Impact of variable sampling on estimates of HIV-1 reservoir formation dates.

Timing of HIV-1 reservoir formation is important for informing HIV cure efforts. It is unclear how much of the variability seen in dating reservoir formation is due to sampling and gene-specific differences. We used a Bayesian extension of root to tip regression (bayroot) to re-estimate formation date distributions in participants from Swedish and South African cohorts, and assessed the impact of variable timing, frequency, and depth of sampling on these estimates. Significant shifts in formation date distributions were only observed with use of faster-evolving genes, while timing, frequency, and depth of sampling had minor or no significant effect on estimates.

Association of oral microbiome with oral human papillomavirus infection: a population study of the National Health and Nutrition Examination Survey, 2009-2012.

BACKGROUND: Oral human papillomavirus(HPV) infection and the oral microbiome are associated with oropharyngeal cancer. However, population-based data on the association of oral microbiome with oral HPV infection are limited. METHOD: We performed a cross-sectional analysis of 5,496 participants aged 20-59 in National Health and Nutrition Examination Surveys(NHANES):2009-2012. The association between either oral microbiome alpha diversity or beta diversity and oral HPV infection was assessed using multivariable logistic regression or principal coordinate analyses(PCoA) and multivariate analysis of variance(PERMANOVA). RESULTS: For alpha diversity, we found a lower number of observed Amplicon sequence variants(ASVs) (adjusted odds ratio[aOR] = 0.996; 95%CI = 0.992-0.999) and reduced Faith's Phylogenetic Diversity(aOR = 0.95; 95%CI = 0.90-0.99) associated with high-risk oral HPV infection in the overall population. This trend was observed in males for both high-risk and any oral HPV infection. Beta diversity showed differentiation of oral microbiome community by high-risk oral HPV infection as measured by Bray-Curtis dissimilarity (R2 = 0.054%; P = .029) and unweighted UniFrac distance (R2 = 0.046%; P = .045) among the overall population, and associations were driven by males. CONCLUSIONS: Both oral microbiome alpha diversity(within-sample richness and phylogenetic diversity) and beta diversity(heterogeneous dispersion of oral microbiome community) are associated with HPV infection. Longitudinal studies are needed to characterize the role of the microbiome in the natural history of oral HPV infection.

Insertive condom-protected and condomless vaginal sex both have a profound impact on the penile immune correlates of HIV susceptibility.

The penis is the primary site of HIV acquisition in heterosexual men. Elevated penile inflammatory cytokines increase sexual acquisition risk, and topically applied cytokines enhance foreskin HIV susceptibility in an explant model. However, the impact of penile-vaginal sex on these immune parameters is undefined. Heterosexual couples were recruited to the Sex, Couples and Science (SECS) Study, with the collection of penile swabs, semen, cervico-vaginal secretions, and blood after a period of abstinence, and repeated sampling up to 72 hours after either condomless (n = 30) or condom-protected (n = 8) penile-vaginal sex. Soluble immune parameters were quantified by multiplex immunoassay. Co-primary immune endpoints were penile levels of IL-8 and MIG, cytokines previously linked to penile HIV acquisition. One hour after sex there were dramatic increases in penile IL-8 and MIG levels, regardless of condom use, with a gradual return to baseline by 72 hours; similar patterns were observed for other chemoattractant chemokines. Penile cytokine changes were similar in circumcised and uncircumcised men, and repeated measures ANOVA and ANCOVA models demonstrated that the degree of change after condomless sex was explained by cytokine levels in their partners' cervico-vaginal secretions. This may have important implications for the biology of penile HIV acquisition.

Gynecological concerns and vaginal practices and exposures among transfeminine individuals who have undergone vaginoplasty.

