Progesterone Levels Associate with a Novel Population of CCR5+CD38+ CD4 T Cells Resident in the Genital Mucosa with Lymphoid Trafficking Potential.

The female genital tract (FGT) provides a means of entry to pathogens, including HIV, yet immune cell populations at this barrier between host and environment are not well defined. We initiated a study of healthy women to characterize resident T cell populations in the lower FGT from lavage and patient-matched peripheral blood to investigate potential mechanisms of HIV sexual transmission. Surprisingly, we observed FGT CD4 T cell populations were primarily CCR7(hi), consistent with a central memory or recirculating memory T cell phenotype. In addition, roughly half of these CCR7(hi) CD4 T cells expressed CD69, consistent with resident memory T cells, whereas the remaining CCR7(hi) CD4 T cells lacked CD69 expression, consistent with recirculating memory CD4 T cells that traffic between peripheral tissues and lymphoid sites. HIV susceptibility markers CCR5 and CD38 were increased on FGT CCR7(hi) CD4 T cells compared with blood, yet migration to the lymphoid homing chemokines CCL19 and CCL21 was maintained. Infection with GFP-HIV showed that FGT CCR7(hi) memory CD4 T cells are susceptible HIV targets, and productive infection of CCR7(hi) memory T cells did not alter chemotaxis to CCL19 and CCL21. Variations of resident CCR7(hi) FGT CD4 T cell populations were detected during the luteal phase of the menstrual cycle, and longitudinal analysis showed the frequency of this population positively correlated to progesterone levels. These data provide evidence women may acquire HIV through local infection of migratory CCR7(hi) CD4 T cells, and progesterone levels predict opportunities for HIV to access these novel target cells.

Role of donor genital tract HIV-1 diversity in the transmission bottleneck.

The predominant mode of HIV-1 infection is heterosexual transmission, where a genetic bottleneck is imposed on the virus quasispecies. To probe whether limited genetic diversity in the genital tract (GT) of the transmitting partner drives this bottleneck, viral envelope sequences from the blood and genital fluids of eight transmission pairs from Rwanda and Zambia were analyzed. The chronically infected transmitting partner's virus population was heterogeneous with distinct genital subpopulations, and the virus populations within the GT of two of four women sampled longitudinally exhibited evidence of stability over time intervals on the order of weeks to months. Surprisingly, the transmitted founder variant was not derived from the predominant GT subpopulations. Rather, in each case, the transmitting variant was phylogenetically distinct from the sampled locally replicating population. Although the exact distribution of the virus population present in the GT at the time of transmission cannot be unambiguously defined in these human studies, it is unlikely, based on these data, that the transmission bottleneck is driven in every case by limited viral diversity in the donor GT or that HIV transmission is solely a stochastic event.

UC781 microbicide gel retains anti-HIV activity in cervicovaginal lavage fluids collected following twice-daily vaginal application.

The potent nonnucleoside reverse transcriptase inhibitor UC781 has been safety tested as a vaginal microbicide gel formulation for prevention of HIV-1 sexual transmission. To investigate whether UC781 retained anti-infective activity following exposure to the female genital tract, we conducted an ex vivo analysis of the UC781 levels and antiviral activity in cervicovaginal lavage (CVL) fluids from 25 Thai women enrolled in a 14-day safety trial of twice-daily vaginal application of two concentrations of the UC781 microbicide gel. CVL samples were collected from women in the 0.1% (n = 5), 0.25% (n = 15), and placebo (n = 5) gel arms following the first application of gel (T(15 min)) and 8 to 24 h after the final application (T(8-24 h)) and separated into cell-free (CVL-s) and pelletable (CVL-p) fractions. As UC781 is highly hydrophobic, there were significantly higher levels of UC781 in the CVL-p samples than in the CVL-s samples for the UC781 gel arms. In T(8-24 h) CVL-p samples, 2/5 and 13/15 samples collected from the 0.1% and 0.25% UC781 gel arms, respectively, efficiently blocked infection with ≥ 4 log(10) 50% tissue culture infective dose (TCID(50)) of a CCR5-tropic CRF01_AE HIV-1 virus stock. Independent of the arm, the 11 CVL-p samples with UC781 levels of ≥ 5 µg/CVL sample reduced infectious HIV by ≥ 4 log(10) TCID(50). Our results suggest that the levels and anti-infective activities of UC781 gel formulations are likely to be associated with a cellular or pelletable component in CVL samples. Therefore, cellular and pelletable fractions should be assayed for drug levels and anti-infective activity in preclinical studies of candidate microbicides.

