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1.  Impact of community antiretroviral therapy coverage on HIV incidence in Kenyan female sex workers: A fifteen year prospective cohort study 
AIDS (London, England)  2015;29(17):2279-2286.
Objective
To test the hypothesis that increasing community ART coverage would be associated with lower HIV incidence in female sex workers (FSWs) in Mombasa District, Kenya.
Design
Prospective cohort study.
Methods
From 1998–2012, HIV-negative FSWs were asked to return monthly for an interview regarding risk behavior and testing for sexually transmitted infections including HIV. We evaluated the association between community ART coverage and FSW’s risk of becoming HIV infected using Cox proportional hazards models adjusted for potential confounding factors.
Results
1,404 FSWs contributed 4,335 woman-years of follow-up, with 145 acquiring HIV infection (incidence 3.35/100 woman-years). The ART rollout began in 2003. By 2012, an estimated 52% of HIV-positive individuals were receiving treatment. Community ART coverage was inversely associated with HIV incidence (adjusted hazard ratio [aHR] 0.77; 95% confidence interval [95%CI] 0.61–0.98; p=0.03), suggesting that each 10% increase in coverage was associated with a 23% reduction in FSWs’ risk of HIV acquisition. Community ART coverage had no impact on HSV-2 incidence (aHR 0.97; 95%CI 0.79–1.20; p=0.8).
Conclusions
Increasing general population ART coverage was associated with lower HIV incidence in FSWs. The association with HIV incidence, but not HSV-2 incidence, suggests that the effect of community ART coverage may be specific to HIV. Interventions such as pre-exposure prophylaxis and antiretroviral-containing microbicides have produced disappointing results in HIV prevention trials with FSWs. These results suggest that FSWs’ risk of acquiring HIV infection might be reduced through the indirect approach of increasing ART coverage in the community.
doi:10.1097/QAD.0000000000000829
PMCID: PMC4640974  PMID: 26237099
Antiretroviral therapy; HIV; transmission; women; sex worker; Africa
2.  HIV Transmission Biology: Translation for HIV Prevention 
AIDS (London, England)  2015;29(17):2219-2227.
Rigorous testing of new HIV prevention strategies is a time-consuming and expensive undertaking. Thus, making well-informed decisions on which candidate prevention approaches are most likely to provide the most benefit is critical to appropriately prioritizing clinical testing. In the case of biological interventions, the decision to test a given prevention approach in human trials rests largely on evidence of protection in pre-clinical studies. The ability of pre-clinical studies to predict efficacy in humans may depend on how well the model recapitulates key biological features of HIV transmission relevant to the question at hand. Here, we review our current understanding of the biology of HIV transmission based on data from animal models, cell culture, and viral sequence analysis from human infection. We summarize studies of the bottleneck in viral transmission; the characteristics of transmitted viruses; the establishment of infection; and the contribution of cell-free and cell-associated virus. We seek to highlight the implications of HIV transmission biology for development of prevention interventions, and to discuss the limitations of existing pre-clinical models.
doi:10.1097/QAD.0000000000000845
PMCID: PMC4640979  PMID: 26418086
HIV transmission; HIV prevention; Early viral events; Transmission bottleneck; Transmitted viruses
3.  HIV Acquisition During Pregnancy and Postpartum is Associated with Genital Infections and Partnership Characteristics: A Cohort Study 
AIDS (London, England)  2015;29(15):2025-2033.
Objective
To determine the risk and cofactors for HIV acquisition during pregnancy and postpartum.
Design
Prospective cohort study
Methods
Pregnant women in western Kenya were enrolled if HIV seronegative at that visit or within 3 months. Serial HIV nucleic acid amplification tests (NAATs) were conducted at 1–3 month intervals to 9 months postpartum. Genital swabs were collected for detection of chlamydia and gonorrhea at baseline, and for trichomonas, bacterial vaginosis (BV), and yeast at baseline and follow-up.
Results
Among 1304 pregnant women, median age was 22 years, 78% were married for a median of 4 years, 66% reported knowing partner HIV status, and 8% reported using condoms. Study retention was 98%. During 1235 person-years of follow-up, HIV incidence was 2.31/100 person-years (95% Confidence Interval [CI]:0.71–4.10). Incident HIV was associated with syphilis (Hazard Ratio [HR] 9.18, 95% CI:2.15–39.3), chlamydia (HR 4.49, 95% CI:1.34–15.0), BV (HR 2.91, 95% CI:1.25–6.76), yeast (HR 3.46, 95% CI:1.46–8.19), STI history (HR 3.48, 95%, CI:1.31–9.27), lifetime number of sex partners, (HR 1.19, 95% CI:1.03–1.37), partner age discordance (HR 1.07 per year, 95% CI:1.02–1.13) and shorter marriage (HR 1.19 per year, 95% CI:1.03–1.38). No women with incident HIV reported an HIV-infected partner. In multivariate analyses, chlamydia, older partners, and yeast infection remained significant; however, power was limited.
Conclusions
Pregnant and lactating women may not perceive HIV risk and rarely used condoms. Prevention and treatment of genital infections and risk stratification to identify women for pre-exposure prophylaxis (PrEP) could decrease HIV acquisition in pregnant/lactating women.