BACKGROUND: Vaginoplasty is a gender-affirming surgery that is medically necessary for some transfeminine individuals. Little research exists describing vaginal health after the initial recovery from surgery, and evidence-based guidelines for vaginal care practices are unavailable. AIM: The study sought to describe self-reported gynecological concerns and vaginal care practices among transfeminine persons who have undergone vaginoplasty. METHODS: A total of 60 transfeminine participants 18+ years of age, living in Canada, and who had undergone vaginoplasty at least 1 year prior were recruited through social media, community groups, healthcare provider referrals, and study recontact. Participants completed a cross-sectional, online questionnaire detailing demographics, gynecological concerns, and genital practices and exposures. Hierarchical clustering was used to group participants based on behavioral practices and exposures. Associations between clusters and gynecological concerns were assessed. OUTCOMES: Outcomes included self-reported gynecological concerns within the past year, recent vulvar or vaginal symptoms (past 30 days), and behavioral practices/exposures, including douching with varied products and dilating. RESULTS: Participants reported a variety of concerns in the past year, including urinary tract infection (13%) and internal hair regrowth (23%). More than half (57%) had experienced at least 1 recent vaginal symptom, most commonly malodor (27%) and vaginal bleeding (21%). Of participants, 48% were dilating weekly and 52% reported douching in the past 30 days. Four distinct clusters of vaginal practices/exposures were identified: limited exposures; dilating, no douching; dilating and douching; and diverse exposures. No significant associations between cluster membership and gynecological concerns were identified, though cluster membership was significantly associated with surgical center (P = .03). Open-text write-ins provided descriptions of symptoms and symptom management strategies. CLINICAL IMPLICATIONS: The results provide insight for clinicians on common patient-reported gynecological concerns and current vaginal care practices and exposures, including symptom management strategies. STRENGTHS AND LIMITATIONS: This was the first study to investigate vaginal health and genital practices/exposures among a community sample of transfeminine individuals. As participants self-enrolled for a detailed survey and swab collection, individuals experiencing concerns were likely overrepresented. CONCLUSION: Transfeminine individuals reported a range of gynecological concerns outside of the surgical healing period. Genital practices/exposures varied across clusters, but no clear associations between clusters and symptoms were identified; instead, practice/exposure clusters were dependent on where the individual underwent vaginoplasty. There is a need for evidence to inform diagnostics, treatments, and vaginal care guidelines to support vaginal health.

How Does Voluntary Medical Male Circumcision Reduce HIV Risk?

PURPOSE OF REVIEW: Voluntary medical male circumcision (VMMC) is a surgical procedure that reduces HIV acquisition risk by almost two-thirds. However, global implementation is lagging, in part due to VMMC hesitancy. A better understanding of the mechanism(s) by which this procedure protects against HIV may increase acceptance of VMMC as an HIV risk reduction approach among health care providers and their clients. RECENT FINDINGS: HIV acquisition in the uncircumcised penis occurs preferentially across the inner foreskin tissues, due to increased susceptibility that is linked to elevated inflammatory cytokine levels in the sub-preputial space and an increased tissue density of HIV-susceptible CD4 + T cells. Inflammation can be caused by sexually transmitted infections, but is more commonly induced by specific anaerobic components of the penile microbiome. Circumcision protects by both directly removing the susceptible tissues of the inner foreskin, and by inducing a less inflammatory residual penile microbiome. VMMC reduces HIV susceptibility by removing susceptible penile tissues, and also through impacts on the penile immune and microbial milieu. Understanding these mechanisms may not only increase VMMC acceptability and reinvigorate global VMMC programs, but may also lead to non-surgical HIV prevention approaches focused on penile immunology and/or microbiota.

Addressing an HIV cure in LMIC.

HIV-1 persists in infected individuals despite years of antiretroviral therapy (ART), due to the formation of a stable and long-lived latent viral reservoir. Early ART can reduce the latent reservoir and is associated with post-treatment control in people living with HIV (PLWH). However, even in post-treatment controllers, ART cessation after a period of time inevitably results in rebound of plasma viraemia, thus lifelong treatment for viral suppression is indicated. Due to the difficulties of sustained life-long treatment in the millions of PLWH worldwide, a cure is undeniably necessary. This requires an in-depth understanding of reservoir formation and dynamics. Differences exist in treatment guidelines and accessibility to treatment as well as social stigma between low- and-middle income countries (LMICs) and high-income countries. In addition, demographic differences exist in PLWH from different geographical regions such as infecting viral subtype and host genetics, which can contribute to differences in the viral reservoir between different populations. Here, we review topics relevant to HIV-1 cure research in LMICs, with a focus on sub-Saharan Africa, the region of the world bearing the greatest burden of HIV-1. We present a summary of ART in LMICs, highlighting challenges that may be experienced in implementing a HIV-1 cure therapeutic. Furthermore, we discuss current research on the HIV-1 latent reservoir in different populations, highlighting research in LMIC and gaps in the research that may facilitate a global cure. Finally, we discuss current experimental cure strategies in the context of their potential application in LMICs.

Quantitation of the latent HIV-1 reservoir from the sequence diversity in viral outgrowth assays.

