A case control study was performed to determine the effects of HIV-1-specific cellular immune responses on the odds of acquiring a second HIV-1 infection (superinfection). Changes in the frequency of cytokine-producing or cytolytic CD8+ or CD4+ T cells were not associated with significant alterations in the odds of superinfection, suggesting that HIV-1 specific cellular immune responses at the level induced by chronic infection do not appear to significantly contribute to protection from HIV-1 superinfection.

HIV-1 is grouped phylogenetically into clades, which may impact rates of HIV-1 disease progression. Clade D infection in particular has been shown to be more pathogenic. Here we confirm in a Nairobi-based prospective female sex worker cohort (1985–2004) that Clade D (n = 54) is associated with a more rapid CD4 decline than clade A1 (n = 150, 20.6% vs 13.4% decline per year, 1.53-fold increase, p = 0.015). This was independent of “protective” HLA and country of origin (p = 0.053), which in turn were also independent predictors of the rate of CD4 decline (p = 0.026 and 0.005, respectively). These data confirm that clade D is more pathogenic than clade A1. The precise reason for this difference is currently unclear, and requires further study. This is first study to demonstrate difference in HIV-1 disease progression between clades while controlling for protective HLA alleles.

Background

Poor susceptibility of Cryptococcus neoformans to fluconazole (FLC) is a matter of concern among clinicians in Africa. The emergence of resistance to FLC was recently reported in Kenya but it is not known if it is widespread. Thus, there is need for more antifungal drug susceptibility studies in Kenya.

Objective

To measure the in vitro antifungal drug susceptibilities of incident C. neoformans isolates from acquired immunodeficiency syndrome patients in Kenya.

Methods

Antifungal susceptibility testing was performed on 67 C. neoformans isolates by broth microdilution method as outlined in the Clinical and Laboratory Standards Institute document M27-A3 using FLC, amphotericin B (AMB), voriconazole (VOR), ravuconazole (RAV), and flucytosine (5-FC). Isolates were grown on L-canavanine glycine bromothymol blue medium for serotype identification.

Results

Six percent of the isolates were identified as C. neoformans var. gattii serotype B or C and 94% as C. neoformans var. neoformans. All isolates tested were susceptible to AMB, VOR and RAV (100%), and high susceptibilities were seen to FLC (97%), and 5-FC (90%). Only 3% and 10% of the isolates’ susceptibility to FLC and 5-FC respectively, was dose dependent or intermediate.

Conclusions

These results demonstrate high susceptibilities of incident C. neoformans isolates to FLC and AMB, antifungals used for treatment of cryptococcal meningitis in Kenya.

Background

Identifying the immune correlates of reduced susceptibility to HIV remains a key goal for the HIV vaccine field, and individuals who are HIV-exposed, seronegative (HESN) may offer important clues. Reduced systemic immune activation has been described in HESN individuals. Conversely, pro-inflammatory T cell subsets, particularly CD4+ T cells producing the cytokine IL17 (Th17 cells), may represent a highly susceptible target for HIV infection after sexual exposure. Therefore, we characterized the cellular pro-inflammatory and IL17/IL22 cytokine immune milieu in the genital mucosa and blood of HESN female sex workers (FSWs).

Methods and Results

Blinded lab personnel characterized basal and mitogen-induced gene and cytokine immune responses in the cervix and blood of HESN FSWs (n = 116) and non-FSW controls (n = 17) using qPCR and ELISA. IL17 and IL22 production was significantly reduced in both the cervix and blood of HESNs, both in resting cells and after mitogen stimulation. In addition, HESN participants demonstrated blunted production of both pro-inflammatory cytokines and β-chemokines.

Discussion and Conclusions

We conclude that HIV exposure without infection was associated with blunted IL17/IL22 and pro-inflammatory responses, both systemically and at the site of mucosal HIV exposure. It will be important for further studies to examine the causal nature of the association and to define the cell subsets responsible for these differences.

Data from a randomized trial of oral periodic presumptive treatment (PPT) to reduce vaginal infections were analyzed to assess the effect of the intervention on a healthy vaginal environment (normal flora confirmed by Gram stain with no candidiasis or trichomoniasis). The incidence of a healthy vaginal environment was 608 cases per 100 person-years in the intervention arm and 454 cases per 100 person-years in the placebo arm (hazard ratio [HR], 1.36; 95% confidence interval [CI], 1.17–1.58). Sustained vaginal health (healthy vaginal environment for ≥3 consecutive visits) was also more frequent in the intervention arm (HR, 1.69; 95% CI, 1.23–2.33). PPT is effective at establishing and sustaining a healthy vaginal environment.

