type 2 (HSV-2) and other genital infections on human immunodeficiency virus type 1 (HIV-1) incidence in a cohort study conducted between 2002 and 2005 among female bar/hotel workers in Moshi, Tanzania., Methods: At baseline and every 3 months thereafter, participants were interviewed, and blood and genital samples were collected. Predictors of HIV-1 incidence were evaluated using a Cox proportional hazards regression model., Results: Of 845 women who were HIV-1 seronegative at baseline, 689 (81.5%) were monitored in the study for a total of 698.6 person-years at risk (PYARs). The overall HIV-1 incidence was 4.6/100 PYARs (95% confidence interval [CI], 3.0-6.2/100 PYARs), and condom use was very low. After adjustment for other risk factors, the risk of HIV-1 was increased among women with HSV-2 at baseline (hazard ratio [HR], 4.3 [95% CI, 1.5-12.4]) and in those who acquired HSV-2 during the study period (HR, 5.5 [95% CI, 1.2-25.4]). Other independent predictors of HIV-1 were baseline chlamydial infection (HR, 5.2), bacterial vaginosis (HR, 2.1), and the occurrence of genital ulcers (HR, 2.7)., Conclusion: HSV-2 and other genital infections were the most important risk factors for HIV-1. Control of these infections could help to reduce HIV-1 incidence in this population.

Results from a randomized clinical trial suggested that vitamin A/beta-carotene supplementation to HIV-1-infected women during pregnancy and lactation may increase the risk of vertical transmission. Limited information is available on the potential role of vitamin A on heterosexual HIV transmission. This is a relevant question in many resource-limited settings where both vitamin A deficiency and HIV infection are highly prevalent. We conducted a case-control study (34 cases and 38 controls) nested within a cohort of HIV-negative women attending family planning clinics in Tanzania, to examine whether low serum concentrations at baseline were associated with the risk of seroconversion. There was not a significant association (OR = 2.14, 95% C I = 0.54, 8.45). In light of these and previous results, we conclude that there is not enough evidence yet to suggest a causal association between vitamin A and heterosexual transmission.

We recently identified an HIV-1 subtype C and D circulating recombinant form (CRF10&#95;CD) in infants in Dar es Salaam, Tanzania. So far, this is the only reported HIV-1 CRF in East Africa. However, evidence for its spread in the adult population is scarce. Here we describe the presence of CRF10&#95;CD in two asymptomatic bar and hotel workers in Moshi, Northern Tanzania. Subgenomic sequences from gag (3'p24-5'p7), env (C2-C5), and the 5' LTR were used for phylogenetic analysis and identification of recombination. Genetic divergence between the CRF10&#95;CD sequences from Moshi suggested that they were contracted from independent sources. A third bar worker was infected with an apparent CRF10&#95;CD/subtype A recombinant virus. Our data indicate that CRF10&#95;CD genomes can be transmitted both vertically and heterosexually.

During the past year, a group has argued that unsafe injections are a major if not the main mode of HIV-1 transmission in sub-Saharan Africa. We review the main arguments used to question the epidemiological interpretations on the lead role of unsafe sex in HIV-1 transmission, and conclude there is no compelling evidence that unsafe injections are a predominant mode of HIV-1 transmission in sub-Saharan Africa. Conversely, though there is a clear need to eliminate all unsafe injections, epidemiological evidence indicates that sexual transmission continues to be by far the major mode of spread of HIV-1 in the region. Increased efforts are needed to reduce sexual transmission of HIV-1.

The HIV-1 prevalence among bar and hotel workers in Tanzania suggests they are a high-risk group for HIV-1 infection. We determined the HIV-1 subtype of 3'-p24/5'-p7 gag and C2-C5 env sequences from 40 individuals representing this population in Moshi. Genetic patterns composed of A(gag)-A(env), C(gag)-C(env), and D(gag)-D(env) were found in 19 (48.0%), 8 (20.0%), and 3 (8.0%) samples, respectively. The remaining 10 samples (25%) had different subtypes in gag and env, indicative of intersubtype recombinants. Among these recombinants, two contained sequences from HIV-1 subsubtype A2, a new genetic variant in Tanzania. Five bar and hotel workers may have been infected with viruses from a common source, based on phylogenetic analysis. The information obtained by surveillance of HIV-1 subtypes in a high-risk population should be useful in the design and evaluation of behavioral, therapeutic, and vaccine trial interventions aimed at reducing HIV-1 transmission.