BACKGROUND: The ability of HIV-1 to integrate into the genomes of quiescent host immune cells, establishing a long-lived latent viral reservoir (LVR), is the primary obstacle to curing these infections. Quantitative viral outgrowth assays (QVOAs) are the gold standard for estimating the size of the replication-competent HIV-1 LVR, measured by the number of infectious units per million (IUPM) cells. QVOAs are time-consuming because they rely on culturing replicate wells to amplify the production of virus antigen or nucleic acid to reproducibly detectable levels. Sequence analysis can reduce the required number of culture wells because the virus genetic diversity within the LVR provides an internal replication and dilution series. Here we develop a Bayesian method to jointly estimate the IUPM and variant frequencies (a measure of clonality) from the sequence diversity of QVOAs. RESULTS: Using simulation experiments, we find our Bayesian approach confers significantly greater accuracy over current methods to estimate the IUPM, particularly for reduced numbers of QVOA replicates and/or increasing actual IUPM. Furthermore, we determine that the improvement in accuracy is greater with increasing genetic diversity in the sample population. We contrast results of these different methods applied to new HIV-1 sequence data derived from QVOAs from two individuals with suppressed viral loads from the Rakai Health Sciences Program in Uganda. CONCLUSIONS: Utilizing sequence variation has the additional benefit of providing information on the contribution of clonality of the LVR, where high clonality (the predominance of a single genetic variant) suggests a role for cell division in the long-term persistence of the reservoir. In addition, our Bayesian approach can be adapted to other limiting dilution assays where positive outcomes can be partitioned by their genetic heterogeneity, such as immune cell populations and other viruses.

Chemokine Levels in the Penile Coronal Sulcus Correlate with HIV-1 Acquisition and Are Reduced by Male Circumcision in Rakai, Uganda.

Individual susceptibility to HIV is heterogeneous, but the biological mechanisms explaining differences are incompletely understood. We hypothesized that penile inflammation may increase HIV susceptibility in men by recruiting permissive CD4 T cells, and that male circumcision may decrease HIV susceptibility in part by reducing genital inflammation. We used multi-array technology to measure levels of seven cytokines in coronal sulcus (penile) swabs collected longitudinally from initially uncircumcised men enrolled in a randomized trial of circumcision in Rakai, Uganda. Coronal sulcus cytokine levels were compared between men who acquired HIV and controls who remained seronegative. Cytokines were also compared within men before and after circumcision, and correlated with CD4 T cells subsets in foreskin tissue. HIV acquisition was associated with detectable coronal sulcus Interleukin-8 (IL-8 aOR 2.26, 95%CI 1.04-6.40) and Monokine Induced by γ-interferon (MIG aOR 2.72, 95%CI 1.15-8.06) at the visit prior to seroconversion, and the odds of seroconversion increased with detection of multiple cytokines. Coronal sulcus chemokine levels were not correlated with those in the vagina of a man's female sex partner. The detection of IL-8 in swabs was significantly reduced 6 months after circumcision (PRR 0.59, 95%CI 0.44-0.87), and continued to decline for at least two years (PRR 0.29, 95%CI 0.16-0.54). Finally, prepuce IL-8 correlated with increased HIV target cell density in foreskin tissues, including highly susceptible CD4 T cells subsets, as well as with tissue neutrophil density. Together, these data suggest that penile inflammation increases HIV susceptibility and is reduced by circumcision.

Reduced Frequency of Cells Latently Infected With Replication-Competent Human Immunodeficiency Virus-1 in Virally Suppressed Individuals Living in Rakai, Uganda.

Background: Human immunodeficiency virus type 1 (HIV-1) persists in latently infected resting CD4+ T cells (rCD4 cells), posing a major barrier to curing HIV-1 infection. Previous studies have quantified this pool of latently infected cells in Americans; however, no study has quantified this reservoir in sub-Saharan Africans, who make up the largest population of HIV-1-infected individuals globally. Methods: Peripheral blood was collected from 70 virally suppressed HIV-1-infected individuals from Rakai District, Uganda, who had initiated antiretroviral therapy (ART) during chronic infection. The quantitative viral outgrowth assay was used to determine frequency of latently infected rCD4 cells containing replication-competent virus. Multivariate regression was used to identify correlates of reservoir size and to compare reservoir size between this Ugandan cohort and a previously studied cohort of individuals from Baltimore, Maryland. Results: The median frequency of latently infected rCD4 cells in this Ugandan cohort was 0.36 infectious units per million cells (IUPM; 95% confidence interval, 0.26-0.55 IUPM), 3-fold lower than the frequency observed in the Baltimore cohort (1.08 IUPM; .72-1.49 IUPM; P < .001). Reservoir size in Ugandans was correlated positively with set-point viral load and negatively with duration of viral suppression. Conclusions: Virally suppressed Ugandans had a 3-fold lower frequency of rCD4 cells latently infected with replication-competent HIV-1, compared with previous observations in a cohort of American patients, also treated with ART during chronic infection. The biological mechanism driving the observed smaller reservoir in Ugandans is of interest and may be of significance to HIV-1 eradication efforts.