Background

Few studies have examined the association between self-reported sexual risk behaviors and biological outcomes in HIV-1-seropositive African adults.

Methods

We conducted a prospective cohort study in 898 HIV-1-seropositive women who reported engaging in transactional sex in Mombasa, Kenya. Primary outcome measures included detection of sperm in genital secretions, pregnancy, and sexually transmitted infections (STIs). Because three outcomes were evaluated, data are presented with odds ratios [OR] and 96.7% confidence intervals [CI] to reflect that we would reject a null hypothesis if a p-value were ≤0.033 (Simes’ methodology).

Results

During 2,404 person-years of follow-up, self-reported unprotected intercourse was associated with significantly higher likelihood of detecting sperm in genital secretions (OR 2.32, 96.7% CI 1.93, 2.81), and pregnancy (OR 2.78, 96.7% CI 1.57, 4.92), but not with detection of STIs (OR 1.20, 96.7% CI 0.98, 1.48). At visits where women reported being sexually active, having >1 sex partner in the past week was associated with lower likelihood of detecting sperm in genital secretions (OR 0.74, 96.7% CI 0.56, 0.98). This association became non-significant after adjustment for reported condom use (adjusted OR 0.81, 96.7% CI 0.60, 1.08).

Conclusions

Combining behavioral and biological outcomes, which provide complementary information, is advantageous for understanding sexual risk behavior in populations at risk for transmitting HIV-1. The paradoxical relationship between higher numbers of sex partners and less frequent identification of sperm in genital secretions highlights the potential importance of context-specific behavior, such as condom use dependent on partner type, when evaluating sexual risk behavior.

Objectives

Although antiretroviral therapy (ART) prolongs life and reduces infectiousness, in some contexts, it has been associated with increased sexual risk taking.

Design

Retrospective case–control study.

Setting

Nairobi-based dedicated female sex worker (FSW) clinic.

Participants

HIV-infected FSWs before and after ART initiation (n=62); HIV-infected and -uninfected control FSWs not starting ART during the same follow-up period (n=40).

Intervention

Initiation of ART.

Primary outcome measures

Self-reported condom use, client numbers and sexually transmitted infection incidence over the study period (before and after ART initiation in cases).

Results

Sexual risk-taking behaviour with casual clients did not increase after ART initiation; condom use increased and sexually transmitted infection incidence decreased in both cases and controls, likely due to successful cohort-wide HIV prevention efforts.

Conclusions

ART provision was not associated with increases in unsafe sex in this FSW population.

Article summary

Article focus

Impact of starting ART on sexual risk-taking behaviour in FSWs, which could have an important impact on HIV transmission to clients.

Key messages

ART initiation was not associated with increased risk taking or sexually transmitted infection incidence.

Both of these declined over time, most likely as a result of risk reduction counselling.

Strengths and limitations of this study

Strengths include a relevant population, longitudinal follow-up and inclusion of biological measures of risk taking (sexually transmitted infection incidence).

Limitations include relatively small study groups and limited sampling time points.

Identifying naturally-occurring neutralizing antibodies (NAb) that are cross-reactive against all global subtypes of HIV-1 is an important step toward the development of a vaccine. Establishing the host and viral determinants for eliciting such broadly NAbs is also critical for immunogen design. NAb breadth has previously been shown to be positively associated with viral diversity. Therefore, we hypothesized that superinfected individuals develop a broad NAb response as a result of increased antigenic stimulation by two distinct viruses. To test this hypothesis, plasma samples from 12 superinfected women each assigned to three singly infected women were tested against a panel of eight viruses representing four different HIV-1 subtypes at matched time points post-superinfection (∼5 years post-initial infection). Here we show superinfected individuals develop significantly broader NAb responses post-superinfection when compared to singly infected individuals (RR = 1.68, CI: 1.23–2.30, p = 0.001). This was true even after controlling for NAb breadth developed prior to superinfection, contemporaneous CD4+ T cell count and viral load. Similarly, both unadjusted and adjusted analyses showed significantly greater potency in superinfected cases compared to controls. Notably, two superinfected individuals were able to neutralize variants from four different subtypes at plasma dilutions >1∶300, suggesting that their NAbs exhibit elite activity. Cross-subtype breadth was detected within a year of superinfection in both of these individuals, which was within 1.5 years of their initial infection. These data suggest that sequential infections lead to augmentation of the NAb response, a process that may provide insight into potential mechanisms that contribute to the development of antibody breadth. Therefore, a successful vaccination strategy that mimics superinfection may lead to the development of broad NAbs in immunized individuals.

