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Problem: Stability over time of systemic and mucosal immunity and their associations with bacterial vaginosis (BV) and HIV-specific parameters were assessed., Method of Study: Immune mediators and HIV viral load in plasma and cervicovaginal lavage (CVL), E. coli inhibition, and Nugent score were measured at three semiannual visits among 94 participants in the Women's Interagency HIV Study. Mixed models identified the factors associated with immune mediators., Results: There was higher E. coli inhibition and lower inflammation over time in the genital tract and systemically. BV was consistently associated with higher CVL inflammatory mediators and lower CVL E. coli inhibition. HIV-infected women with higher CD4 counts had lower systemic and genital inflammatory mediators, and genital HIV shedding was associated with higher CVL inflammatory mediators. Use of antiretroviral therapy (ART) was associated with lower plasma and CVL mediators, but higher E. coli inhibition., Conclusion: HIV and BV are linked to inflammation, and ART may be associated with improved vaginal health., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: The lack of biomarkers that are predictive of safety is a critical gap in the development of microbicides. The present experiments were designed to evaluate the predictive value of in vitro models of microbicide safety., Methods: Changes in the epithelial barrier were evaluated by measuring transepithelial electrical resistance (TER) after exposure of human epithelial cells to candidate microbicides in a dual-chamber system. The significance of observed changes was addressed by challenging cultures with human immunodeficiency virus (HIV) and measuring the ability of virus to cross the epithelium and infect target T cells cultured in the lower chamber., Results: Exposure to nonoxynol-9 (N-9) or cellulose sulfate (CS), but not 9-[2-(phosphonomethoxy)propyl]adenine (also referred to as tenofovir) or PRO2000, resulted in a rapid and sustained reduction in TER and a marked increase in HIV infection of T cells cultured in the lower chamber. Moreover, CS triggered nuclear factor kappaB activation in peripheral blood mononuclear cells and increased HIV replication in chronically infected U1 cells., Conclusions: Epithelial barrier disruption and enhanced viral replication may have contributed to the increased risk of HIV acquisition observed in phase 3 trials of N-9 and CS. Expansion of in vitro safety testing to include these models would provide a more stringent preclinical assessment of microbicide safety and may prove to be more predictive of clinical outcomes.

BackgroundPrimary human papillomavirus (HPV) screening (PHS) utilizes oncogenic human papillomavirus (oncHPV) testing as the initial cervical cancer screening method and typically, if positive, additional reflex-triage (eg, HPV16/18-genotyping, Pap testing). While US guidelines support PHS usage in the general population, PHS has been little studied in women living with HIV (WLWH).MethodsWe enrolled n = 865 WLWH (323 from the Women's Interagency HIV Study [WIHS] and 542 from WIHS-affiliated colposcopy clinics). All participants underwent Pap and oncHPV testing, including HPV16/18-genotyping. WIHS WLWH who tested oncHPV[+] or had cytologic atypical squamous cells of undetermined significance or worse (ASC-US+) underwent colposcopy, as did a random 21% of WLWH who were oncHPV[-]/Pap[-] (controls). Most participants additionally underwent p16/Ki-67 immunocytochemistry.ResultsMean age was 46 years, median CD4 was 592 cells/µL, 95% used antiretroviral therapy. Seventy WLWH had histologically-determined cervical intraepithelial neoplasia grade 2 or greater (CIN-2+), of which 33 were defined as precancer (ie, [i] CIN-3+ or [ii] CIN-2 if concurrent with cytologic high grade squamous intraepithelial lesions [HSILs]). PHS had 87% sensitivity (Se) for precancer, 9% positive predictive value (PPV), and a 35% colposcopy referral rate (Colpo). "PHS with reflex HPV16/18-genotyping and Pap testing" had 84% Se, 16% PPV, 30% Colpo. PHS with only HPV16/18-genotyping had 24% Colpo. "Concurrent oncHPV and Pap Testing" (Co-Testing) had 91% Se, 12% PPV, 40% Colpo. p16/Ki-67 immunochemistry had the highest PPV, 20%, but 13% specimen inadequacy.ConclusionsPHS with reflex HPV16/18-genotyping had fewer unnecessary colposcopies and (if confirmed) could be a potential alternative to Co-Testing in WLWH.