Penile Immune Activation and Risk of HIV Shedding: A Prospective Cohort Study.

Background: Genital immune activation is suspected to modulate local human immunodeficiency virus (HIV) RNA levels and the risk of sexual HIV transmission. Methods: A prospective, observational cohort study of 221 HIV-infected men undergoing male circumcision (MC) was conducted in Rakai, Uganda. Penile lavage samples collected from the coronal sulcus at baseline and 4 weekly visits after MC were assayed for pro-inflammatory cytokines and HIV RNA. The main analysis was limited to 175 men with detectable HIV plasma viral load (VL > 400 copies/mL; n = 808 visits). The primary exposures of interest were individual and total cytokine detection at the previous postoperative visit. Adjusted prevalence risk ratios (adjPRR) of detectable HIV shedding (VL > 40 copies/mL) were estimated by Poisson regression models with generalized estimating equations and robust variance estimators and included adjustment for plasma HIV VL. Findings: Among men with a detectable plasma VL, penile HIV shedding was detected at 136 visits (16.8%). Detectable interleukin (IL)-1ß (adjPRR = 2.14; 95% confidence interval (CI) = 1.02-4.48), IL-6 (adjPRR = 2.24; 95% CI = 1.28-3.90), IL-8 (adjPRR = 2.42; 95% CI = 1.15-5.08), IL-10 (adjPRR = 2.51; 95% CI = 1.67-3.80), and IL-13 (adjPRR = 1.87; 95% CI = 1.15-3.03) were associated with penile HIV shedding at the subsequent visit. Men with 2-4 (adjPRR = 2.36; 95% CI = 1.08-5.14) and 5-7 (adjPRR = 3.00; 95% CI = 1.28-7.01) detectable cytokines had a greater likelihood of detectable penile HIV shedding at the subsequent visit, compared to men with ≤ 1 detectable cytokine. The total number of detectable cytokines was also associated with a higher penile log10 HIV VL at the subsequent visit among HIV shedders. Interpretation: Pro-inflammatory cytokine production had a dose-dependent and temporal association with penile HIV shedding, suggesting that genital immune activation may increase the risk of sexual HIV transmission by driving local HIV replication.

The biology of how circumcision reduces HIV susceptibility: broader implications for the prevention field.

Circumcision reduces heterosexual HIV-1 acquisition in men by at least 60%. However, the biological mechanisms by which circumcision is protective remain incompletely understood. We test the hypothesis that the sub-preputial microenvironment created by the foreskin drives immune activation in adjacent foreskin tissues, facilitating HIV-1 infection through a combination of epithelial barrier disruption, enhanced dendritic cell maturation, and the recruitment/activation of neutrophils and susceptible CD4 T cell subsets such as Th17 cells. Furthermore, we provide evidence that the genital microbiome may be an important driver of this immune activation. This suggests that new modalities to reduce genital immune activation and/or alter the genital microbiome, used alone or in combination with topical microbicides, may be of significant benefit to HIV prevention.

Genital Anaerobic Bacterial Overgrowth and the PrePex Male Circumcision Device, Rakai, Uganda.