Author Summary

A broad and potent antibody response is considered essential for an effective HIV-1 vaccine that will protect against diverse circulating strains. Consequently, there is great interest in both the host and viral factors that impact the development of the neutralizing antibody (NAb) response in natural HIV-1 infections. HIV-infected individuals who become superinfected with a second virus from a different source partner represent unique cases for studying the antibody response, as superinfection reflects exposure to different HIV-1 antigenic variants, and hence may provide insight into the development of broadly NAbs. In support of this model, we show here that superinfected individuals develop broader and more potent NAb responses than singly infected individuals, a result that is likely due to the increased antigenic stimulation from two viruses compared to one. Our findings remained unchanged after controlling for other factors that have been shown to influence the NAbs response, such as CD4+ T cell count and viral load. This study demonstrates that superinfection yields antibodies that have the capacity to recognize diverse circulating HIV-1 variants. Therefore, further characterization of these superinfected individuals' NAb responses could lead to novel insights into pathways that elicit broadly NAbs.

Objectives

Genital ulcer disease (GUD) is associated with increased HIV-1 RNA shedding in antiretroviral therapy (ART)-naïve women. The effect of GUD on HIV-1 shedding among ART-treated women is not known. Our objective was to test the hypothesis that genital ulcerations increase genital HIV-1 RNA shedding in women receiving ART.

Methods

Eligible women initiated ART and attended monthly visits with inspection for genital lesions and collection of genital swabs. GUD cases diagnosed after ≥2 months on ART were included for analysis and served as their own controls. HIV-1 RNA was quantitated in specimens collected before, during, and after GUD for all cases. The lower limit of quantitation was 100 HIV-1 RNA copies/swab. Using the pre-GUD visit as the reference, we compared detection of genital HIV-1 RNA before versus during and after GUD episodes.

Results

Thirty-six women had GUD episodes after ART initiation. HIV-1 RNA was detected before, during, and after GUD in cervical secretions from 4 (11%), 1 (3%), and 6 (17%) women respectively, and in vaginal secretions from 3 (8%), 4 (11%) and 4 (11%) women respectively. After adjustment for time on ART, there was no difference in detection of cervical HIV-1 RNA before versus during GUD (aOR 0.22, 95% CI 0.04–1.23). Likewise, GUD did not increase HIV-1 detection in vaginal secretions (adjusted odds ratio [aOR] 1.32, 95% CI 0.29–5.92).

Conclusions

GUD did not significantly increase cervical or vaginal HIV-1 shedding. Our results suggest that ART maintains its effectiveness for genital HIV-1 suppression despite GUD episodes.

Resistance to HIV infection in a cohort of commercial sex workers living in Nairobi, Kenya, is linked to mucosal and antiinflammatory factors that may be influenced by the vaginal microbiota. Since bacterial vaginosis (BV), a polymicrobial dysbiosis characterized by low levels of protective Lactobacillus organisms, is an established risk factor for HIV infection, we investigated whether vaginal microbiology was associated with HIV-exposed seronegative (HESN) or HIV-seropositive (HIV+) status in this cohort. A subset of 44 individuals was selected for deep-sequencing analysis based on the chaperonin 60 (cpn60) universal target (UT), including HESN individuals (n = 16), other HIV-seronegative controls (HIV-N, n = 16), and HIV+ individuals (n = 12). Our findings indicate exceptionally high phylogenetic resolution of the cpn60 UT using reads as short as 200 bp, with 54 species in 29 genera detected in this group. Contrary to our initial hypothesis, few differences between HESN and HIV-N women were observed. Several HIV+ women had distinct profiles dominated by Escherichia coli. The deep-sequencing phylogenetic profile of the vaginal microbiota corresponds closely to BV+ and BV− diagnoses by microscopy, elucidating BV at the molecular level. A cluster of samples with intermediate abundance of Lactobacillus and dominant Gardnerella was identified, defining a distinct BV phenotype that may represent a transitional stage between BV+ and BV−. Several alpha- and betaproteobacteria, including the recently described species Variovorax paradoxus, were found to correlate positively with increased Lactobacillus levels that define the BV− (“normal”) phenotype. We conclude that cpn60 UT is ideally suited to next-generation sequencing technologies for further investigation of microbial community dynamics and mucosal immunity underlying HIV resistance in this cohort.