Background: Transmission of HIV-1 through breastfeeding is a major problem, although its timing is not well characterized., Methods: The authors examined the timing and correlates of HIV-1 transmission through breastfeeding among 1078 HIV-infected pregnant women from Dar es Salaam, Tanzania enrolled in a trial to examine the effect of vitamin A and other vitamin supplements on mother-to-child transmission of HIV-1 and other health outcomes. Cumulative incidence was measured among children of women not randomized to vitamin A (n = 312), given the higher risk of infection observed among those in the vitamin A arm. For analyses of correlates, data from all children not infected by age 6 weeks were used (p = 659)., Results: Mean duration of breastfeeding was 20.3 months (SD = 4.4 months; median = 20.5 months). Thirty-seven infections were observed during 4372 child-months of follow-up evaluation, or 10.2 cases per 100 child-years. Infection risk by age 4 months was 3.8% (95% confidence interval [CI], 1.6%-6.1%) and increased to 17.9% (95% CI, 11.2%-24.5%) by age 24 months. In a multivariate proportional hazards model, high maternal viral load (p =.0001), low CD4 cell count (p =.004), and high maternal erythrocyte sedimentation rate (ESR; p=.004) were significant predictors of transmission of HIV-1 through breastfeeding. Mothers who had breast lesions during pregnancy were 2.00 times more likely to transmit the virus during breastfeeding than mothers without these lesions (95% CI, 1.29-3.08; p=.002)., Conclusions: The rate of breastfeeding transmission of HIV-1 is high, and early weaning is likely to be associated with reduced transmission. Antiretroviral drugs given to HIV-infected mothers are likely to reduce the risk of breastfeeding transmission. In their absence, interventions that enhance immune reconstitution, such as micronutrient supplements, may be beneficial against transmission. Methods to prevent and treat nipple cracks and mastitis may also be important.

Background: HIV-1 transmission through breastfeeding is a global problem and has been associated with poor maternal micronutrient status., Methods: A total of 1078 HIV-infected pregnant women from Tanzania were randomly assigned to vitamin A or multivitamins excluding A from approximately 20 weeks' gestation and throughout lactation., Results: Multivitamins excluding A had no effect on the total risk of HIV-1 transmission (RR 1.04, 95% CI 0.82-1.32, P= 0.76). Vitamin A increased the risk of transmission (RR 1.38, 95% CI 1.09-1.76, P = 0.009). Multivitamins were associated with non-statistically significant reductions in transmission through breastfeeding, and mortality by 24 months among those alive and not infected at 6 weeks. Multivitamins significantly reduced breastfeeding transmission in infants of mothers with low baseline lymphocyte counts (RR 0.37; 95% CI 0.16-0.85, P = 0.02) compared with infants of mothers with higher counts (RR 0.99, 95% CI 0.68-1.45, P = 0.97; -for-interaction 0.03). Multivitamins also protected against transmission among mothers with a high erythrocyte sedimentation rate (P-for-interaction 0.06), low hemoglobin (P-for-interaction 0.06), and low birthweight babies (P-for-interaction 0.04). Multivitamins reduced death and prolonged HIV-free survival significantly among children born to women with low maternal immunological or nutritional status. Vitamin A alone increased breastfeeding transmission but had no effect on mortality by 24 months., Conclusion: Vitamin A increased the risk of HIV-1 transmission. Multivitamin (B, C, and E) supplementation of breastfeeding mothers reduced child mortality and HIV-1 transmission through breastfeeding among immunologically and nutritionally compromised women. The provision of these supplements to HIV-infected lactating women should be considered.

This study was conducted to determine the predictors of plasma HIV-1 RNA viral load in HIV-1-positive pregnant women (N = 151) participating in a clinical trial in Tanzania. Viral load was measured at randomization, delivery, and approximately 7 months after delivery. The median viral load was 20,400 copies/mL at baseline, 20,216 copies/mL at delivery, and 19,100 copies/mL 7 months after delivery. The absolute CD4+ lymphocyte count had a strong negative correlation with HIV-1 RNA viral load at baseline (r = -.38), time of delivery (r = -.36), and 7 months after delivery (r = -.53). The association between CD4+ lymphocyte count and HIV-1 RNA viral load was modified by the per capita daily food expenditure in the household, although the difference in viral load became small as the food expenditure in the household increased and was marginally significant at the 75th percentile of the per capita food expenditure. The presence of malaria parasites at baseline was associated with an approximate 116% higher viral load at the three evaluation points (p =.007). Although the long-term effects of malaria on viral load are unknown, prevention of malaria among people living with HIV-1 should be given the highest priority.