The PrePex circumcision device causes ischemic necrosis of the foreskin, raising concerns of anaerobic overgrowth. We compared the subpreputial microbiome of 2 men 7 days after PrePex device placement to that of 145 uncircumcised men in Rakai, Uganda, using 16S ribosomal (rRNA) RNA gene-based quantitative polymerase chain reaction analysis and sequencing. PrePex users had higher absolute abundance of all bacteria than uncircumcised men (P = .001), largely due to increased numbers of the following anaerobes: Porphyromonas (5.2 × 10(7) 16S rRNA gene copies/swab in the PrePex group and 1.1 × 10(6) 16S rRNA gene copies/swab in uncircumcised men; P = .002), Peptoniphilus (1.0 × 10(7) and 1.8 × 10(6) 16S rRNA gene copies/swab, respectively; P < .05), Anaerococcus (1.0 × 10(7) and 1.1 × 10(6) 16S rRNA gene copies/swab, respectively; P < .001), and Campylobacter ureolyticus (1.7 × 10(5) and 1.6 × 10(7)16S rRNA gene copies/swab, respectively; P < .001). The PrePex-associated increase in anaerobes may account for unpleasant odor and a possible heightened risk of tetanus.

HIV acquisition is associated with increased antimicrobial peptides and reduced HIV neutralizing IgA in the foreskin prepuce of uncircumcised men.

BACKGROUND: The foreskin is the site of most HIV acquisition in uncircumcised heterosexual men. Although HIV-exposed, seronegative (HESN) uncircumcised men demonstrate HIV-neutralizing IgA and increased antimicrobial peptides (AMPs) in the foreskin prepuce, no prospective studies have examined the mucosal immune correlates of HIV acquisition. METHODS: To assess the association of foreskin immune parameters with HIV acquisition, antimicrobial peptides and IgA with the capacity to neutralize a primary clade C HIV strain were quantified by blinded investigators, using sub-preputial swabs collected longitudinally during a randomized trial of male circumcision for HIV prevention in Rakai, Uganda. RESULTS: Participants were 99 men who acquired HIV (cases) and 109 randomly selected controls who remained HIV seronegative. At enrollment, 44.4% of cases vs. 69.7% of controls demonstrated IgA neutralization (adjusted OR = 0.31; 95% CI, 0.16-0.61). IgA neutralization was detected in 38.7% of cases and 70.7% of controls at the last seronegative case visit prior to HIV acquisition and the comparable control visit (adjusted OR 0.21; 95% CI, 0.11-0.39). Levels of the α-defensins and secretory leukocyte protease inhibitor (SLPI) were over ten-fold higher in the foreskin prepuce of cases who acquired HIV, both at enrollment (mean 4.43 vs. 3.03 and 5.98 vs. 4.61 log(n) pg/mL, P = 0.005 and 0.009, respectively), and at the last seronegative visit (mean 4.81 vs. 3.15 and 6.46 vs. 5.20 log(n) pg/mL, P = 0.0002 and 0.013). CONCLUSIONS: This prospective, blinded analysis is the first to assess the immune correlates of HIV acquisition in the foreskin. HIV-neutralizing IgA, previously associated with the HESN phenotype, was a biomarker of HIV protection, but other HESN associations correlated with increased HIV acquisition. This emphasizes the importance of prospective epidemiological studies or in vitro tissue studies to define the impact of mucosal parameters on HIV risk.

HIV Infection in Uncircumcised Men Is Associated With Altered CD8 T-cell Function But Normal CD4 T-cell Numbers in the Foreskin.

BACKGROUND: Human immunodeficiency virus (HIV)-infected (HIV+) men are more susceptible to sexually transmitted infections, and may be superinfected by HIV. We hypothesized that HIV induces immune alterations in the foreskin that may impact the subsequent acquisition/clearance of genital coinfections. METHODS: Foreskin tissue and blood were obtained from 70 HIV-uninfected and 20 HIV+ men undergoing circumcision. T cells were characterized by flow cytometry, immunohistochemistry, and polymerase chain reaction. RESULTS: There was substantial influx of CD8 T-cells into the foreskins of HIV+ men (108.8 vs 23.1 cells/mm(2); P < .001); but foreskin CD4 T-cell density was unchanged (43.0 vs 33.7/mm(2); P = .67), despite substantial blood depletion (409.0 vs 877.8 cells/µL; P < .001). While frequencies of foreskin C-C chemokine receptor type 5(+) (CCR5(+)) T cells, T regulatory cells, and T-helper 17 cells were unaltered in HIV+ men, CD8 T-cell production of tumor necrosis factor α (TNFα) was decreased. HIV-specific CD8 T cells were present in the foreskins of HIV+ men, although their frequency and function was reduced compared to the blood. CONCLUSIONS: Foreskin CD4 T-cell density and CCR5 expression were not reduced during HIV infection, perhaps explaining susceptibility to HIV superinfection. Foreskin CD8 T-cell density was increased, but decreased production of TNFα may enhance susceptibility to genital coinfections in HIV+ men.