doi:10.1097/QAD.0000000000000793
PMCID: PMC4692052  PMID: 26352880
HIV; incidence; mother-to-child transmission; acute; seroconversion
4.  Nevirapine Resistance in Previously Nevirapine-Unexposed HIV-1-Infected Kenyan Infants Initiating Early Antiretroviral Therapy 
Abstract
Nevirapine (NVP) resistance occurs frequently in infants following NVP use in prevention of mother-to-child transmission (PMTCT) regimens. However, among previously NVP-unexposed infants treated with NVP-antiretroviral therapy (ART), the development and impact of NVP resistance have not been well characterized. In a prospective clinical trial providing early ART to HIV-infected infants<5 months of age in Kenya (OPH03 study), we followed NVP-unexposed infants who initiated NVP-ART for 12 months. Viral loads were assessed and resistance determined using a population-based genotypic resistance assay. Of 99 infants screened, 33 had no prior NVP exposure, 22 of whom were initiated on NVP-ART. Among 19 infants with follow-up, seven (37%) infants developed resistance: one at 3 months and six at 6 months after ART initiation. The cumulative probability of NVP resistance was 5.9% at 3 months and 43.5% at 6 months. Baseline HIV RNA levels (p=0.7) and other characteristics were not associated with developing resistance. Post-ART, higher virus levels at visits preceding the detection of resistance were significantly associated with increased detection of resistance (p=0.004). Virus levels after 6 and 12 months of ART were significantly higher in infants with resistance than those without (p=0.007, p=0.030, respectively). Among infants without previous NVP exposure, development of NVP resistance was frequent and was associated with virologic failure during the first year of ART. Earlier development of NVP resistance in infants than in adults initiating NVP-ART may be due to longer viremia following ART or inadequate NVP levels resulting from NVP lead-in dosing. The development of NVP resistance may, in part, explain the superiority of protease inhibitor-based ART in infants.
doi:10.1089/aid.2014.0370
PMCID: PMC4533021  PMID: 25819584
5.  Adapting SHIVs In Vivo Selects for Envelope-Mediated Interferon-α Resistance 
PLoS Pathogens  2016;12(7):e1005727.
Lentiviruses are able to establish persistent infection in their respective hosts despite a potent type-I interferon (IFN-I) response following transmission. A number of IFN-I-induced host factors that are able to inhibit lentiviral replication in vitro have been identified, and these studies suggest a role for IFN-induced factors as barriers to cross-species transmission. However, the ability of these factors to inhibit viral replication in vivo has not been well characterized, nor have the viral determinants that contribute to evasion or antagonism of the host IFN-I response. In this study, we hypothesized that the host IFN-I response serves as a strong selective pressure in the context of SIV/HIV chimeric virus (SHIV) infection of macaques and sought to identify the viral determinants that contribute to IFN-I resistance. We assessed the ability of SHIVs encoding HIV-1 sequences adapted by serial passage in macaques versus SHIVs encoding HIV sequences isolated directly from infected individuals to replicate in the presence of IFNα in macaque lymphocytes. We demonstrate that passage in macaques selects for IFNα resistant viruses that have higher replication kinetics and increased envelope content. SHIVs that encode HIV-1 sequences derived directly from infected humans were sensitive to IFNα –induced inhibition whereas SHIVs obtained after passage in macaques were not. This evolutionary process was directly observed in viruses that were serially passaged during the first few months of infection–a time when the IFNα response is high. Differences in IFNα sensitivity mapped to HIV-1 envelope and were associated with increased envelope levels despite similar mRNA expression, suggesting a post-transcriptional mechanism. These studies highlight critical differences in IFNα sensitivity between HIV-1 sequences in infected people and those used in SHIV models.
Author Summary
The innate immune system is an important host defense against viral infection. Recently, there has been significant interest in characterizing the innate immune response to HIV-1 infection, in particular the role of type-I interferon (IFN-I). Understanding the interaction of HIV-1 with the innate immune system is particularly important for the development of animal models of infection as innate host factors present potential species-specific barriers to the establishment of persistent infection. One of the most commonly used animal models of HIV-1 infection is chimeric SIV/HIV (SHIV) infection of macaques. Here, we demonstrate that the process of adapting SHIVs for replication in macaques selects for viruses that are resistant to the IFNα response, and we identity important viral determinants that contribute to this resistance. This improved understanding of virus interactions with the innate immune response may facilitate the development of improved animal models of HIV-1 infection.
doi:10.1371/journal.ppat.1005727
PMCID: PMC4939950  PMID: 27399306
6.  Changes in the Contribution of Genital Tract Infections to HIV acquisition among Kenyan High-Risk Women from 1993 to 2012 
AIDS (London, England)  2015;29(9):1077-1085.
OBJECTIVE
To understand temporal trends in the contribution of different genital tract infections to HIV incidence over 20 years of follow-up in a cohort of high-risk women.
DESIGN
Prospective cohort study.
METHODS
We performed monthly evaluations for HIV, vaginal yeast, bacterial vaginosis (BV), Trichomonas vaginalis, Neisseria gonorrhoeae, non-specific cervicitis, herpes simplex virus type 2 (HSV-2), genital ulcer disease (GUD) and genital warts. We used Cox regression to evaluate the association between STIs and HIV acquisition over 4 time periods (1993–1997, 1998–2002, 2003–2007, 2008–2012). Models were adjusted for age, workplace, sexual risk behavior, hormonal contraceptive use, and other STIs. The resulting hazard ratios were used to calculate population attributable risk percent (PAR%).
RESULTS
Between 1993 and 2012, 1,964 women contributed 6,135 person-years of follow-up. The overall PAR% for each infection was: prevalent HSV-2 (48.3%), incident HSV-2 (4.5%), BV (15.1%), intermediate microbiota (7.5%), vaginal yeast (6.4%), T. vaginalis (1.1%), N. gonorrhoeae (0.9%), non-specific cervicitis (0.7%), GUD (0.8%), and genital warts (−0.2%). Across the four time periods, the PAR% for prevalent HSV-2 (40.4%, 61.8%, 58.4%, 48.3%) and BV (17.1%, 19.5%, 14.7%, 17.1%), remained relatively high and had no significant trend for change over time. The PAR% for trichomoniasis, gonorrhea, GUD and genital warts remained <3% across the four periods.