ObjectiveRecent studies reported a lower human papillomavirus 16 (HPV16) prevalence in cervical precancer among African American than Caucasian women in the general population. We assessed this relationship in women with HIV.DesignWomen living with or at risk for HIV in the Women's Interagency HIV Study were followed semi-annually with Pap tests, colposcopy/histology (if indicated), and collection of cervicovaginal lavage samples for HPV testing by PCR. Racial and ethnic groups were defined using genomic Ancestry Informative Markers (AIMs).ResultsAmong 175 cases of cervical intraepithelial neoplasia 3 or worse (CIN-3+), 154 were diagnosed in women with HIV. African American (27%) and Hispanic (37%) cases were significantly less likely than Caucasian (62%) women to test positive for HPV16 (P = 0.01). In multivariate logistic regression models, these associations remained significant for African Americans (odds ratio = 0.13; 95% confidence interval (CI) 0.04-0.44; P = 0.001) but not Hispanics, after controlling for HIV status, CD4 count, history of AIDS, age, smoking, and sexual behavior. Limiting the analysis to women with HIV did not change the findings.ConclusionHPV16 prevalence is lower in African American compared with Caucasian women with HIV and cervical precancer, independent of immune status. Future studies to determine why these racial differences exist are warranted, and whether there are similar associations between race and invasive cervical cancer in women with HIV. Further, HPV types not covered by quadrivalent and bivalent vaccines may play an especially important role in cervical precancer among HIV-positive African American women, a possible advantage to using nonavalent HPV vaccine in this population.

ObjectiveWe suggested cervical cancer screening strategies for women living with HIV (WLHIV) by comparing their precancer risks to general population women, and then compared our suggestions with current Centers for Disease Control and Prevention (CDC) guidelines.DesignWe compared risks of biopsy-confirmed cervical high-grade squamous intraepithelial neoplasia or worse (bHSIL+), calculated among WLHIV in the Women's Interagency HIV Study, to 'risk benchmarks' for specific management strategies in the general population.MethodsWe applied parametric survival models among 2423 WLHIV with negative or atypical squamous cell of undetermined significance (ASC-US) cytology during 2000-2015. Separately, we synthesized published general population bHSIL+ risks to generate 3-year risk benchmarks for a 3-year return (after negative cytology, i.e. 'rescreening threshold'), a 6-12-month return (after ASC-US), and immediate colposcopy [after low-grade squamous intraepithelial lesion (LSIL)].ResultsAverage 3-year bHSIL+ risks among general population women ('risk benchmarks') were 0.69% for a 3-year return (after negative cytology), 8.8% for a 6-12-month return (after ASC-US), and 14.4% for colposcopy (after LSIL). Most CDC guidelines for WLHIV were supported by comparing risks in WLHIV to these benchmarks, including a 3-year return with CD4 greater than 500 cells/μl and after either three negative cytology tests or a negative cytology/oncogenic human papillomavirus cotest (all 3-year risks≤1.3%); a 1-year return after negative cytology with either positive oncogenic human papillomavirus cotest (1-year risk = 1.0%) or CD4 cell count less than 500 cells/μl (1-year risk = 1.1%); and a 6-12-month return after ASC-US (3-year risk = 8.2% if CD4 cell count at least 500 cells/μl; 10.4% if CD4 cell count = 350-499 cells/μl). Other suggestions differed modestly from current guidelines, including colposcopy (vs. 6-12 month return) for WLHIV with ASC-US and CD4 cell count less than 350 cells/μl (3-year risk = 16.4%) and a lengthened 2-year (vs. 1-year) interval after negative cytology with CD4 cell count at least 500 cells/μl (2-year risk = 0.98%).ConclusionsCurrent cervical cancer screening guidelines for WLHIV are largely appropriate. CD4 cell count may inform risk-tailored strategies.