Healthcare access and barriers to utilization among transgender and gender diverse people in Africa: a systematic review.

Background: Transgender and gender diverse (TGD) people face significant challenges in accessing timely, culturally competent, and adequate healthcare due to structural and systemic barriers, yet there is a lack of research exploring the access and utilization of healthcare services within African TGD communities. To address this gap, this systematic review explored: (1) barriers to accessing healthcare services and gender-affirming hormone therapy (GAHT) faced by TGD people, (2) demographic and societal factors correlated with the utilization of healthcare services and GAHT, (3) common healthcare and support services utilized by TGD people, and (4) patterns of accessing healthcare services and GAHT within TGD communities. Methods: A systematic literature search was conducted in PubMed, Embase, and Scopus in September 2023. Eligible studies included peer-reviewed original research, reports, and summaries published in the English language assessing health service accessibility and utilization of TGD people in Africa between January 2016 and December 2023. Results: From 2072 potentially relevant articles, 159 were assessed for eligibility following duplicate removal, and 49 were included for analysis. Forty-five articles addressed barriers to accessing healthcare services and GAHT, seven focused on demographic and societal factors correlated with the utilization of healthcare services and GAHT, 16 covered common healthcare and support services utilized by TGD people, and seven examined patterns of accessing healthcare services and GAHT. Findings suggested a limited availability of health services, inadequate knowledge of TGD healthcare needs among healthcare providers, a lack of recognition of TGD people in healthcare settings, healthcare-related stigma, and financial constraints within African TGD communities. An absence of studies conducted in Northern and Central Africa was identified. Conclusions: TGD people in Africa encounter significant barriers when seeking healthcare services, leading to disparity in the utilization of healthcare and resulting in a disproportionate burden of health risks. The implications of these barriers highlight the urgent need for more high-quality evidence to promote health equity for African TGD people. Trial registration: PROSPERO CRD42024532405. Supplementary Information: The online version contains supplementary material available at 10.1186/s44263-024-00073-2.

Comparison between a deep-learning and a pixel-based approach for the automated quantification of HIV target cells in foreskin tissue.

The availability of target cells expressing the HIV receptors CD4 and CCR5 in genital tissue is a critical determinant of HIV susceptibility during sexual transmission. Quantification of immune cells in genital tissue is therefore an important outcome for studies on HIV susceptibility and prevention. Immunofluorescence microscopy allows for precise visualization of immune cells in mucosal tissues; however, this technique is limited in clinical studies by the lack of an accurate, unbiased, high-throughput image analysis method. Current pixel-based thresholding methods for cell counting struggle in tissue regions with high cell density and autofluorescence, both of which are common features in genital tissue. We describe a deep-learning approach using the publicly available StarDist method to count cells in immunofluorescence microscopy images of foreskin stained for nuclei, CD3, CD4, and CCR5. The accuracy of the model was comparable to manual counting (gold standard) and surpassed the capability of a previously described pixel-based cell counting method. We show that the performance of our deep-learning model is robust in tissue regions with high cell density and high autofluorescence. Moreover, we show that this deep-learning analysis method is both easy to implement and to adapt for the identification of other cell types in genital mucosal tissue.

Effect of cryopreservation on CD4+ T cell subsets in foreskin tissue.

Voluntary medical male circumcision (VMMC) reduces HIV acquisition by at least 60%, but the determinants of HIV susceptibility in foreskin tissues are incompletely understood. Flow cytometry is a powerful tool that helps us understand tissue immune defenses in mucosal tissue like the inner foreskin, but foreskin flow cytometry has only been validated using fresh tissue samples. This restricts immune analyses to timepoints immediately after surgical acquisition and hinders research in this area. We compared fresh analysis with whole tissue cryopreservation and later thawing and digestion to analyze CD4+ T cell populations relevant to HIV susceptibility (CCR5, CD25, CD127, CCR4, CXCR3, CCR6, CCR10, HLA-DR, and CD38). Eight foreskin samples from HIV-negative males aged >18 years were collected after VMMC. For each sample, half the foreskin was immediately cryopreserved for later digestion and flow cytometry analysis, while the remaining tissues were analyzed fresh. We demonstrate no significant impact of cryopreservation on CD4+ T cell expression of CD25, CCR4, CCR6, HLA-DR, CCR10, or CD127. Although expression levels of CCR5, CD38, and CXCR3 were increased after cryopreservation, the relative ranking of participants was retained. In conclusion, cryopreserved foreskin tissues may be suitable for subsequent digestion and flow cytometry phenotyping of HIV-susceptible T cell populations.