CONCLUSIONS
Bacterial vaginosis and HSV-2 have consistently been the largest contributors to HIV acquisition risk in the Mombasa Cohort over the past 20 years. Interventions that prevent these conditions would benefit women’s health, and could reduce their risk of becoming infected with HIV.
doi:10.1097/QAD.0000000000000646
PMCID: PMC4576156  PMID: 26125141
Genital tract infections; HIV; women; Africa; population attributable risk percent
7.  Risk of Drug Resistance Among Persons Acquiring HIV Within a Randomized Clinical Trial of Single- or Dual-Agent Preexposure Prophylaxis 
The Journal of Infectious Diseases  2015;211(8):1211-1218.
Background. Preexposure prophylaxis (PrEP) with emtricitabine plus tenofovir disoproxil fumarate (FTC/TDF) or TDF alone reduces the risk of human immunodeficiency virus (HIV) acquisition. Understanding the risk of antiretroviral resistance selected by PrEP during breakthrough infections is important because of the risk of treatment failure during subsequent antiretroviral use.
Methods. Within the largest randomized trial of FTC/TDF versus TDF as PrEP, plasma samples were tested for HIV with resistance mutations associated with FTC (K65R and M184IV) and TDF (K65R and K70E), using 454 sequencing.
Results. Of 121 HIV seroconverters, 25 received FTC/TDF, 38 received TDF, and 58 received placebo. Plasma drug levels in 26 individuals indicated PrEP use during or after HIV acquisition, of which 5 had virus with resistance mutations associated with their PrEP regimen. Among those with PrEP drug detected during infection, resistance was more frequent in the FTC/TDF arm (4 of 7 [57%]), compared with the TDF arm (1 of 19 [5.3%]; P = .01), owing to the FTC-associated mutation M184IV. Of these cases, 3 had unrecognized acute infection at PrEP randomization, and 2 were HIV negative at enrollment.
Conclusions. These results suggest that resistance selected by PrEP is rare but can occur both with PrEP initiation during acute seronegative HIV infection and in PrEP breakthrough infections and that FTC is associated with a greater frequency of resistance mutations than TDF.
doi:10.1093/infdis/jiu677
PMCID: PMC4402339  PMID: 25587020
HIV; pre-exposure prophylaxis; antiretroviral resistance; HIV prevention
8.  Passively acquired antibody-dependent cellular cytotoxicity (ADCC) activity in HIV-infected infants is associated with reduced mortality 
Cell host & microbe  2015;17(4):500-506.
SUMMARY
In addition to direct effects on virus infectivity, antibodies mediate antibody-dependent cellular cytotoxicity (ADCC), the killing of an antibody-coated virus-infected cell by cytotoxic effector cells. Although ADCC has been suggested to protect against HIV, the relationship between HIV-specific ADCC antibodies at the time of HIV exposure and infection outcome in humans remains to be assessed. We evaluated the ADCC activity of passively acquired antibodies in infants born to HIV-infected mothers. ADCC levels were higher in uninfected than infected infants, although not significantly. Increase in ADCC antibody activity in infected infants was associated with reduced mortality risk. Infant ADCC positively correlated with the magnitude of IgG1 binding and IgG1 levels were associated with survival in infected infants. Infant IgG3-binding antibodies were not associated with infected infant survival. These data suggest a therapeutic benefit of pre-existing HIV-specific ADCC antibodies and support a role for eliciting ADCC-mediating IgG1 in HIV vaccines.
doi:10.1016/j.chom.2015.03.002
PMCID: PMC4392343  PMID: 25856755
9.  Short Communication: Fc Gamma Receptors IIa and IIIa Genetic Polymorphisms Do Not Predict HIV-1 Disease Progression in Kenyan Women 
Abstract
Genetic polymorphisms of the Fc gamma receptors (FcγR) IIa and IIIa have been implicated in the rate of HIV-1 disease progression, but results are inconsistent. We aimed to determine the association between these polymorphisms and disease progression in a cohort of HIV-1 seroconverters from Mombasa, Kenya. Neither FcγRIIa nor FcγRIIIa genotypes were predictive of set point viral load, viral load increase, CD4 decline, or HIV-1 disease progression (time to CD4 count <200 cells/mm3, death, or treatment initiation). Our results suggest that FcγR polymorphisms might not be an important indicator of viral control and disease progression in this population.
doi:10.1089/aid.2014.0209
PMCID: PMC4348085  PMID: 25312792
10.  Identification of Owl Monkey CD4 Receptors Broadly Compatible with Early-Stage HIV-1 Isolates 
Journal of Virology  2015;89(16):8611-8622.
ABSTRACT
Most HIV-1 variants isolated from early-stage human infections do not use nonhuman primate versions of the CD4 receptor for cellular entry, or they do so poorly. We and others have previously shown that CD4 has experienced strong natural selection over the course of primate speciation, but it is unclear whether this selection has influenced the functional characteristics of CD4 as an HIV-1 receptor. Surprisingly, we find that selection on CD4 has been most intense in the New World monkeys, animals that have never been found to harbor lentiviruses related to HIV-1. Based on this, we sampled CD4 genetic diversity within populations of individuals from seven different species, including five species of New World monkeys. We found that some, but not all, CD4 alleles found in Spix's owl monkeys (Aotus vociferans) encode functional receptors for early-stage human HIV-1 isolates representing all of the major group M clades (A, B, C, and D). However, only some isolates of HIV-1 subtype C can use the CD4 receptor encoded by permissive Spix's owl monkey alleles. We characterized the prevalence of functional CD4 alleles in a colony of captive Spix's owl monkeys and found that 88% of surveyed individuals are homozygous for permissive CD4 alleles, which encode an asparagine at position 39 of the receptor. We found that the CD4 receptors encoded by two other species of owl monkeys (Aotus azarae and Aotus nancymaae) also serve as functional entry receptors for early-stage isolates of HIV-1.