BackgroundDetermining cervical precancer risk among human immunodeficiency virus (HIV)-infected women who despite a normal Pap test are positive for oncogenic human papillomavirus (oncHPV) types is important for setting screening practices.MethodsA total of 2791 HIV-infected and 975 HIV-uninfected women in the Women's Interagency HIV Study were followed semiannually with Pap tests and colposcopy. Cumulative risks of cervical intraepithelial neoplasia grade 2 or greater (CIN-2+; threshold used for CIN treatment) and grade 3 or greater (CIN-3+; threshold to set screening practices) were measured in HIV-infected and HIV-uninfected women with normal Pap tests, stratified by baseline HPV results, and also in HIV-infected women with a low-grade squamous intraepithelial lesion (LSIL; benchmark indication for colposcopy).ResultsAt baseline, 1021 HIV-infected and 518 HIV-uninfected women had normal Pap tests, of whom 154 (15%) and 27 (5%), respectively, tested oncHPV positive. The 5-year CIN-2+ cumulative risk in the HIV-infected oncHPV-positive women was 22% (95% confidence interval [CI], 9%-34%), 12% (95% CI, 0%-22%), and 14% (95% CI, 2%-25%) among those with CD4 counts

ObjectiveTo understand how regional body composition affects bone mineral density (BMD) in HIV-infected and HIV-uninfected women.MethodsDual energy x-ray absorptiometry was used to measure regional lean and fat mass and BMD at lumbar spine (LS), total hip (TH), and femoral neck (FN) in 318 HIV-infected and 122 HIV-uninfected Women's Interagency HIV Study participants at baseline and 2 and 5 years later. Total lean and fat mass were measured using bioimpedance analysis. Multivariate marginal linear regression models assessed the association of HIV status and body composition on BMD change.ResultsCompared with HIV-uninfected women, HIV-infected women were older (44 vs. 37 years), more likely to be Hepatitis C virus-infected (32% vs. 14%), and postmenopausal (26% vs. 3%) and had lower baseline total fat mass, trunk fat, and leg fat. In multivariate models, increased total lean mass was independently associated with increased BMD at LS, TH, and FN, and total fat mass was associated with increased BMD at TH and FN (all P < 0.05). When total fat was replaced in multivariate models with trunk fat and leg fat, increased trunk fat (and not leg fat) was associated with increased TH and FN BMD (P < 0.001).ConclusionsTotal fat and lean mass are strong independent predictors of TH and FN BMD, and lean mass was associated with greater LS BMD. Regardless of HIV status, greater trunk fat (and not leg fat) was associated with increased TH and FN BMD, suggesting that weight-bearing fat may be a more important predictor of BMD in the hip.

Problem: Bacterial vaginosis (BV) disproportionally impacts Black and Hispanic women, placing them at risk for HIV, sexually transmitted infections and preterm birth. It is unknown whether there are differences by genetic ancestry in BV risk or whether polymorphisms associated with BV risk differ by ancestry. Methods: Women's Interagency HIV Study (WIHS) participants with longitudinal Nugent scores were dichotomized as having (n = 319, Nugent 7–10) or not having BV (n = 367, Nugent 0–3). Genetic ancestry was defined by clustering of principal components from ancestry informative markers and further stratified by BV status. 627 single nucleotide polymorphisms (SNPs) across 41 genes important in mucosal defense were identified in the WIHS GWAS. A logistic regression analysis was adjusted for nongenetic predictors of BV and self‐reported race/ethnicity to assess associations between genetic ancestry and genotype. Results: Self‐reported race and genetic ancestry were associated with BV risk after adjustment for behavioral factors. Polymorphisms in mucosal defense genes including syndecans, cytokines and toll‐like receptors (TLRs) were associated with BV in all ancestral groups. Conclusions: The common association of syndecan, cytokine and TLR genes and the importance of immune function and inflammatory pathways in BV, suggests these should be targeted for further research on BV pathogenesis and therapeutics. [ABSTRACT FROM AUTHOR]