We conducted this study to determine the prevalence and risk factors for HIV-1 infection among women (N = 312) who were working in the bars and hotels in Moshi, a town in northern Tanzania. Study subjects were interviewed to obtain information about HIV-1 risk factors and examined to collect samples for the diagnosis of sexually transmitted diseases (STDs). The prevalence of HIV-1 was 26.3% (95% confidence interval [CI], 21.4%-31.2%). In multivariate analyses, the risk of HIV-1 increased with increasing age (p value, test for linear trend <.001) and the number of sexual partners during the last 5 years (p value, test for linear trend <.03). Other significant predictors were having a male partner with other sexual partners (Adjusted odds ratio [AOR], 1.92; 95% CI, 1.03-3.60), and consuming alcohol >2 days per week (AOR, 2.56; 95% CI, 1.12-5.88). The risk of HIV-1 was also significantly increased in women with bacterial vaginosis (AOR, 2.37; 95% CI, 1.09-5.13) and in study subjects with herpes simplex virus (HSV)-2 antibodies (AOR, 2.48; 95% CI, 1.24-4.98). These results indicate that women working in these settings were at increased risk of HIV-1. Programs aiming at promoting safer sexual practices and control of other STDs are urgently needed in this population. Such programs should address the underlying conditions that facilitate risk behaviors and create obstacles for these women who wish to protect themselves against HIV-1.

We recently reported that the risk of sexually acquired HIV-1 infection is increased significantly by variants in the gene encoding CD101, a protein thought to modify inflammatory responses. Using blood samples from individuals with and without these variants, we demonstrate that CD101 variants modify the prevalence of circulating inflammatory cell types and show that CD101 variants are associated with increased proinflammatory cytokine production by circulating T cells. One category of CD101 variants is associated with a reduced capacity of regulatory T cells to suppress T cell cytokine production, resulting in a reduction in the baseline level of immune quiescence. These data are supported by transcriptomics data revealing alterations in the intrinsic regulation of antiviral pathways and HIV resistance genes in individuals with CD101 variants. Our data support the hypothesis that CD101 contributes to homeostatic regulation of bystander inflammation, with CD101 variants altering heterosexual HIV-1 acquisition by facilitating increased prevalence and altered function of T cell subsets.

BackgroundA heightened proinflammatory state has been hypothesized to enhance human immunodeficiency virus type 1 (HIV-1) transmission - both susceptibility of HIV-1-exposed persons and infectiousness of HIV-1-infected persons.MethodsUsing prospective data from heterosexual African couples with HIV-1 serodiscordance, we conducted a nested case-control analysis to assess the relationship between cytokine concentrations and the risk of HIV-1 acquisition. Case couples (n = 120) were initially serodiscordant couples in which HIV-1 was transmitted to the seronegative partner during the study; control couples (n = 321) were serodiscordant couples in which HIV-1 was not transmitted to the seronegative partner. Differences in a panel of 30 cytokines were measured using plasma specimens from both HIV-1-susceptible and HIV-1-infected partners. Plasma was collected before seroconversion for cases.ResultsFor both HIV-1-infected and HIV-1-susceptible partners, cases and controls had significantly different mean responses in cytokine panels (P < .001, by the Hotelling T(2) test), suggesting a broadly different pattern of immune activation for couples in which HIV-1 was transmitted, compared with couples without transmission. Individually, log10 mean concentrations of interleukin 10 (IL-10) and CXCL10 were significantly higher for both HIV-1-susceptible and HIV-1-infected case partners, compared with HIV-1-susceptible and HIV-1-infected control partners (P < .01 for all comparisons). In multivariate analysis, HIV-1 transmission was significantly associated with elevated CXCL10 concentrations in HIV-1-susceptible partners (P = .001) and with elevated IL-10 concentrations in HIV-1-infected partners (P = .02).ConclusionsImmune activation, as measured by levels of cytokine markers, particularly elevated levels of IL-10 and CXCL1, are associated with increased HIV-1 susceptibility and infectiousness.