IMPORTANCE Nonhuman primates, particularly macaques, are used for preclinical evaluation of HIV-1 vaccine candidates. However, a significant limitation of the macaque model is the fact that most circulating HIV-1 variants cannot use the macaque CD4 receptor to enter cells and have to be adapted to these species. This is particularly true for viral variants from early stages of infection, which represent the most relevant vaccine targets. In this study, we found that some individuals from captive owl monkey populations harbor CD4 alleles that are compatible with a broad collection of HIV-1 isolates, including those isolated from early in infection in highly affected populations and representing diverse subtypes.
doi:10.1128/JVI.00890-15
PMCID: PMC4524261  PMID: 26063421
11.  Maternal Neutralization-Resistant Virus Variants Do Not Predict Infant HIV Infection Risk 
mBio  2016;7(1):e02221-15.
ABSTRACT
Mother-to-child transmission (MTCT) of HIV provides a setting for studying immune correlates of protection. Neutralizing antibodies (NAbs) are suggested to contribute to a viral bottleneck during MTCT, but their role in blocking transmission is unclear, as studies comparing the NAb sensitivities of maternal viruses have yielded disparate results. We sought to determine whether transmitting mothers differ from nontransmitting mothers in the ability to neutralize individual autologous virus variants present at transmission. Ten transmitting and 10 nontransmitting HIV-infected mothers at high risk of MTCT were included in this study. Full-length HIV envelope genes (n = 100) were cloned from peripheral blood mononuclear cells obtained near transmission from transmitting mothers and at similar time points from nontransmitting mothers. Envelope clones were tested as pseudoviruses against contemporaneous, autologous maternal plasma in neutralization assays. The association between transmission and the log2 50% inhibitory concentration (IC50) for multiple virus variants per mother was estimated by using logistic regression with clustered standard errors. t tests were used to compare proportions of neutralization-resistant viruses. Overall, transmitting mothers had a median IC50 of 317 (interquartile range [IQR], 202 to 521), and nontransmitting mothers had a median IC50 of 243 (IQR, 95 to 594). Transmission risk was not significantly associated with autologous NAb activity (odds ratio, 1.25; P = 0.3). Compared to nontransmitting mothers, transmitting mothers had similar numbers of or fewer neutralization-resistant virus variants, depending on the IC50 neutralization resistance cutoff. In conclusion, HIV-infected mothers harbor mostly neutralization-sensitive viruses, although resistant variants were detected in both transmitting and nontransmitting mothers. These results suggest that MTCT during the breastfeeding period is not driven solely by the presence of maternal neutralization escape variants.
IMPORTANCE
There are limited data demonstrating whether NAbs can prevent HIV transmission and infection in humans, and for this reason, NAbs have been studied in MTCT, where maternal antibodies are present at the time of transmission. Results of these studies have varied, perhaps because of differences in methods. Importantly, studies often used cultured viruses and samples from time points outside the window of transmission, which could confound findings. Here, we considered the role of maternal NAbs against individual maternal virus variants near the time of transmission. We found no evidence that NAbs are associated with protection from infection. In fact, depending on the cutoff used to define neutralization resistance, we found evidence that nontransmitting mothers have more neutralization-resistant virus variants. These results suggest that lack of virus transmission in the early breastfeeding period is not simply due to an absence of maternal neutralization escape variants and likely includes multiple factors.
doi:10.1128/mBio.02221-15
PMCID: PMC4742716  PMID: 26838723
12.  Genital Shedding of Resistant Human Immunodeficiency Virus-1 Among Women Diagnosed With Treatment Failure by Clinical and Immunologic Monitoring 
Open Forum Infectious Diseases  2016;3(1):ofw019.
Detection of resistant HIV-1 in genital secretions of women failing first-line therapy was associated with a greater number of resistance mutations in plasma. While genital resistance emerged later than plasma resistance, genital shedding could increase risk for transmitted drug resistance.
Background. The accumulation of human immunodeficiency virus (HIV) resistance mutations can compromise treatment outcomes and promote transmission of drug-resistant virus. We conducted a study to determine the duration and evolution of genotypic drug resistance in the female genital tract among HIV-1-infected women failing first-line therapy.
Methods. Treatment failure was diagnosed based on World Health Organization (WHO) clinical or immunologic criteria, and second-line therapy was initiated. Stored plasma and genital samples were tested to determine the presence and timing of virologic failure and emergence of drug resistance. The median duration of genital shedding of genotypically resistant virus prior to regimen switch was estimated.
Results. Nineteen of 184 women were diagnosed with treatment failure, of whom 12 (63.2%) had confirmed virologic failure at the switch date. All 12 women with virologic failure (viral load, 5855–1 086 500 copies/mL) had dual-class resistance in plasma. Seven of the 12 (58.3%) had genital HIV-1 RNA levels high enough to amplify (673–116 494 copies/swab), all with dual-class resistance. The median time from detection of resistance in stored samples to regimen switch was 895 days (95% confidence interval [CI], 130–1414 days) for plasma and 629 days (95% CI, 341–984 days) for genital tract secretions.
Conclusions. Among women diagnosed with treatment failure using WHO clinical or immunologic criteria, over half had virologic failure confirmed in stored samples. Resistant HIV-1 RNA was shed in the genital tract at detectable levels for ≈1.7 years before failure diagnosis, with steady accumulation of mutations. These findings add urgency to the ongoing scale-up of viral load testing in resource-limited settings.
doi:10.1093/ofid/ofw019
PMCID: PMC4784013  PMID: 26966695
antiretroviral therapy; female; HIV; viral drug resistance; virus shedding
13.  HIV Neutralizing Antibodies Induced by Native-like Envelope Trimers 
Science (New York, N.Y.)  2015;349(6244):aac4223.