Background: Mechanisms linking herpes simplex virus type 2 (HSV-2) with human immunodeficiency virus (HIV) are not fully defined. We tested the hypothesis that HSV-2 and HIV dual infection is associated with cervicovaginal inflammation and/or vaginal dysbiosis.Methods: Genital tract samples were obtained weekly over a 12-week period from 30 women seropositive (+) for HIV and HSV-2 and 15 women each who were seropositive for one or seronegative (-) for both viruses. Immune mediators, antimicrobial activity, and microbial composition and diversity were compared.Results: Significant differences in the concentrations of interferon-γ (P = .002), tumor necrosis factor-α (P = .03), human beta defensin 1 (P = .001), secretory leukocyte protease inhibitor (P = .01), and lysozyme (P = .03) were observed across the 4 groups (Kruskal-Wallis). There were also significant differences in vaginal microbial alpha diversity (Simpson index) (P = .0046). Specifically, when comparing HIV-1+/HSV-2+ to HIV-1-/HSV-2- women, a decrease in Lactobacillus crispatus and increase in diverse anaerobes was observed. The number of genital HSV outbreaks was greater in HIV+ versus HIV- women (39 versus 12) (P = .04), but there were no significant differences when comparing outbreak to non-outbreak visits.Conclusions: Increased microbial diversity and cervicovaginal inflammation in HIV and HSV-2 dually infected women may adversely impact genital health and, in the absence of antiretroviral therapy, facilitate HIV shedding. [ABSTRACT FROM AUTHOR]

Background: Herpes simplex virus type 2 (HSV-2; herpes) exacerbates human immunodeficiency virus type 1 (HIV) by unclear mechanisms. These studies tested the impact of HSV-2 on systemic T-cells and HIV reservoirs.Methods: Peripheral blood mononuclear cells from HIV-infected women on antiretroviral therapy who were HSV-2 seropositive or seronegative and HIV-uninfected controls were analyzed by flow cytometry. Cell-associated HIV DNA and RNA were quantified in the absence or presence of activating stimuli, recombinant interleukin 32γ (IL-32γ), and a RUNX1 inhibitor. RNA was assessed by nanostring.Results: CD4, but not CD8, T-cell phenotypes differed in HIV+/HSV-2+ versus HIV+/HSV-2- (overall P = .002) with increased frequency of CCR5+, CXCR4+, PD-1+, and CD69+ and decreased frequency of CCR10+ and CCR6+ T-cells. The changes were associated with higher HIV DNA. Paradoxically, IL-32, a proinflammatory cytokine, was lower in subpopulations of CD4+ T-cells in HSV-2+ versus HSV-2- women. Recombinant IL-32γ blocked HIV reactivation in CD4+ T-cells and was associated with an increase in RUNX1 expression; the blockade was overcome by a RUNX1 inhibitor.Conclusions: Herpes is associated with phenotypic changes in CD4+ T-cells, including a decrease in IL-32, which may contribute to increased HIV reservoirs. Blocking IL-32 may facilitate HIV reactivation to improve shock and kill strategies. [ABSTRACT FROM AUTHOR]

Systemic and mucosal inflammation may play a role in HIV control. A cross-sectional comparison was conducted among women in the Women's Interagency HIV Study to explore the hypothesis that compared with HIV-uninfected participants, women with HIV, and, in particular, those with high plasma viral load (PVL) have increased levels of mucosal and systemic inflammatory mediators and impaired mucosal endogenous antimicrobial activity.Nineteen HIV-uninfected, 40 HIV-infected on antiretroviral therapy (ART) with PVL  2600 copies/mL (low viral load) (HIV+-LVL), and 19 HIV-infected on or off ART with PVL >10,000 (high viral load) (HIV+-HVL) were evaluated. Immune mediators and viral RNA were quantified in plasma and cervicovaginal lavage (CVL). The CVL antimicrobial activity was also determined.Compared to HIV-uninfected participants, HIV+-HVL women had higher levels of mucosal but not systemic proinflammatory cytokines and chemokines, higher Nugent scores, and lower Escherichia coli bactericidal activity. In contrast, there were no significant differences between HIV+-LVL and HIV-uninfected controls. After adjusting for PVL, HIV genital tract shedding was significantly associated with higher CVL concentrations of IL-6, IL-1β, MIP-1α, and CCL5 (RANTES) and higher plasma concentrations of MIP-1α. High PVL was associated with higher CVL levels of IL-1β and RANTES, as well as with higher Nugent scores, lower E. coli bactericidal activity, smoking, and lower CD4 counts; smoking and CD4 count retained statistical significance in a multivariate model.Further study is needed to determine if the relationship between mucosal inflammation and PVL is causal and to determine if reducing mucosal inflammation is beneficial. [ABSTRACT FROM AUTHOR]