Persistent genital HIV-1 shedding among women taking antiretroviral therapy (ART) may present a transmission risk. We investigated associations between genital HIV-1 suppression after ART initiation and adherence, resistance, pre-treatment CD4 count, and hormonal contraceptive use. First-line ART was initiated in 102 women. Plasma and genital HIV-1 RNA were measured at months 0, 3, and 6. Adherence was a strong and consistent predictor of genital HIV-1 suppression (p<0.001), while genotypic resistance was associated with higher vaginal HIV-1 RNA at 6 months (p=0.04). These results emphasize the importance of adherence to optimize the potential benefits of ART for reducing HIV-1 transmission risk.

Background

Cervicitis increases the quantity of HIV-1 RNA in cervical secretions when women are not taking antiretroviral therapy (ART), and successful treatment of cervicitis reduces HIV-1 shedding in this setting.

Objective

To determine the effect of acquisition and treatment of cervical infections on genital HIV-1 shedding in women receiving ART.

Design

Prospective cohort study.

Methods

We followed 147 women on ART monthly for incident non-specific cervicitis, gonorrhea, and chlamydia. Cervical swabs for HIV-1 RNA quantitation were collected at every visit. The lower limit for linear quantitation was 100 copies/swab. We compared the prevalence of HIV-1 RNA detection before (baseline) versus during and after treatment of cervical infections.

Results

Thirty women contributed a total of 31 successfully treated episodes of non-specific cervicitis (N=13), gonorrhea (N=17), and chlamydia (N=1). HIV-1 RNA was detected in cervical secretions before, during, and after cervicitis at 1 (3.2%), 5 (16.1%), and 3 (9.7%) visits respectively. Compared to baseline, detection of HIV-1 RNA was increased when cervical infections were present (adjusted odds ratio 5.7, 95% confidence interval 1.0–30.3, P=0.04). However, even in the subset of women with cervical HIV-1 RNA levels above the threshold for quantitation, most had low concentrations during cervical infections (median 115, range 100–820 copies/swab).

Conclusions

While these data show a statistically significant increase in cervical HIV-1 RNA detection when cervical infections are present, most cervical HIV-1 RNA concentrations were near the threshold for detection, suggesting that infectivity remains low. Antiretroviral therapy appears to limit increases in genital HIV-1 shedding caused by cervical infections.

Background

Trichomonas vaginalis has been associated with increased vaginal HIV-1 RNA shedding in antiretroviral therapy (ART)-naïve women. The effect of trichomoniasis on vaginal HIV-1 shedding in ART-treated women has not been characterized. We tested the hypothesis that T. vaginalis infection would increase vaginal HIV-1 RNA shedding in women on ART, and that successful treatment would reduce vaginal HIV-1 RNA levels.

Methods

We conducted a prospective cohort study including monthly follow-up of 147 women receiving ART in Mombasa, Kenya. Those with T. vaginalis infection, defined by the presence of motile trichomonads on vaginal saline wet mount, received treatment with single dose metronidazole (2 g). Test of cure was performed at the next monthly visit. Using the pre-infection visit as the reference category, we compared detection of vaginal HIV-1 RNA before versus during and after infection using generalized estimating equations. A cut-off of 100 HIV-1 RNA copies/swab was used as the lower limit for linear quantitation.

Results

Among 31 women treated for trichomoniasis, the concentration of vaginal HIV-1 RNA was above the limit for quantitation before, during, and after T. vaginalis infection in 4 (13% [95% CI 4% - 30%]), 4 (13% [95% CI 4% - 30%]), and 5 (16% [95% confidence interval {CI} 5% - 34%]) women respectively. After adjusting for potential confounding factors, we could detect no difference in the likelihood of detecting vaginal HIV-1 RNA before versus during infection (odds ratio [OR] 1.41, 95% CI 0.23 - 8.79, p = 0.7). In addition, detection of HIV-1 RNA was similar before infection versus after successful treatment (OR 0.68, 95% CI (0.13 - 3.45), p = 0.6).