HIV-1 RNA rectal shedding is reduced in men with low plasma HIV-1 RNA viral loads and is not enhanced by sexually transmitted bacterial infections of the rectum.

BACKGROUND: Among human immunodeficiency virus (HIV)-infected men who have sex with men (MSM) taking combination antiretroviral therapy (cART), the impact of rectal sexually transmitted infections (STIs) on rectal HIV-1 shedding is unknown. METHODS: Human immunodeficiency virus type 1 (HIV-1) RNA was quantified from rectal swabs collected for Neisseria gonorrhoeae (GC) and Chlamydia trachomatis (CT) screening of HIV-1-infected MSM. Correlations of STIs with rectal viral load were explored using multinomial regression modeling. HIV-1 coreceptor tropism was predicted from sequencing in a subset of men. RESULTS: Thirty-one (39%) of 80 men (59 prescribed combination antiretroviral therapy [cART]) had HIV detected in 38 (42%) of 91 rectal swabs. Rectal HIV detection was associated with plasma virus loads above 3.15 log10 copies/mL (95% confidence limit [CL] 2.73, 3.55) and paired rectal viral loads and plasma viral loads were correlated (Kendall's tau [τ] 0.68, Spearman rho [P] = .77). Rectal STIs and abnormal anal cytology were not associated with rectal viral load. HIV coreceptor distribution was very similar between the plasma and rectum in 3 of 4 men. CONCLUSIONS: Plasma and rectal viral load were correlated, and rectal STIs did not increase the likelihood of detecting HIV in the rectal secretions in MSM, including those with low or undetectable plasma viral load. Suppressing plasma viral load is likely to reduce risk of HIV transmission to insertive partners.

Impact of etonogestrel implant use on T-cell and cytokine profiles in the female genital tract and blood.

BACKGROUND: While prior epidemiologic studies have suggested that injectable progestin-based contraceptive depot medroxyprogesterone acetate (DMPA) use may increase a woman's risk of acquiring HIV, recent data have suggested that DMPA users may be at a similar risk for HIV acquisition as users of the copper intrauterine device and levonorgestrel implant. Use of the etonogestrel Implant (Eng-Implant) is increasing but there are currently no studies evaluating its effect on HIV acquisition risk. OBJECTIVE: Evaluate the potential effect of the Eng-Implant use on HIV acquisition risk by analyzing HIV target cells and cytokine profiles in the lower genital tract and blood of adult premenopausal HIV-negative women using the Eng-Implant. METHODS: We prospectively obtained paired cervicovaginal lavage (CVL) and blood samples at 4 study visits over 16 weeks from women between ages 18-45, with normal menses (22-35 day intervals), HIV uninfected with no recent hormonal contraceptive or copper intrauterine device (IUD) use, no clinical signs of a sexually transmitted infection at enrollment and who were medically eligible to initiate Eng-Implant. Participants attended pre-Eng-Implant study visits (week -2, week 0) with the Eng-Implant inserted at the end of the week 0 study visit and returned for study visits at weeks 12 and 14. Genital tract leukocytes (enriched from CVL) and peripheral blood mononuclear cells (PBMC) from the study visits were evaluated for markers of activation (CD38, HLA-DR), retention (CD103) and trafficking (CCR7) on HIV target cells (CCR5+CD4+ T cells) using multicolor flow cytometry. Cytokines and chemokines in the CVL supernatant and blood plasma were measured in a Luminex assay. We estimated and compared study endpoints among the samples collected before and after contraception initiation with repeated-measures analyses using linear mixed models. RESULTS: Fifteen of 18 women who received an Eng-Implant completed all 4 study visits. The percentage of CD4+ T cells in CVL was not increased after implant placement but the percentage of CD4+ T cells expressing the HIV co-receptor CCR5 did increase after implant placement (p = 0.02). In addition, the percentage of central memory CD4+ T-cells (CCR7+) in CVL increased after implant placement (p = 0.004). The percentage of CVL CD4+, CCR5+ HIV target cells expressing activation markers after implant placement was either reduced (HLA-DR+, p = 0.01) or unchanged (CD38+, p = 0.45). Most CVL cytokine and chemokine concentrations were not significantly different after implant placement except for a higher level of the soluble lymphocyte activation marker (sCD40L; p = 0.04) and lower levels of IL12p70 (p = 0.02) and G-CSF (p<0.001). In systemic blood, none of the changes noted in CVL after implant placement occurred except for decreases in the percentage CD4 T-cells expressing HLA-DR+ T cells (p = 0.006) and G-CSF (p = 0.02). CONCLUSIONS: Eng-Implant use was associated with a moderate increase in the availability of HIV target cells in the genital tract, however the percentage of these cells that were activated did not increase and there were minimal shifts in the overall immune environment. Given the mixed nature of these findings, it is unclear if these implant-induced changes alter HIV risk.