A challenge for HIV-1 immunogen design is inducing neutralizing antibodies (NAbs) against neutralization-resistant (Tier-2) viruses that dominate human transmissions. We show that a soluble recombinant HIV-1 envelope glycoprotein trimer that adopts a native conformation (BG505 SOSIP.664) induced NAbs potently against the sequence-matched Tier-2 virus in rabbits and similar but weaker responses in macaques. The trimer also consistently induced cross-reactive NAbs against more sensitive (Tier-1) viruses. Tier-2 NAbs recognized conformational epitopes that differed between animals and in some cases overlapped with those recognized by broadly neutralizing antibodies (bNAbs), whereas Tier-1 responses targeted linear V3 epitopes. A second trimer, B41 SOSIP.664, also induced a strong autologous Tier-2 NAb response in rabbits. Thus, native-like trimers represent a promising starting point for developing HIV-1 vaccines aimed at inducing bNAbs.
doi:10.1126/science.aac4223
PMCID: PMC4498988  PMID: 26089353
14.  Structure/Function Studies Involving the V3 Region of the HIV-1 Envelope Delineate Multiple Factors That Affect Neutralization Sensitivity 
Journal of Virology  2015;90(2):636-649.
ABSTRACT
Antibodies (Abs) specific for the V3 loop of the HIV-1 gp120 envelope neutralize most tier 1 and many tier 2 viruses and are present in essentially all HIV-infected individuals as well as immunized humans and animals. Vaccine-induced V3 Abs are associated with reduced HIV infection rates in humans and affect the nature of transmitted viruses in infected vaccinees, despite the fact that V3 is often occluded in the envelope trimer. Here, we link structural and experimental data showing how conformational alterations of the envelope trimer render viruses exceptionally sensitive to V3 Abs. The experiments interrogated the neutralization sensitivity of pseudoviruses with single amino acid mutations in various regions of gp120 that were predicted to alter packing of the V3 loop in the Env trimer. The results indicate that the V3 loop is metastable in the envelope trimer on the virion surface, flickering between states in which V3 is either occluded or available for binding to chemokine receptors (leading to infection) and to V3 Abs (leading to virus neutralization). The spring-loaded V3 in the envelope trimer is easily released by disruption of the stability of the V3 pocket in the unliganded trimer or disruption of favorable V3/pocket interactions. Formation of the V3 pocket requires appropriate positioning of the V1V2 domain, which is, in turn, dependent on the conformation of the bridging sheet and on the stability of the V1V2 B-C strand-connecting loop.
IMPORTANCE The levels of antibodies to the third variable region (V3) of the HIV envelope protein correlate with reduced HIV infection rates. Previous studies showed that V3 is often occluded, as it sits in a pocket of the envelope trimer on the surface of virions; however, the trimer is flexible, allowing occluded portions of the envelope (like V3) to flicker into an exposed position that binds antibodies. Here we provide a systematic interrogation of mechanisms by which single amino acid changes in various regions of gp120 (i) render viruses sensitive to neutralization by V3 antibodies, (ii) result in altered packing of the V3 loop, and (iii) activate an open conformation that exposes V3 to the effects of V3 Abs. Taken together, these and previous studies explain how V3 antibodies can protect against HIV-1 infection and why they should be one of the targets of vaccine-induced antibodies.
doi:10.1128/JVI.01645-15
PMCID: PMC4702699  PMID: 26491157
15.  The Role of Cell-Associated Virus in Mother-to-Child HIV Transmission 
The Journal of Infectious Diseases  2014;210(Suppl 3):S631-S640.
Mother-to-child transmission (MTCT) of human immunodeficiency virus (HIV) continues to contribute to the global burden of disease despite great advances in antiretroviral (ARV) treatment and prophylaxis. In this review, we discuss the proposed mechanisms of MTCT, evidence for cell-free and cell-associated transmission in different routes of MTCT, and the impact of ARVs on virus levels and transmission. Many population-based studies support a role for cell-associated virus in transmission and in vitro studies also provide some support for this mode of transmission. However, animal model studies provide proof-of-principle that cell-free virus can establish infection in infants, and studies of ARVs in HIV-infected pregnant women show a strong correlation with reduction in cell-free virus levels and protection. ARV treatment in MTCT potentially provides opportunities to better define the infectious form of virus, but these studies will require better tools to measure the infectious cell reservoir.
doi:10.1093/infdis/jiu344
PMCID: PMC4303081  PMID: 25414417
breast milk; cell-associated virus; cell-free virus; genital secretions; HIV-1; infected leukocytes; mother-to-child transmission
16.  Oligonucleotide Ligation Assay Detects HIV Drug Resistance Associated with Virologic Failure among Antiretroviral-Naïve Adults in Kenya 
Background
Transmitted drug resistance (TDR) is increasing in some areas of Africa. Detection of TDR may predict virologic failure of first-line non-nucleoside reverse-transcriptase inhibitor (NNRTI)-based antiretroviral therapy (ART). We evaluated the utility of a relatively inexpensive oligonucleotide ligation assay (OLA) to detect clinically relevant TDR at time of ART initiation.
Methods
Pre-ART plasmas from ART-naive Kenyans initiating an NNRTI-based fixed-dose combination ART in a randomized adherence trial conducted in 2006 were retrospectively analyzed by OLA for mutations conferring resistance to NNRTI (K103N, Y181C, and G190A) and lamivudine (M184V). Post-ART plasmas were analyzed for virologic failure (≥1,000 copies/mL) at 6 month intervals over 18-month follow-up. Pre-ART plasmas of those with virologic failure were evaluated for drug resistance by consensus and 454-pyrosequencing.
Results
Among 386 participants, TDR was detected by OLA in 3.89% [95% Confidence Interval (CI), 2.19-6.33], and was associated with a 10-fold higher rate of virologic failure [Hazard Ratio (HR), 10.39; 95% CI, 3.23-32.41; p<0.001) compared to those without TDR. OLA detected 24 TDR mutations (K103N, n=13; Y181C, n=5; G190A, n=3; M184V, n=3) in 15 subjects (NNRTI, n=15; 3TC, n=3). Among 51 participants who developed virologic failure, consensus sequencing did not detect additional TDR mutations conferring high-level resistance, and pyrosequencing only detected additional mutations at frequencies <2%. Mutant frequencies <2% at ART initiation were significantly less likely to be found at the time of virologic failure compared to frequencies ≥2% (22% vs. 63%; p<0.001).