Conclusion

Detection of vaginal HIV-1 RNA during ART was uncommon at visits before, during and after T. vaginalis infection.

Objective

The objective of this study was to test the hypothesis that sexual risk behaviour would increase following initiation of antiretroviral therapy (ART) in Kenyan female sex workers (FSWs).

Design

Prospective cohort study.

Setting

FSW cohort in Mombasa, Kenya, 1993-2008.

Subjects

898 women contributed HIV-1-seropositive follow-up visits, of whom 129 initiated ART.

Intervention

Beginning in March 2004, ART was provided to women qualifying for treatment according to Kenyan National Guidelines. Participants received sexual risk reduction education and free condoms at every visit.

Main Outcome Measures

Main outcome measures included unprotected intercourse, abstinence, 100% condom use, number of sexual partners, and frequency of sex. Outcomes were evaluated at monthly follow-up visits using a one week recall interval.

Results

Compared to non-ART-exposed follow-up, visits following ART initiation were not associated with an increase in unprotected sex (adjusted odds ratio [AOR] 0.86, 95% confidence interval [CI] 0.62-1.19, P=0.4). There was a non-significant decrease in abstinence (AOR 0.81, 95% CI 0.65-1.01, P=0.07), which was offset by a substantial increase in 100% condom use (AOR 1.54, 95% CI 1.07-2.20, P=0.02). Numbers of sex partners and frequency of sex were similar before versus after starting ART. A trend for decreased sexually transmitted infections following ART initiation provides additional support for the validity of the self-reported behavioural outcomes (AOR 0.67, 95% CI 0.44-1.02, P=0.06).

Conclusions

In the setting of ongoing risk reduction education and provision of free condoms, initiation of ART was not associated with increased sexual risk behaviour in this cohort of Kenyan FSWs.

The origin of broadly neutralizing HIV-specific antibodies and their relation to HIV evolution are not well defined. Here we examined virus evolution and neutralizing antibody escape in a subtype A infected individual with a broad, cross subtype, antibody response. The majority of envelope variants isolated over the first ~ 5 years post-infection were poorly neutralized by contemporaneous plasma that neutralized variants from earlier in infection, consistent with a dynamic process of escape. The majority of variants could be neutralized by later plasma, suggesting these evolving variants may have contributed to the elicitation of new antibody responses. However, some variants from later in infection were recognized by plasma from earlier in infection, including one notably neutralization-sensitive variant that was sensitive due to a proline at position 199 in V2. These studies suggest a complex pattern of virus evolution in this individual with a broad NAb response, including persistence of neutralization-sensitive viruses.

Background

With the persistent challenges towards controlling the HIV epidemic, there is an ongoing need for research into HIV vaccines and drugs. Sub-Saharan African countries - worst affected by the HIV pandemic - have participated in the conduct of clinical trials for HIV vaccines. In Kenya, the Kenya AIDS Vaccine Initiative (KAVI) at the University of Nairobi has conducted HIV vaccine clinical trials since 2001.

Methodology

Participants were recruited after an extensive informed consent process followed by screening to determine eligibility. Screening included an assessment of risk behavior, medical history and physical examination, and if clinically healthy, laboratory testing. In the absence of locally derived laboratory reference ranges, the ranges used in these trials were derived from populations in the West.

Principal findings

Two hundred eighty-one participants were screened between 2003 and 2006 for two clinical trials. Of these, 167 (59.4%) met the inclusion/exclusion criteria. Overall, laboratory abnormalities based on the non-indigenous laboratory references used were the most frequent reasons (61.4%) for ineligibility. Medical abnormalities contributed 30.7% of the total reasons for ineligibility. Based on the laboratory reference intervals now developed from East and Southern Africa, those ineligible due to laboratory abnormalities would have been 46.3%. Of the eligible participants, 18.6% declined enrolment.

Conclusions

Participant recruitment for HIV vaccine clinical trials is a rigorous and time-consuming exercise. Over 61% of the screening exclusions in clinically healthy people were due to laboratory abnormalities. It is essential that laboratory reference ranges generated from local populations for laboratory values be used in the conduct of clinical trials to avoid unnecessary exclusion of willing participants and to avoid over-reporting of adverse events for enrolled participants.

