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.

HIV-1 genital shedding is suppressed in the setting of high genital antiretroviral drug concentrations throughout the menstrual cycle.

BACKGROUND: It is not known if fluctuations in genital tract antiretroviral drug concentrations correlate with genital virus shedding in human immunodeficiency virus (HIV)-infected women on antiretroviral therapy (ART). METHODS: Among 20 HIV-infected women on ART (tenofovir [TFV], emtricitabine [FTC], and ritonavir-boosted atazanavir [ATV]) with suppressed plasma virus loads, blood and cervicovaginal samples collected twice weekly for 3 weeks were tested for antiretroviral concentrations, HIV-1 RNA, and proviral DNA. RESULTS: Cervicovaginal:plasma antiretroviral concentration ratios were highest for FTC (11.9, 95% confidence interval [CI], 8.66-16.3), then TFV (3.52, 95% CI, 2.27-5.48), and ATV (2.39, 95% CI, 1.69-3.38). Within- and between-person variations in plasma and genital antiretroviral concentrations were observed. Low amounts of genital HIV-1 RNA (<50 copies/mL) were detected in 45% of women at 16% of visits. Genital HIV-1 DNA was detected in 70% of women at 35% of visits. Genital virus detection was associated with higher concentrations of mucosal leukocytes but not with genital antiretroviral concentrations, menstrual cycle phase, bacterial vaginosis, genital bleeding, or plasma virus detection. CONCLUSIONS: Standard doses of ART achieved higher genital than plasma concentrations across the menstrual cycle. Therapeutic ART suppresses genital virus shedding throughout the menstrual cycle, even in the presence of factors reported to increase virus shedding.

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.

Novel preclinical models of topical PrEP pharmacodynamics provide rationale for combination of drugs with complementary properties.

BACKGROUND: The limited success of recent HIV topical pre-exposure prophylaxis clinical trials highlights the need for more predictive models of drug efficacy that better simulate what may happen during sexual exposure. To address this gap, we developed complementary in vitro models to evaluate the ability of drugs to retain anti-HIV activity if cells were washed with seminal plasma (simulating what may happen following exposure to ejaculate), and to protect drug-naive T cells (representing newly recruited immune cells) co-cultured with explants that had been pretreated with drug. We focused on tenofovir disoproxil fumarate (TDF), the non-nucleoside reverse transcriptase inhibitors dapivirine (DPV) and IQP-0528, and the entry inhibitors maraviroc (MVC) and the D-peptide chol-PIE-12 trimer (PIE12). Studies were extended to macaques and the ability of cervical biopsies obtained from animals treated with an intravaginal ring formulation of IQP-0528 to protect ex vivo co-cultured T cells was determined. The antiviral activity of cervicovaginal lavage samples against a primary Clade C isolate was also measured and correlated with drug levels. RESULTS: Cells exposed to TDF were equally protected from HIV whether or not the drug-treated cells were washed with medium or seminal plasma prior to challenge. In contrast, several-fold higher concentrations of NNRTIs and entry inhibitors were needed to attain similar levels of HIV inhibition following a wash with seminal plasma. Conversely, the NNRTIs and PIE12, but not TDF or MVC, were effectively transferred from ex vivo treated explants and protected co-cultured T cells. Biopsies obtained from IQP-0528 ring-treated macaques also protected co-cultured T cells with viral inhibition ranging from 42-72%. Antiviral activity correlated with the concentration of drug recovered. Combinations of TDF with IQP-0528 protected in both in vitro models. CONCLUSIONS: Together, these models suggest that intracellularly retained drugs such as TDF may protect resident immune cells following coitus but sustained delivery may be required to protect immune cells subsequently recruited into the genital tract. Sustained delivery may also be critical for NNRTIs, which are rapidly transported out of cells and could be lost following sexual intercourse. An ideal approach may be a combination of drugs with complementary bioavailability profiles formulated for sustained delivery.

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.

Residual hormone levels in used contraceptive rings as a measurement of adherence to vaginal ring use.

OBJECTIVE: This study sought to measure residual contraceptive hormone levels in vaginal rings as an adherence marker for monitoring product use in clinical trials. STUDY DESIGN: Residual etonogestrel and ethinyl estradiol levels from used NuvaRings® of 26 self-reported adherent women enrolled in a clinical trial of vaginal ring acceptability were compared to those from 16 women who used NuvaRing® as their contraceptive choice. RESULTS: Twenty-one (81%) clinical trial rings had contraceptive hormone levels within the range of those used as a contraceptive choice. Five returned rings had unused or discordant levels of residual contraceptive hormones. CONCLUSION: Residual vaginal ring drug levels could help assess adherence in clinical trials.

Self-collected genital swabs compared with cervicovaginal lavage for measuring HIV-1 and HSV-2 and the effect of acyclovir on viral shedding.

HIV-1 and HSV-2 are frequent genital co-infections in women. To determine how self-collected genital swabs compare to provider-collected cervicovaginal lavage, paired self-collected genital swabs and cervicovaginal lavage from women co-infected with HIV-1 and HSV-2 were evaluated. Women were in an acyclovir clinical trial and their samples were tested for HIV-1 RNA (361 samples) and HSV-2 DNA (378 samples). Virus shedding, quantity and acyclovir effect were compared. HIV-1 and HSV-2 were more frequently detected in self-collected genital swabs: 74.5% of self-collected genital swabs and 63.6% of cervicovaginal lavage had detectable HIV-1 (p ≤ 0.001, Fisher's exact test) and 29.7% of self-collected genital swabs and 19.3% of cervicovaginal lavage had detectable HSV-2 (p ≤ 0.001) in the placebo month. Cervicovaginal lavage and self-collected genital swabs virus levels were correlated (Spearman's rho, 0.68 for HIV; 0.61 for HSV-2) and self-collected genital swabs levels were generally higher. In multivariate modeling, self-collected genital swabs and cervicovaginal lavage could equally detect the virus-suppressive effect of acyclovir: for HIV-1, proportional odds ratios were 0.42 and 0.47 and for HSV-2, they were 0.10 and 0.03 for self-collected genital swabs and cervicovaginal lavage, respectively. Self-collected genital swabs should be considered for detection and measurement of HIV-1 and HSV-2 in clinical trials and other studies as they are a sensitive method to detect virus and can be collected in the home with frequent sampling.

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.

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.

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.