Conclusions
Detection of TDR by a point mutation assay may prevent use of sub-optimal ART.
doi:10.1097/QAI.0000000000000312
PMCID: PMC4197120  PMID: 25140907
transmitted drug resistance; oligonucleotide ligation assay; HIV; Kenya; antiretroviral therapy
17.  FCGR2A and FCGR3A Genotypes in Human Immunodeficiency Virus Mother-to-Child Transmission 
Open Forum Infectious Diseases  2015;2(4):ofv149.
Host FcγR polymorphisms may influence HIV infection and disease progression. Here, we examine FCGR2A and FCGR3A genotypes in HIV mother-to-child transmission. Infant genotypes did not impact infection or progression, but the maternal FCGR3A genotype may influence early breastfeeding transmission risk.
Background. Fc-mediated effector functions have been suggested to influence human immunodeficiency virus (HIV) acquisition and disease progression. Analyzing the role of host Fc gamma receptor (FcγR) polymorphisms on HIV outcome in mother-to-child transmission (MTCT) will increase our understanding of how host genetics may alter immune responses in prevention, therapy, and disease. This study analyzed the impact of FCGR2A and FCGR3A genotypes on MTCT in a cohort in which Fc-mediated antibody functions are predictive of infant HIV outcome.
Methods. Human immunodeficiency virus-positive mothers and their infants from a historical MTCT cohort were genotyped for FCGR2A and FCGR3A. We assessed the impact of these genotypes on transmission and acquisition of HIV and disease progression using χ2 tests, survival analyses, and logistic regression.
Results. Among 379 mother-infant pairs, infant FCGR2A and FCGR3A genotypes were not associated with infant HIV infection or disease progression. Maternal FCGR2A was not associated with transmission, but there was a trend between maternal FCGR3A genotype and transmission (P = .07). When dichotomizing mothers into FCGR3A homozygotes and heterozygotes, heterozygotes had a 64.5% higher risk of transmission compared with homozygotes (P = .02). This risk was most evident in the early breastfeeding window, but a trend was only observed when restricting analyses to breastfeeding mothers (hazards ratio, 1.64; P = .064).
Conclusions. Infant FCGR2A and FCGR3A genotypes were not associated with HIV infection or disease progression, and, thus, host FcγR genotype may not significantly impact vaccination or therapeutic regimens that depend on Fc-mediated antibody functions. Maternal FCGR3A genotype may influence early breastfeeding transmission risk, but more studies should be conducted to clarify this association and its mechanism.
doi:10.1093/ofid/ofv149
PMCID: PMC4653957  PMID: 26613093
Fc gamma receptors (FcγR); FCGR2A; FCGR3A; HIV; mother-to-child transmission (MTCT)
18.  Elevation of Soluble Intercellular Adhesion Molecule-1 Levels, but Not Angiopoietin 2, in the Plasma of Human Immunodeficiency Virus–Infected African Women with Clinical Kaposi Sarcoma 
Circulating levels of endothelial activation biomarkers are elevated in during infection with human immunodeficiency virus 1 (HIV-1) and may also be increased in Kaposi sarcoma (KS). We compared 23 HIV-1-seropositive women with clinically diagnosed KS with 46 randomly selected controls matched for visit year, CD4 count, and antiretroviral therapy status. Conditional logistic regression was used to identify differences between cases and controls. The odds of clinical KS increased with increasing plasma viral load and with intercellular adhesion molecule 1 (ICAM-1) levels above or equal to the median. There was a borderline association between increasing plasma angiopoietin 2 levels and KS. In multivariable modeling including plasma viral load, angiopoietin 2, and ICAM-1, plasma ICAM-1 levels above or equal to the median remained associated with clinical KS (odds ratio = 14.2, 95% confidence interval = 2.3–87.7). Circulating ICAM-1 levels should be evaluated as a potential biomarker for disease progression and treatment response among HIV-infected KS patients.
doi:10.4269/ajtmh.14-0209
PMCID: PMC4183391  PMID: 25002294
19.  HIV-specific CD4-induced Antibodies Mediate Broad and Potent Antibody-dependent Cellular Cytotoxicity Activity and are Commonly Detected in Plasma from HIV-infected Humans 
EBioMedicine  2015;2(10):1464-1477.
HIV-specific antibodies (Abs) can reduce viral burden by blocking new rounds of infection or by destroying infected cells via activation of effector cells through Fc–FcR interaction. This latter process, referred to as antibody-dependent cellular cytotoxicity (ADCC), has been associated with viral control and improved clinical outcome following both HIV and SIV infections. Here we describe an HIV viral-like particle (VLP)-based sorting strategy that led to identification of HIV-specific memory B cells encoding Abs that mediate ADCC from a subtype A-infected Kenyan woman at 914 days post-infection. Using this strategy, 12 HIV-envelope-specific monoclonal antibodies (mAbs) were isolated and three mediated potent ADCC activity when compared to well-characterized ADCC mAbs. The ADCC-mediating Abs also mediated antibody-dependent cell-mediated virus inhibition (ADCVI), which provides a net measure of Fc receptor-triggered effects against replicating virus. Two of the three ADCC-mediating Abs targeted a CD4-induced (CD4i) epitope also bound by the mAb C11; the third antibody targeted the N-terminus of V3. Both CD4i Abs identified here demonstrated strong cross-clade breadth with activity against 10 of 11 envelopes tested, including those from clades A, B, C, A/D and C/D, whereas the V3-specific antibody showed more limited breadth. Variants of these CD4i, C11-like mAbs engineered to interrupt binding to FcγRs inhibited a measurable percentage of the donor's ADCC activity starting as early as 189 days post-infection. C11-like antibodies also accounted for between 18–78% of ADCC activity in 9 chronically infected individuals from the same cohort study. Further, the two CD4i Abs originated from unique B cells, suggesting that antibodies targeting this epitope can be commonly produced. Taken together, these data provide strong evidence that CD4i, C11-like antibodies develop within the first 6 months of infection and they can arise from unique B-cell lineages in the same individual. Further, these mAbs mediate potent plasma IgG-specific ADCC breadth and potency and contribute to ADCC activity in other HIV-infected individuals.