Trial registration

Protocol IAVI VRC V001 [1]. ClinicalTrials.gov NCT00124007 Protocol IAVI 010 [2] (registration with ClincalTrials.gov is in progress)

Protocols IAVI 002 and IAVI 004 are Phase 1 trials only mentioned in introductory paragraphs; details will not be reported. Registration was not required when they were conducted.

Introduction

Genital ulcer disease (GUD) is common in HIV-1-infected women, and a small number of studies have suggested increased GUD risk after antiretroviral therapy (ART) initiation. To better define this risk, we monitored 134 women at ART initiation and monthly thereafter.

Methods

Women were evaluated monthly for genital ulcers. Syphilis serology was tested quarterly, and chancroid culture performed on ulcers that were felt to be clinically consistent with a diagnosis of chancroid. A logistic model with generalized estimating equations was used to analyze predictors of GUD from baseline until 6 months after ART initiation.

Results

During the study period, GUD occurred in 54 women (40.3%) at 85 visits (10.0%). GUD prevalence was 9.7% at baseline, increased to 16.7% at month 1 (adjusted odds ratio [aOR] 1.9 [1.0 – 3.6], p = 0.04), then decreased to 6.4% by month 6. History of GUD (aOR 3.8 [1.9 – 7.7], p < 0.001) and CD4 count <100 (aOR 1.8 [1.0 – 3.4, p = 0.06) were associated with increased risk of GUD after ART initiation.

Discussion

Women experience increased risk of GUD in the first month after ART initiation, particularly if they have low CD4 counts or a history of GUD.

Objectives

Several studies have demonstrated an association between herpes simplex virus type 2 (HSV-2) and human immunodeficiency virus type 1 (HIV-1), but limited data on risk factors for HSV-2 acquisition are available. The objective of this analysis was to determine the incidence and risk factors for HSV-2 acquisition among HIV-1-seronegative Kenyan female sex workers.

Methods

Between February 1993 and December 2006, HIV-1-seronegative women attending a municipal sexually transmitted infections (STI) clinic were invited to enroll in a prospective cohort study. Screening for HIV-1 and STIs were done at monthly follow-up visits. Archived blood samples were tested for HSV-2.

Results

Of 1527 HIV-1-seronegative women enrolled, 302 (20%) were HSV-2-seronegative at baseline, of whom, 297 had at least one follow-up visit. HSV-2 incidence was high (23 cases/100 person-years; 115 cases). In multivariate analysis, HSV-2 was significantly associated with more recent entry into sex work, workplace, and higher number of sex partners per week. Condom use was protective, although this was statistically significant only for the intermediate strata (25–75% condom use, HR 0.43, p=0.05). There were statistical trends for bacterial vaginosis to increase HSV-2 risk (HR 1.56, p=0.07) and for oral contraceptive use to decrease risk (HR 0.50, p=0.08). The 23% annual HSV-2 incidence in this study is among the highest reported anywhere in the world.

Conclusions

Women were at increased risk if they had recently entered sex work, had a higher number of sex partners, or worked in bars. HSV-2 risk reduction interventions are urgently needed among high-risk African women.

Several candidate HIV vaccines aim to induce virus-specific cellular immunity particularly in the genital tract, typically the initial site of HIV acquisition. However, standardized and sensitive methods for evaluating HIV-specific immune responses at the genital level are lacking. Therefore we evaluated real-time quantitative PCR (qPCR) as a potential platform to measure these responses. β-Actin and GAPDH were identified as the most stable housekeeping reference genes in peripheral blood mononuclear cells (PBMCs) and cervical mononuclear cells (CMCs) respectively and were used for normalizing transcript mRNA expression. HIV-specific cellular T cell immune responses to a pool of optimized CD8+ HIV epitopes (HIV epitope pool) and Staphylococcal enterotoxin B (SEB) superantigen control were assayed in HIV infected PBMC by qPCR, with parallel assessment of cytokine protein production. Peak HIV-specific mRNA expression of IFNγ, IL-2 and TNFα occurred after 3, 5 and 12 hours respectively. PBMCs were titrated to cervical appropriate cell numbers to determine minimum required assay input cell numbers; qPCR retained sensitivity with input of at least 2.5×104 PBMCs. This optimized qPCR assay was then used to assess HIV-specific cellular T cell responses in cytobrush-derived cervical T cells from HIV positive individuals. SEB induced IFNγ mRNA transcription was detected in CMCs and correlated positively with IFNγ protein production. However, qPCR was unable to detect HIV-induced cytokine mRNA production in the cervix of HIV-infected women despite robust detection of gene induction in PBMCs. In conclusion, although qPCR can be used to measure ex vivo cellular immune responses to HIV in blood, HIV-specific responses in the cervix may fall below the threshold of qPCR detection. Nonetheless, this platform may have a potential role in measuring mitogen-induced immune responses in the genital tract.