Repeated rectal application of a hyperosmolar lubricant is associated with microbiota shifts but does not affect PrEP drug concentrations: results from a randomized trial in men who have sex with men.

INTRODUCTION: Oral pre-exposure prophylaxis (PrEP) with tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) is highly effective in preventing HIV infection among men who have sex with men (MSM). The effects of consistent personal lubricant use in the rectum on tissue PrEP drug concentrations and the rectal microbiota are unknown. We investigated rectal PrEP drug concentrations and the microbiota in MSM before and after repeated rectal application of a hyperosmolar lubricant. METHODS: We randomized 60 HIV-negative MSM to apply 4 mL of hyperosmolar rectal lubricant daily (n = 20), take daily oral TDF/FTC (n = 19), or both (n = 21) for seven days. Blood, rectal biopsies and rectal secretions were collected via rigid sigmoidoscopy before and on day 8 after product use. Tenofovir (TFV) and FTC as well as their intracellular metabolites tenofovir-diphosphate (TFV-DP), FTC-triphosphate (FTC-TP) were measured by HPLC-mass spectrometry. Rectal mucosal microbiota was sequenced with 16S rRNA sequencing using Illumina MiSeq. RESULTS: Seven days of lubricant application was not associated with differences in PrEP drug concentrations in rectal tissue or secretions. Lubricant use was associated with a decrease in the relative abundance of the Bacteroides genus (p = 0.01) and a non-significant increase in the Prevotella genus (p = 0.09) in the rectum. PrEP drug concentrations in rectal tissue and secretions were not associated with microbiota composition or diversity either before or after lubricant use. CONCLUSIONS: Repeated rectal application of a hyperosmolar lubricant does not affect mucosal PrEP drug concentrations but is associated with changes in the rectal microbiome.

Mucosal innate immune factors in the female genital tract are associated with vaginal HIV-1 shedding independent of plasma viral load.

Recent studies indicate that mucosal innate immune factors modulate HIV-1 infection in vitro. Our interest was to examine the levels of innate mucosal factors for their potential association with HIV-1 shedding in the female genital tract. Vaginal lavages were collected from HIV-1-infected women who had vaginal viral loads (VVL) that were below, within, or above the 90% confidence interval (CI) predicted by their matched plasma viral loads. Innate immune factors [cathepsin D, lactoferrin (Lf), myeloid related protein (MRP)-8, MRP-8/14, secretory leukocyte protease inhibitor, and gp340], cytokines (IL-1beta and TNF-alpha), and chemokines (MIP-1alpha, MIP-1beta, RANTES, and SDF-1alpha) were quantified by ELISA. Leukocyte levels were determined using a leukocyte reagent strip for urinalysis. Lf, MRP-8/14, gp340, and IL-1beta levels were significantly higher in vaginal lavages above the 90% CI and generally correlated with each other and with VVL. Leukocyte levels were significantly higher in the lavages that had virus shedding above the 90% CI and correlated strongly with Lf levels and VVL. In this group of women, these results suggest that the levels of certain innate immune factors are more closely associated with HIV-1 shedding in the genital mucosa than plasma virus concentrations.

Female genital tract shedding of CXCR4-tropic HIV Type 1 is associated with a majority population of CXCR4-tropic HIV Type 1 in blood and declining CD4(+) cell counts.