Highlights
•We identified new HIV-specific mAbs that mediate broad and potent ADCC & ADCVI activities.•Two potent mAbs targeted CD4i, C11-like epitopes and originated from unique B cells.•C11-like Abs were common and constituted 18–78% of plasma ADCC activity.•C11-like antibodies could be measured in plasma as early as 189 days post-HIV-infection.
Following HIV infection, an individual mounts an immune response that includes virus-specific antibodies, some of which destroy HIV-infected cells via antibody-dependent cellular cytotoxicity (ADCC). Studies have identified correlations between ADCC-mediating antibodies and improved clinical outcome in HIV infection. Here, we identified and characterized three monoclonal antibodies that mediated potent ADCC against diverse HIV strains. Two antibodies contributed significantly to the donor's ADCC activity early after infection and similar antibody responses were produced by other HIV-infected individuals from the same cohort. These data suggest that potent ADCC-mediating Abs are commonly made and therefore may be a target benchmark for vaccine strategies.
doi:10.1016/j.ebiom.2015.09.001
PMCID: PMC4634620  PMID: 26629541
ADCC; RF-ADCC; Antibody; HIV; CD4-induced; CD4i; C11; Potency; Breadth
20.  A Prospective Cohort Study of the Effect of Depot Medroxyprogesterone Acetate on Detection of Plasma and Cervical HIV-1 in Women Initiating and Continuing Antiretroviral Therapy 
Depot medroxyprogesterone acetate (DMPA) use among HIV-1 infected women may increase transmission by increasing plasma and genital HIV-1 RNA shedding. We investigated associations between DMPA use and HIV-1 RNA in plasma and cervical secretions. 102 women initiated ART, contributing 925 follow-up visits over a median of 34 months. Compared to visits with no hormonal contraception exposure, DMPA exposure did not increase detection of plasma (adjusted odds ratio (AOR) 0.81, 95% CI 0.47–1.39) or cervical HIV-1 RNA (AOR 1.41, 95% CI 0.54–3.67). Our results suggest that DMPA is unlikely to increase infectivity in HIV-positive women who are adherent to effective ART.
doi:10.1097/QAI.0000000000000187
PMCID: PMC4419746  PMID: 24798764
hormonal contraception; HIV-1; antiretroviral therapy; DMPA
21.  Mutations in HIV-1 Envelope That Enhance Entry with the Macaque CD4 Receptor Alter Antibody Recognition by Disrupting Quaternary Interactions within the Trimer 
Journal of Virology  2014;89(2):894-907.
ABSTRACT
Chimeric simian immunodeficiency virus (SIV)/human immunodeficiency virus (HIV) (SHIV) infection of macaques is commonly used to model HIV type 1 (HIV-1) transmission and pathogenesis in humans. Despite the fact that SHIVs encode SIV antagonists of the known macaque host restriction factors, these viruses require additional adaptation for replication in macaques to establish a persistent infection. Additional adaptation may be required in part because macaque CD4 (mCD4) is a suboptimal receptor for most HIV-1 envelope glycoprotein (Env) variants. This requirement raises the possibility that adaptation of HIV-1 Env to the macaque host leads to selection of variants that lack important biological and antigenic properties of the viruses responsible for the HIV-1 pandemic in humans. Here, we investigated whether this adaptation process leads to changes in the antigenicity and structure of HIV-1 Env. For this purpose, we examined how two independent mutations that enhance mCD4-mediated entry, A204E and G312V, impact antibody recognition in the context of seven different parental HIV-1 Env proteins from diverse subtypes. We also examined HIV-1 Env variants from three SHIVs that had been adapted for increased replication in macaques. Our results indicate that these different macaque-adapted variants had features in common, including resistance to antibodies directed to quaternary epitopes and sensitivity to antibodies directed to epitopes in the variable domains (V2 and V3) that are buried in the parental, unadapted Env proteins. Collectively, these findings suggest that adaptation to mCD4 results in conformational changes that expose epitopes in the variable domains and disrupt quaternary epitopes in the native Env trimer.
IMPORTANCE These findings indicate the antigenic consequences of adapting HIV-1 Env to mCD4. They also suggest that to best mimic HIV-1 infection in humans when using the SHIV/macaque model, HIV-1 Env proteins should be identified that use mCD4 as a functional receptor and preserve quaternary epitopes characteristic of HIV-1 Env.
doi:10.1128/JVI.02680-14
PMCID: PMC4300673  PMID: 25378497
22.  HIV-1 superinfection is associated with accelerated viral load increase but has limited impact on disease progression 
AIDS (London, England)  2014;28(15):2281-2286.
Objective
HIV-1 superinfection occurs frequently in high-risk populations, but its clinical consequences remain poorly characterized. We undertook this study to determine the impact of HIV-1 superinfection on disease progression.
Design/methods
In the largest prospective cohort study of superinfection to date, we compared measures of HIV-1 progression in women who acquired superinfection with those who did not. Clinical and laboratory data were collected at quarterly intervals. Linear mixed effects models were used to compare post-acute viral load and CD4 T-cell counts over time in singly infected and superinfected women. Cox proportional hazards analysis was used to determine the effect of superinfection on time to clinical progression (CD4 < 200, ART initiation or death).
Results
Among 144 women, 21 of whom acquired superinfection during follow-up, the rate of viral load increase was higher in superinfected than singly infected women (p=0.0008). In adjusted analysis, superinfected women had lower baseline viral load before superinfection (p=0.05) and a trend for increased viral load at superinfection acquisition (p=0.09). We also observed a borderline association of superinfection with accelerated CD4 decline (p=0.06). However, there was no significant difference in time to clinical progression events.