Background

We conducted a double-blind, randomized, placebo-controlled Phase I study of a recombinant replication-defective adenovirus type 5 (rAd5) vector expressing HIV-1 Gag and Pol from subtype B and Env from subtypes A, B and C, given alone or as boost following a DNA plasmid vaccine expressing the same HIV-1 proteins plus Nef, in 114 healthy HIV-uninfected African adults.

Methodology/Principal Findings

Volunteers were randomized to 4 groups receiving the rAd5 vaccine intramuscularly at dosage levels of 1×1010 or 1×1011 particle units (PU) either alone or as boost following 3 injections of the DNA vaccine given at 4 mg/dose intramuscularly by needle-free injection using Biojector® 2000. Safety and immunogenicity were evaluated for 12 months. Both vaccines were well-tolerated. Overall, 62% and 86% of vaccine recipients in the rAd5 alone and DNA prime - rAd5 boost groups, respectively, responded to the HIV-1 proteins by an interferon-gamma (IFN-γ) ELISPOT. The frequency of immune responses was independent of rAd5 dosage levels. The highest frequency of responses after rAd5 alone was detected at 6 weeks; after DNA prime - rAd5 boost, at 6 months (end of study). At baseline, neutralizing antibodies against Ad5 were present in 81% of volunteers; the distribution was similar across the 4 groups. Pre-existing immunity to Ad5 did not appear to have a significant impact on reactogenicity or immune response rates to HIV antigens by IFN-γ ELISPOT. Binding antibodies against Env were detected in up to 100% recipients of DNA prime - rAd5 boost. One volunteer acquired HIV infection after the study ended, two years after receipt of rAd5 alone.

Conclusions/Significance

The HIV-1 rAd5 vaccine, either alone or as a boost following HIV-1 DNA vaccine, was well-tolerated and immunogenic in African adults. DNA priming increased the frequency and magnitude of cellular and humoral immune responses, but there was no effect of rAd5 dosage on immunogenicity endpoints.

Trial Registration

ClinicalTrials.gov NCT00124007

Objective

Vaginal colonization with Lactobacillus species is characteristic of normal vaginal ecology. The absence of vaginal lactobacilli, particularly hydrogen peroxide (H2O2)-producing isolates, has been associated with symptomatic bacterial vaginosis (BV) and increased risk for HIV-1 acquisition. Identification of factors associated with vaginal Lactobacillus colonization may suggest interventions to improve vaginal health.

Methods

We conducted a prospective cohort study of correlates of vaginal Lactobacillus colonization among Kenyan HIV-1 seronegative female sex workers. At monthly follow-up visits, vaginal Lactobacillus cultures were obtained. Generalized estimating equations were used to examine demographic, behavioral, and medical correlates of Lactobacillus isolation, including isolation of H2O2-producing strains.

Results

Lactobacillus cultures were obtained from 1020 women who completed a total of 8896 follow-up visits. Vaginal washing, typically with water alone or with soap and water, was associated with an approximately 40% decreased likelihood of Lactobacillus isolation, including isolation of H2O2-producing strains. Recent antibiotic use, excluding metronidazole and treatments for vaginal candidiasis, reduced Lactobacillus isolation by ~30%. H2O2-producing lactobacilli were significantly less common among women with Trichomonas vaginalis infection and those who were seropositive for herpes simplex virus type 2. In contrast, H2O2-producing lactobacilli were significantly more common among women with concurrent vaginal candidiasis.

Conclusions

Modifiable biologic and behavioral factors are associated with Lactobacillus colonization in African women. Our results suggest intervention strategies to improve vaginal health in women at high risk for HIV-1.