Herold BC, Mesquita PM, Madan RP, Keller MJ. Female genital tract secretions and semen impact the development of microbicides for the prevention of human immunodeficiency virus (HIV) and other sexually transmitted infections. Am J Reprod Immunol 2011; 65: 325-333 Pharmacologic strategies for the prevention of HIV include vaccines, post-exposure prophylaxis with antiretroviral therapy, and topical microbicides. Vaginal microbicides have the potential to augment innate defenses in the genital tract but may also disrupt endogenous protection and increase HIV acquisition risk, as observed in clinical trials of nonoxynol-9. The initially disappointing results of microbicide clinical trials stimulated the development of more sensitive and comprehensive pre-clinical safety studies, which include dual-chamber culture systems to model the epithelial barrier and post-coital studies to evaluate the effects of semen and sexual intercourse on microbicide efficacy. This review discusses the key factors that contribute to a healthy female genital tract environment, the impact of semen on mucosal defense, and how our understanding of these mediators informs the development of effective vaginal microbicides. [ABSTRACT FROM AUTHOR]

Background: Preclinical and early phase clinical microbicide studies have not consistently predicted the outcome of efficacy trials. To address this gap, candidate biomarkers of microbicide pharmacodynamics and safety were evaluated in a doubleblind, placebo-controlled trial of tenofovir gel, the first microbicide to demonstrate significant protection against HIV acquisition. Methods: 30 women were randomized to apply a single daily dose of tenofovir or placebo gel for 14 consecutive days. Anti- HIV activity was measured in cervicovaginal lavage (CVL) on Days 0, 3, 7, 14 and 21 by luciferase assay as a surrogate marker of pharmacodynamics. Endogenous activity against E. coli and HSV-2 and concentrations of immune mediators were quantified in CVL as candidate biomarkers of safety. Tenofovir levels were measured in CVL and blood. Results: A significant increase in anti-HIV activity was detected in CVL from women who applied tenofovir gel compared to their endogenous anti-HIV activity in genital tract secretions on Day 0 and compared to activity in CVL from women in the placebo group. The activity correlated significantly with CVL concentration of tenofovir (r = 0.6, p<0.001) and fit a sigmoid Emax pharmacodynamic model. Anti-HIV activity in CVL from women who applied tenofovir persisted when virus was introduced in semen, whereas endogenous anti-HIV activity decreased. Tenofovir did not trigger an inflammatory response or induce sustained loss in endogenous antimicrobial activity or immune mediators. Conclusions: Tenofovir gel had no deleterious impact on soluble mucosal immunity. The increased anti-HIV activity in CVL, which persisted in the presence of semen and correlated with tenofovir concentration, is consistent with the efficacy observed in a recent clinical trial. These results promote quantified CVL anti-HIV activity as a surrogate of tissue pharmacodynamics and as a potential biomarker of adherence to product. This simple, feasible and inexpensive bioassay may promote the development of models more predictive of microbicide efficacy. Trial Registration: ClinicalTrials.gov NCT00594373 [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus, genital herpes, and other sexually transmitted infections are a critical national and global health priority requiring the rapid development of safe and effective control methods. Topical microbicides, self-administered agents designed for vaginal use, that block transmission at the mucosal surface may provide a realistic method of intervention that could be distributed worldwide. An optimal microbicide should protect against infection but must also be safe, without adversely affecting the mucosal environment, including mediators of host defense. Thus, a critical component in microbicides development is to identify optimal assays that could serve as surrogate markers to predict safety of microbicides prior to embarking on large-scale clinical trials. This will require a greater understanding of the mediators of mucosal immunity in the female genital tract. [ABSTRACT FROM AUTHOR]