This study compared HIV-1 genotypes shed over time (≤3.5 years) in the vaginal secretions (VS) and blood plasma (BP) of 15 chronically infected women. Analysis of predicted coreceptor tropism (CCR5=R5, CXCR4=X4) for quasispecies shedding revealed three patterns: (1) viral quasispecies shed in both VS and BP were restricted to R5-tropism at all time points, (2) quasispecies shed in VS were restricted to R5-tropism at all time points but X4 quasispecies were identified in the BP at one or more time points, and (3) quasispecies shed in matched VS and BP both contained X4-tropic viruses. Overall, the frequency of X4 quasispecies circulation in VS was 2-fold less than in BP and detection of X4 virus in VS was more likely to occur when X4 quasispecies comprised more than 50% of BP viruses (p=0.01) and when declines in blood CD4(+) lymphocyte levels were the greatest (p=0.038). Additionally, the mean number of predicted N-glycosylation sites between matched VS and BP samples was strongly correlated (r=0.86, p<0.0001) with glycosylation densities in the following order (VS R5=BP R5 > BP X4 > VS X4). The X4 glycosylation densities may result from compartmentalization pressures in the female genital tract or the delayed appearance of these viruses in VS. Our results suggest that the presence of X4 virus in VS is associated with a threshold population of X4 quasispecies in BP, which are increasing during the HIV-induced failure of the human immune system.

Lack of effect of compartmentalized drug resistance mutations on HIV-1 pol divergence in antiretroviral-experienced women.

OBJECTIVE: To examine the persistence of compartmentalized HIV drug resistance mutations (DRM) over time in the female genital tract and its effect on pol gene divergence compared to that in blood. DESIGN: Longitudinal cohort of 22 antiretroviral-experienced women in the Emory Vaginal Ecology study. METHODS: Blood and vaginal secretions were collected at serial clinic visits. DRM in the HIV reverse transcriptase and protease regions of pol were determined using population based sequencing. Kimura-2 pairwise DNA distances were calculated to measure blood and vaginal secretions divergence in the intervals between clinic visits. RESULTS: Only eight (36%) women had compartmentalized DRM detected at 14 (31%) of their 45 clinic visits. This compartmentalized resistance was transient; 13 of 14 mutations in blood and all 12 mutations in vaginal secretions were compartmentalized for only one clinic visit. Over time, divergence of both reverse transcriptase and protease were greater in vaginal secretions than in blood. However, divergence in blood, but not in vaginal secretions, increased significantly in the presence of drug resistance or compartmentalized drug resistance. CONCLUSION: Compartmentalized DRM between the blood and vaginal secretions are transient in nature, and the presence of DRM does not affect pol gene divergence in the vaginal secretions. Our results provide new evidence that the genital mucosa does not support an independently evolving subpopulation of HIV-1 genomes.

Field experience in implementing ISO 15189 in Kisumu, Kenya.

Quality medical laboratory services are an integral part of routine health care, medical research, and public health systems. Despite this vital role, quality laboratory services in Africa are scarce. The crucial need for expanding quality laboratory services throughout sub-Saharan Africa is especially critical because of the region's burden of disease. Fortunately, several plans from supporting international partners are underway to help strengthen laboratory infrastructure in this region. A key component of these initiatives is the enforcement of quality assurance services through accreditation by international standards such as the International Organization for Standardization (ISO) 15189. However, acquisition and maintenance of these standards are a significant challenge, especially in resource-limited settings. The most common limiting factors can include funding, government support, equipment, training opportunities, and poor procurement infrastructure. In this article, we discuss the challenges and benefits accrued in pursuing and sustaining ISO 15189 accreditation for the Kenya Medical Research Institute/Centre for Disease Control HIV-Research Laboratory in Kisumu, Kenya.

Carraguard Vaginal Gel Safety in HIV-Positive Women and Men in South Africa.