Background:

It has been suggested that vaginal lactobacilli may reduce the risk of vulvovaginal candidiasis (VVC), but supporting data are limited. Our objective was to determine the relationship between vaginal bacterial flora and VVC.

Methods:

We conducted a prospective cohort analysis among 151 Kenyan sex workers. At monthly follow-up, VVC was defined as the presence of yeast buds, pseudohyphae, or both on vaginal wet preparation or KOH preparation. Generalized estimating equations were used to identify correlates of VVC.

Results:

Participants returned for a median of 12 (interquartile range 11-12) visits. Vulvovaginal candidiasis was present at 162 visits, including 26 with symptomatic VVC. Bacterial vaginosis (BV) was associated with fewer episodes of VVC (adjusted odds ratio [aOR] 0.29, 95% confidence interval [CI] 0.16-0.50). After excluding women with concurrent BV, another possible cause of vaginal symptoms, the likelihood of symptomatic VVC was higher in those with yeast on vaginal wet preparation in the past 60 days (aOR 4.06, 95% CI 1.12-14.74) and those with concurrent vaginal Lactobacillus colonization (aOR 3.75, 95% CI 1.30-10.83).

Conclusions:

Contrary to a commonly posed hypothesis of a protective effect, we found that vaginal Lactobacillus colonization was associated with a >4-fold increase in the likelihood of symptomatic VVC.

Bacterial vaginosis (BV) is a recurrent condition that is associated with a range of negative outcomes, including the acquisition of human immunodeficiency virus and other sexually transmitted diseases, preterm births, and pelvic inflammatory disease. In contrast to the Lactobacillus-dominated normal vaginal microbiota, BV is characterized by a lack of lactobacilli and an abundance of anaerobic and gram-negative organisms, including Gardnerella vaginalis and Atopobium vaginae. To date, the laboratory diagnosis of BV has relied upon the fulfillment of criteria determined by microscopic observation of Gram-stained vaginal swabs. We describe a molecular-based method for the easy determination of the species profile within the vaginal microbiota based on the amplification of the chaperonin-60 genes of all bacteria present in the swab and hybridization of the amplicon to species-specific oligonucleotide-coupled fluorescent beads that are identified by flow cytometry with a Luminex instrument. We designed a nineplex Luminex array for characterization of the vaginal microbiota and applied it to the analysis of vaginal swabs from individuals from Africa and North America. Using the presence of A. vaginae or G. vaginalis, or both, as the defining criterion for BV, we found that the method was highly specific and sensitive for the diagnosis of BV using microscopy as a gold standard.

HLA-B*57-mediated selection pressure leads to a typical escape pathway in human immunodeficiency virus type 1 (HIV-1) CD8 epitopes such as TW10. Whether this T242N pathway is shared by all clades remains unknown. We therefore assessed the nature of HLA-B*57 selection in a large, observational Kenyan cohort where clades A1 and D predominate. While T242N was ubiquitous in clade D HLA-B*57+ subjects, this mutation was rare (15%) in clade A1. Instead, P243T and I247L were selected by clade A1-infected HLA-B*57 subjects but not by HLA-B*5801+ subjects. Our data suggest that clade A1 consensus proline at Gag residue 243 might represent an inherent block to T242N escape in clade A1. We confirmed immunologically that P243T and I247L likely represent escape mutations. HLA-B*57 evolution also differed between clades in the KF11 and IW9 epitopes. A better understanding of clade-specific evolution is important for the development of HIV vaccines in regions with multiple clades.

The determinants of a broad neutralizing antibody (NAb) response and its effect on human immunodeficiency virus type 1 (HIV-1) disease progression are not well defined, partly because most prior studies of a broad NAb response were cross-sectional. We examined correlates of NAb response breadth among 70 HIV-infected, antiretroviral-naïve Kenyan women from a longitudinal seroincident cohort. NAb response breadth was measured 5 years after infection against five subtype A viruses and one subtype B virus. Greater NAb response breadth was associated with a higher viral load set point and greater HIV-1 env diversity early in infection. However, greater NAb response breadth was not associated with a delayed time to a CD4+ T-cell count of <200, antiretroviral therapy, or death. Thus, a broad NAb response results from a high level of antigenic stimulation early in infection, which likely accounts for prior observations that greater NAb response breadth is associated with a higher viral load later in infection.