Background. Microbicides used to prevent the transmission of human immunodeficiency virus (HIV) are advancing to clinical trials on the basis of activity observed in vitro and in animal models. However, no data demonstrate activity of microbicides after application in humans. This study was designed to determine the antiviral activity in cervicovaginal lavage (CVL) samples collected after intravaginal application of 0.5% PRO 2000 gel (Indevus). Methods. A randomized, double-blind study was conducted to assess the anti-HIV and anti-herpes simplex virus (HSV) activity of PRO 2000 in CVL samples obtained at screening (48 hours before) and 1 hour after application of study or placebo gel. HeLa cells or human macrophages were inoculated with CVL samples spiked with replication-defective HIV containing a luciferase indicator gene and pseudotyped with an R5 envelope. Human cervical epithelial cells were inoculated with CVL samples and challenged with HSV-2(G), and the virus titer was then determined. Results. CVL samples obtained after application of PRO 2000 gel significantly inhibited HIV and HSV infection by at least 1000-fold, compared with CVL samples obtained at screening (P<.001). There were no differences in cytokine levels between the drug and placebo groups. Conclusions. PRO 2000 gel (0.5%) is sufficiently bioavailable and retains substantial antiviral activity after intravaginal application. This strategy provides a mechanism for testing the efficacy of a microbicide before embarking on large-scale clinical trials. [ABSTRACT FROM AUTHOR]

Background: Plasmodium falciparum infection has been reported to increase human immunodeficiency virus (HIV) viral load (VL), which can facilitate HIV transmission. We prospectively studied the impact of mild P falciparum coinfection on HIV VL in Rwanda. Methods: We measured plasma HIV VL at presentation with malaria infection and weekly for 4 weeks after artemether-lumefantrine treatment in Rwandan adults infected with HIV with P falciparum malaria. Regression analyses were used to examine associations between malaria infection and HIV VL changes. Samples with detectable virus underwent genotypic drug-resistance testing. Results: We enrolled 28 HIV-malaria coinfected patients and observed 27 of them for 5 weeks. Three patients (11%) were newly diagnosed with HIV. Acute P falciparum infection had no significant effect on HIV VL slope over 28 days of follow-up. Ten patients with VL <40 copies/mL at enrollment maintained viral suppression throughout. Seventeen patients had a detectable VL at enrollment including 9 (53%) who reported 100% adherence to ARVs; 3 of these had detectable genotypic drug resistance. Conclusions: Unlike studies from highly malaria-endemic areas, we did not identify an effect of P falciparum infection on HIV VL; therefore, malaria is not likely to increase HIV-transmission risk in our setting. However, routine HIV testing should be offered to adults presenting with acute malaria in Rwanda. Most importantly, we identified a large percentage of patients with detectable HIV VL despite antiretroviral (ARV) therapy. Some of these patients had HIV genotypic drug resistance. Larger studies are needed to define the prevalence and factors associated with detectable HIV VL in patients prescribed ARVs in Rwanda.

A structurally novel candidate microbicide, PPCM, which is formed from the reaction of D,L-mandelic acid with sulfuric acid, provides activity against human immunodeficiency virus (HIV) and herpes simplex virus (HSV) and is not cytotoxic. The objectives of the current studies were to comprehensively evaluate the activity of PPCM in cell and explant cultures, explore the possibility of combining PPCM with HIV-specific reverse transcriptase inhibitors, and evaluate the efficacy of a formulated gel against genital herpes in a murine model. PPCM inhibited infection by laboratory and clinical R5 and X4 clade B and clade C HIV strains in cell culture. Ectocervical and endocervical tissue explants exposed to HIV-1BaL in the presence of PPCM were protected (50% inhibitory concentrations [IC50] of 3.9 μg/ml for ectocervix and 3.1 μg/ml for endocervix), and transfer of virus to target T cells via migratory cells was significantly impaired (IC50 of 35.7 μg/ml for ectocervix and 54.6 μg/ml for endocervix). The drug also blocked infection by cell-associated virus. Combinations of PPCM with UC-781 or PMPA in vitro exhibited additive anti-HIV activity. PPCM was incorporated into stable, low-pH gel formulations at concentrations of 0.4% and 4%. Both gels prevented genital herpesvirus infection in mice, even when virus was introduced in human seminal plasma. The abilities of PPCM to inhibit primary HIV isolates, reduce infection by cell-associated virus, and transfer of HIV from migratory to T cells, combined with the complete protection provided by formulated gel against genital herpes, indicate that this drug is an excellent candidate for inclusion in a combination microbicide and would provide protection against both HIV and HSV. [ABSTRACT FROM AUTHOR]