OBJECTIVE: To assess the safety of the candidate microbicide Carraguard gel in HIV-positive women and men. DESIGN: A randomized, placebo-controlled, triple-blinded clinical trial of Carraguard gel when applied vaginally once per day for 14 intermenstrual days by sexually abstinent and sexually active HIV-positive women; and when applied directly to the penis once per day for 7 days by sexually abstinent HIV-positive men. METHODS: In each cohort (n = 20 per cohort), participants were randomized to Carraguard, methylcellulose placebo, or no product (1:1:1). In addition to traditional microbicide trial safety endpoints, the effects of microbicide use on vaginal shedding of HIV-1 RNA and markers of genital inflammation, epithelial sloughing, and microhemorrhage were also explored. RESULTS: Gel compliance was high in both gel-use groups in the 3 cohorts. Carraguard use was not associated with abnormal genital findings, other abnormal clinical findings, markers of genital inflammation, epithelial sloughing or microhemorrhage, or self-reported symptoms in women and men, or with abnormal vaginal flora or genital shedding of HIV-1 RNA in women. Adverse events were mostly mild, not attributed to gel use, and similarly distributed between groups. CONCLUSIONS: Once-daily use of Carraguard for 7 to 14 days appeared to be safe in HIV-positive women and men.

Diversity, divergence, and evolution of cell-free human immunodeficiency virus type 1 in vaginal secretions and blood of chronically infected women: associations with immune status.

Most human immunodeficiency virus type 1 (HIV-1) infections are believed to be the result of exposure to the virus in genital secretions. However, prevention and therapeutic strategies are usually based on characterizations of HIV-1 in blood. To understand better the dynamics between HIV-1 quasispecies in the genital tract and blood, we performed heteroduplex assays on amplified env products from cell-free viral RNA in paired vaginal secretion (VS) and blood plasma (BP) samples of 14 women followed for 1.5 to 3.5 years. Diversity and divergence were less in VS than in BP (P = 0.03 and P < 0.01, respectively), and divergence at both sites was correlated with blood CD4(+) cell levels (VS, P = 0.05; BP, P = 0.01). Evolution of quasispecies was observed in 58% of the women; the loss or gain of quasispecies in VS or BP was always accompanied by such changes at the other site. In addition, sustained compartmentalization of quasispecies in VS was found for four women, even as CD4(+) cell levels decreased to low levels (<50 cells/microl). Quasispecies changes over time were associated with fluctuations in CD4(+) cell levels; concordant increases or decreases in VS and BP divergence had greater CD4(+) cell level changes than intervals with discordant changes (P = 0.05), and women with evolving quasispecies had greater decreases in CD4(+) cell levels compared to that for women who maintained the same quasispecies (P < 0.05). Thus, diversity, divergence, and evolution of cell-free HIV-1 in VS can be different from that in BP, and dynamics between their respective quasispecies are associated with changes in CD4(+) cell levels.

A human colorectal explant culture to evaluate topical microbicides for the prevention of HIV infection.

A human colorectal explant culture was developed to assess the safety and efficacy of topical microbicides proposed for use in humans. Because any product marketed for vaginal application will likely be used for anal intercourse, it is important to evaluate these products in colorectal explant tissue. Microbicides tested included cellulose acetate 1,2-benzenedicarboxylate (CAP), PRO 2000, SPL7013, Vena Gel, and UC781, along with their accompanying placebos. Colorectal tissues were exposed to microbicides overnight and either fixed in formalin to evaluate toxicity by histological analysis or placed in 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) to quantitatively determine tissue viability. Histological analysis showed minimal toxicity for CAP, UC781, and Vena Gel. Shedding of epithelium with intact lamina propria occurred for the PRO 2000 and SPL7013 products, and shedding of epithelium and necrosis of the lamina propria occurred in explants cultured with nonoxynol-9. The MTT assay confirmed these results for PRO 2000 (4% and 0.5%), SPL7013 (and placebo), and nonoxynol-9 but also demonstrated reduced viability for CAP. However, viability of tissues treated with all products was not significantly different from that of the medium control. Efficacy of the microbicides was evaluated by measuring human immunodeficiency virus type 1 (HIV-1) infection of explants in the absence or presence of products. All microbicide formulations tested were highly effective in preventing HIV infection. However, explants treated with some of the placebo formulations also exhibited a lower level of infection. Most of the products developed for vaginal application showed minimal toxicity and were effective in reducing HIV-1 infection in colorectal tissues. These results suggest that this model is useful for evaluating the safety and efficacy of topical microbicides when used rectally.

In vitro comparison of topical microbicides for prevention of human immunodeficiency virus type 1 transmission.