Conclusions
These data suggest that superinfection is associated with accelerated progression in laboratory measures of HIV-1 disease, but has limited impact on the occurrence of clinical events. Our observation that superinfected individuals have lower baseline viral load prior to superinfection suggests there may be host or viral determinants of susceptibility to superinfection.
doi:10.1097/QAD.0000000000000422
PMCID: PMC4503239  PMID: 25102090
HIV-1; women; superinfection; progression; pathogenesis
23.  The Broad Neutralizing Antibody Responses after HIV-1 Superinfection Are Not Dominated by Antibodies Directed to Epitopes Common in Single Infection 
PLoS Pathogens  2015;11(7):e1004973.
HIV-1 vaccines designed to date have failed to elicit neutralizing antibodies (Nabs) that are capable of protecting against globally diverse HIV-1 subtypes. One relevant setting to study the development of a strong, cross-reactive Nab response is HIV-1 superinfection (SI), defined as sequential infections from different source partners. SI has previously been shown to lead to a broader and more potent Nab response when compared to single infection, but it is unclear whether SI also impacts epitope specificity and if the epitopes targeted after SI differ from those targeted after single infection. Here the post-SI Nab responses were examined from 21 Kenyan women collectively exposed to subtypes A, C, and D and superinfected after a median time of ~1.07 years following initial infection. Plasma samples chosen for analysis were collected at a median time point ~2.72 years post-SI. Because previous studies of singly infected populations with broad and potent Nab responses have shown that the majority of their neutralizing activity can be mapped to 4 main epitopes on the HIV-1 Envelope, we focused on these targets, which include the CD4-binding site, a V1/V2 glycan, the N332 supersite in V3, and the membrane proximal external region of gp41. Using standard epitope mapping techniques that were applied to the previous cohorts, the present study demonstrates that SI did not induce a dominant Nab response to any one of these epitopes in the 21 women. Computational sera delineation analyses also suggested that 20 of the 21 superinfected women’s Nab responses could not be ascribed a single specificity with high confidence. These data are consistent with a model in which SI with diverse subtypes promotes the development of a broad polyclonal Nab response, and thus would provide support for vaccine designs using multivalent HIV immunogens to elicit a diverse repertoire of Nabs.
Author Summary
Learning how to elicit a potent, cross-reactive neutralizing antibody (Nab) response capable of protecting against globally diverse human immunodeficiency virus-1 (HIV-1) subtypes is critical to the development of an HIV-1 vaccine. We and others have previously shown that HIV-1 superinfection (SI), or sequential infections from different partners, broadens and strengthens the Nab response. However, until now it was unclear whether SI also impacts the specificity, or epitope targets, of the antibody response. Previous studies have shown that the majority of singly infected individuals with broad and potent responses develop Nabs to 4 main epitopes on the HIV-1 Envelope. In contrast, here we show that none of the 21 SI cases in our Kenyan cohort developed Nabs that strongly target these epitopes. Our study helps to inform vaccine design by highlighting the prospect of eliciting broad and diverse HIV-specific Nab responses through sequential exposure to different HIV antigens.
doi:10.1371/journal.ppat.1004973
PMCID: PMC4497680  PMID: 26158467
24.  Systemic Cytokine Levels Show Limited Correlation with Risk of HIV-1 Acquisition 
It has been hypothesized that immune activation and inflammation may increase HIV-1 susceptibility and that cytokines may be useful biomarkers for risk. Within a prospective cohort, we conducted a nested case-control analysis of plasma cytokine levels among women who acquired HIV-1 <3 months after sampling, compared to three different control groups. We observed associations between lower IL-6 and IL-10 and higher IL-7 levels with HIV-1 acquisition, however these associations were inconsistent when comparing to different control groups. Inconsistent results within our study and among prior studies suggest that reproducible findings are needed before cytokines are useful biomarkers for HIV-1 susceptibility.
doi:10.1097/QAI.0000000000000104
PMCID: PMC4020985  PMID: 24413043
cytokines; HIV; acquisition; susceptibility; immune activation
25.  Natural killer cell and T-cell subset distributions and activation influence susceptibility to perinatal HIV-1 infection 
AIDS (London, England)  2014;28(8):1115-1124.
Objective
To determine neonatal immunologic factors that correlate with mother-to-child-transmission of HIV-1.
Design
This case–control study compared cord blood natural killer (NK) and T-cell populations of HIV-1 exposed infants who subsequently acquired infection by 1 month (cases) to those who remained uninfected by 1 year of life (controls). Control specimens were selected by proportional match on maternal viral load.
Methods
Cryopreserved cord blood mononuclear cells (CBMCs) were thawed and stained for multiparameter flow cytometry to detect NK and T-cell subsets and activation status. CBMCs were also used in a viral suppression assay to evaluate NK cell inhibition of HIV-1 replication in autologous CD4+ T cells.
Results
Cord blood from cases contained a skewed NK cell repertoire characterized by an increased proportion of CD16−CD56+ NK cells. In addition, cases displayed less-activated CD16−CD56+ NK cells and CD8+ T cells, based on HLA-DR+CD38+ costaining. NK cell suppression of HIV-1 replication ex vivo correlated with the proportion of acutely activated CD68+CD16−CD56+ NK cells. Finally, we detected a higher proportion of CD27−CD45RA− effector memory CD4+ and CD8+ T cells in cord blood from cases compared with controls.
Conclusion
When controlled for maternal viral load, cord blood from infants who acquired HIV-1 had a higher proportion of CD16−CD56+ NK cells, lower NK cell activation and higher levels of mature T cells (potential HIV-1 targets) than control infants who remained uninfected. Our data provide evidence that infant HIV-1 acquisition may be influenced by both innate and adaptive immune cell phenotypes and activation status.
doi:10.1097/QAD.0000000000000263
PMCID: PMC4365995  PMID: 24752082
CD38; HLA-DR; Kenya; mother-to-child HIV-1 transmission; natural killer cell; Tem; viral suppression assays

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