Insertive vaginal sex is associated with altered penile immunology and enrichment of Gardnerella vaginalis in uncircumcised Ugandan men.

PROBLEM: HIV susceptibility is linked to the penile immune milieu (particularly IL-8 levels) and microbiome. The effects of insertive vaginal sex itself on penile immunology and microbiota are not well described. METHOD OF STUDY: We compared the immune milieu and microbiology of the coronal sulcus (CS) and distal urethra in 47 uncircumcised Ugandan men reporting ever (n = 42) or never (n = 5) having had vaginal intercourse. Soluble immune factors were assayed by multiplex ELISA, and penile bacteria abundance by 16S rRNA qPCR and sequencing. Co-primary endpoints were penile levels of IL-8 and soluble E-cadherin. RESULTS: Independent of classical STIs, men reporting prior vaginal sex demonstrated elevated IL-8 levels in both the coronal sulcus (1.78 vs. 0.81 log10 pg/mL, p = .021) and urethra (2.93 vs. 2.30 log10 pg/mL; p = .003), with a strong inverse relationship between urethral IL-8 levels and the time from last vaginal sex (r = -0.436; p = .004). Vaginal sex was also associated with elevated penile IL-1α/ß and soluble E-cadherin (sEcad), a marker of epithelial disruption. Gardnerella vaginalis (Gv) was only present in the penile microbiome of men reporting prior vaginal sex, and urethral Gv absolute abundance was strongly associated with urethral inflammation (r = 0.556; p < .001); corynebacteria were enriched in the CS of men reporting no prior vaginal sex and were associated with reduced CS inflammation. CONCLUSIONS: Sexual intercourse was associated with sustained changes in penile immunology, potentially mediated through microbial alterations, in particular the urethral abundance of G. vaginalis. Future studies should further characterize the effects of sexual debut on penile bacteria and immunology.

Effective and targeted latency reversal in CD4+ T cells from individuals on long term combined antiretroviral therapy initiated during chronic HIV-1 infection.

To date, an affordable, effective treatment for an HIV-1 cure remains only a concept with most "latency reversal" agents (LRAs) lacking specificity for the latent HIV-1 reservoir and failing in early clinical trials. We assessed HIV-1 latency reversal using a multivalent HIV-1-derived virus-like particle (HLP) to treat samples from 32 people living with HIV-1 (PLWH) in Uganda, US and Canada who initiated combined antiretroviral therapy (cART) during chronic infection. Even after 5-20 years on stable cART, HLP could target CD4+ T cells harbouring latent HIV-1 reservoir resulting in 100-fold more HIV-1 release into culture supernatant than by common recall antigens, and 1000-fold more than by chemotherapeutic LRAs. HLP induced release of a divergent and replication-competent HIV-1 population from PLWH on cART. These findings suggest HLP provides a targeted approach to reactivate the majority of latent HIV-1 proviruses among individuals infected with HIV-1.

HIV-1 subtype A1, D, and recombinant proviral genome landscapes during long-term suppressive therapy.

The primary obstacle to curing HIV-1 is a reservoir of CD4+ cells that contain stably integrated provirus. Previous studies characterizing the proviral landscape, which have been predominantly conducted in males in the United States and Europe living with HIV-1 subtype B, have revealed that most proviruses that persist during antiretroviral therapy (ART) are defective. In contrast, less is known about proviral landscapes in females with non-B subtypes, which represents the largest group of individuals living with HIV-1. Here, we analyze genomic DNA from resting CD4+ T-cells from 16 female and seven male Ugandans with HIV-1 receiving suppressive ART (n = 23). We perform near-full-length proviral sequencing at limiting dilution to examine the proviral genetic landscape, yielding 607 HIV-1 subtype A1, D, and recombinant proviral sequences (mean 26/person). We observe that intact genomes are relatively rare and clonal expansion occurs in both intact and defective genomes. Our modification of the primers and probes of the Intact Proviral DNA Assay (IPDA), developed for subtype B, rescues intact provirus detection in Ugandan samples for which the original IPDA fails. This work will facilitate research on HIV-1 persistence and cure strategies in Africa, where the burden of HIV-1 is heaviest.