A standardized protocol was used to compare cellular toxicities and anti-human immunodeficiency virus type 1 (HIV-1) activities of candidate microbicides formulated for human use. The microbicides evaluated were cellulose acetate phthalate (CAP), Carraguard, K-Y plus nonoxynol-9 (KY-N9), PRO 2000 (0.5 and 4%), SPL7013 (5%), UC781 (0.1 and 1%), and Vena Gel, along with their accompanying placebos. Products were evaluated for toxicity on cervical and colorectal epithelial cell lines, peripheral blood mononuclear cells (PBMCs), and macrophages (MPhi) by using an ATP release assay, and they were tested for their effect on transepithelial resistance (TER) of polarized epithelial monolayers. Anti-HIV-1 activity was evaluated in assays for transfer of infectious HIV-1 from epithelial cells to activated PBMCs and for PBMC and MPhi infection. CAP, Carraguard, PRO 2000, SPL7013, and UC781 along with their placebos were 20- to 50-fold less toxic than KY-N9 and Vena Gel. None of the nontoxic product concentrations disrupted the TER. Transfer of HIV-1(Ba-L) from epithelial cells to PBMCs and PBMC and MPhi infection with laboratory-adapted HIV-1(Ba-L) and HIV-1(LAI) isolates were inhibited by all products except Carraguard, KY-N9, and Vena Gel. KY-N9, Vena Gel, and Carraguard were not effective in blocking PBMC infection with primary HIV-1(A), HIV-1(C), and HIV-1(CRF01-AE) isolates. The concordance of these toxicity results with those previously reported indicates that our protocol may be useful for predicting toxicity in vivo. Moreover, our systematic anti-HIV-1 testing provides a rational basis for making better informed decisions about which products to consider for clinical trials.

Detection of infectious human immunodeficiency virus type 1 in female genital secretions by a short-term culture method.

Infectious human immunodeficiency virus type 1 (HIV-1) is difficult to detect in female genital secretions by standard virus culture techniques. To improve detection of cell-free HIV-1 in female genital secretions, we adapted a short-term assay that uses the multinuclear-activation galactosidase indicator (MAGI) assay. When vaginal lavages from HIV-1-infected women were tested with the adapted MAGI assay, 25 (64%) of 39 lavages with detectable, cell-free HIV-1 RNA were shown to have infectious virus. No infectious virus was found in 10 vaginal lavages from HIV-1-infected women with undetectable vaginal viral loads. Significantly (P < 0.01) more lavages from HIV-1-infected women tested positive for infectious virus by the MAGI assay than by standard peripheral blood mononuclear cell (PBMC) coculture, which detected infectious virus in only 6 (17%) of 35 vaginal lavages. Lavages with viral loads of >10,000 copies per lavage yielded significantly (P < 0.01) more positive cultures than those with <10,000 copies by using the MAGI assay. Detection of infectious HIV-1 in vaginal lavages was not associated with the presence of genital tract infections or CD4(+)-T-cell counts. However, although the results were not significant (P = 0.08), the MAGI assay detected infectious virus from more vaginal lavages at a vaginal pH of >/=4.5 than at a pH of <4.5. These results indicate that the MAGI assay is more sensitive than PBMC culture methods for detecting infectious virus in female genital secretions. Accurate measurements of infectious virus in genital secretions will improve studies that evaluate sexual transmission of HIV-1.

The menstrual cycle does not affect human immunodeficiency virus type 1 levels in vaginal secretions.

To determine whether the menstrual cycle affects human immunodeficiency virus (HIV) type 1 levels in vaginal secretions, vaginal lavage samples were collected at 7, 14, and 21 days after initiation of menses, to compare virus levels during the follicular, ovulatory, and luteal phases. During 33 menstrual cycles in 25 women, HIV-1 RNA levels in vaginal secretions ranged from <1000 to 5.3x10(7) copies per lavage, and weekly changes ranged from <0.5 to 2.5 log(10) copies per lavage. HIV-1 RNA levels in vaginal lavage samples from days 7, 14, and 21 were not significantly different. No discernible pattern was found in changes of vaginal virus loads (VVLs) during the menstrual cycle. VVLs were not correlated with plasma estradiol or progesterone levels (P>.05). These results suggest that hormonal changes during the menstrual cycle do not have a significant effect on HIV-1 RNA levels in vaginal secretions.