Temporary increase in circulating replication-competent latent HIV-infected resting CD4+ T cells after switch to an integrase inhibitor based antiretroviral regimen.

BACKGROUND: The principal barrier to an HIV cure is the presence of the latent viral reservoir (LVR), which has been understudied in African populations. From 2018 to 2019, Uganda instituted a nationwide rollout of ART consisting of Dolutegravir (DTG) with two NRTI, which replaced the previous regimen of one NNRTI and the same two NRTI. METHODS: Changes in the inducible replication-competent LVR (RC-LVR) of ART-suppressed Ugandans with HIV (n = 88) from 2015 to 2020 were examined using the quantitative viral outgrowth assay. Outgrowth viruses were examined for viral evolution. Changes in the RC-LVR were analyzed using three versions of a Bayesian model that estimated the decay rate over time as a single, linear rate (model A), or allowing for a change at time of DTG initiation (model B&C). FINDINGS: Model A estimated the slope of RC-LVR change as a non-significant positive increase, which was due to a temporary spike in the RC-LVR that occurred 0-12 months post-DTG initiation (p < 0.005). This was confirmed with models B and C; for instance, model B estimated a significant decay pre-DTG initiation with a half-life of 6.9 years, and an ∼1.7-fold increase in the size of the RC-LVR post-DTG initiation. There was no evidence of viral failure or consistent evolution in the cohort. INTERPRETATION: These data suggest that the change from NNRTI- to DTG-based ART is associated with a significant temporary increase in the circulating RC-LVR. FUNDING: Supported by the NIH (grant 1-UM1AI164565); Gilead HIV Cure Grants Program (90072171); Canadian Institutes of Health Research (PJT-155990); and Ontario Genomics-Canadian Statistical Sciences Institute.

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.

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.

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.

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.

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.

Dating reservoir formation in virologically suppressed people living with HIV-1 in Rakai, Uganda.

The timing of the establishment of the HIV latent viral reservoir (LVR) is of particular interest, as there is evidence that proviruses are preferentially archived at the time of antiretroviral therapy (ART) initiation. Quantitative viral outgrowth assays (QVOAs) were performed using Peripheral Blood Mononuclear Cells (PBMC) collected from Ugandans living with HIV who were virally suppressed on ART for >1 year, had known seroconversion windows, and at least two archived ART-naïve plasma samples. QVOA outgrowth populations and pre-ART plasma samples were deep sequenced for the pol and gp41 genes. The bayroot program was used to estimate the date that each outgrowth virus was incorporated into the reservoir. Bayroot was also applied to previously published data from a South African cohort. In the Ugandan cohort (n = 11), 87.9 per cent pre-ART and 56.3 per cent viral outgrowth sequences were unique. Integration dates were estimated to be relatively evenly distributed throughout viremia in 9/11 participants. In contrast, sequences from the South African cohort (n = 9) were more commonly estimated to have entered the LVR close to ART initiation, as previously reported. Timing of LVR establishment is variable between populations and potentially viral subtypes, which could limit the effectiveness of interventions that target the LVR only at ART initiation.

Temporary increase in circulating replication-competent latent HIV-infected resting CD4+ T cells after switch to an integrase inhibitor based antiretroviral regimen.

The principal barrier to an HIV cure is the presence of a latent viral reservoir (LVR) made up primarily of latently infected resting CD4+ (rCD4) T-cells. Studies in the United States have shown that the LVR decays slowly (half-life=3.8 years), but this rate in African populations has been understudied. This study examined longitudinal changes in the inducible replication competent LVR (RC-LVR) of ART-suppressed Ugandans living with HIV (n=88) from 2015-2020 using the quantitative viral outgrowth assay, which measures infectious units per million (IUPM) rCD4 T-cells. In addition, outgrowth viruses were examined with site-directed next-generation sequencing to assess for possible ongoing viral evolution. During the study period (2018-19), Uganda instituted a nationwide rollout of first-line ART consisting of Dolutegravir (DTG) with two NRTI, which replaced the previous regimen that consisted of one NNRTI and the same two NRTI. Changes in the RC-LVR were analyzed using two versions of a novel Bayesian model that estimated the decay rate over time on ART as a single, linear rate (model A) or allowing for an inflection at time of DTG initiation (model B). Model A estimated the population-level slope of RC-LVR change as a non-significant positive increase. This positive slope was due to a temporary increase in the RC-LVR that occurred 0-12 months post-DTG initiation (p<0.0001). This was confirmed with model B, which estimated a significant decay pre-DTG initiation with a half-life of 7.7 years, but a significant positive slope post-DTG initiation leading to a transient estimated doubling-time of 8.1 years. There was no evidence of viral failure in the cohort, or consistent evolution in the outgrowth sequences associated with DTG initiation. These data suggest that either the initiation of DTG, or cessation of NNRTI use, is associated with a significant temporary increase in the circulating RC-LVR.

Soluble E-cadherin: A marker of genital epithelial disruption.

PROBLEM: The genital epithelial barrier is a crucial first line of defence against HIV, and epithelial disruption may enhance HIV susceptibility. Assessment of genital epithelial integrity requires biopsies, but their collection is not practical in many research settings. A validated biomarker of genital epithelial barrier integrity would therefore be useful. The purpose of this study was to evaluate soluble E-cadherin (sE-cad) as a marker of genital epithelial disruption. METHOD OF STUDY: Using in vitro models of endocervical and foreskin epithelial cells, we assessed changes in sE-cad, IL-6, IL-1ß, and IL-1α levels following mechanical disruption. We also assessed changes in sE-cad levels in vivo in cervicovaginal secretions after epithelial disruption by endocervical cytobrush sampling in Canadian women, and assessed the relationship between levels of sE-cad in coronal sulcus swabs to membrane-bound E-cadherin in the overlying foreskin tissue in Ugandan men. RESULTS: sE-cad levels immediately increased after in vitro epithelial physical disruption with the degree of elevation dependent on the extent of disruption, as did levels of IL-1ß and IL-1α; this was followed by a delayed increase in IL-6 levels. In vivo results confirmed that sE-cad levels in cervicovaginal secretions were elevated 6 h after cytobrush sampling when compared to baseline. Furthermore, levels of sE-cad in the prepuce were inversely correlated with the amount of membrane-bound E-cadherin of overlying tissue. CONCLUSION: Our results validate the use of sE-cad as a marker of epithelial disruption and demonstrate that the processes of physical disruption and inflammation in the genital tract are strongly intertwined.

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.

The Penis, the Vagina and HIV Risk: Key Differences (Aside from the Obvious).

Globally, most Human Immunodeficiency Virus type 1 (HIV) transmission occurs through vaginal-penile sex (heterosexual transmission). The local immune environment at the site of HIV exposure is an important determinant of whether exposure during sex will lead to productive infection, and the vaginal and penile immune milieus are each critically shaped by the local microbiome. However, there are key differences in the microbial drivers of inflammation and immune quiescence at these tissue sites. In both, a high abundance of anaerobic taxa (e.g., Prevotella) is associated with an increased local density of HIV target cells and an increased risk of acquiring HIV through sex. However, the taxa that have been associated to date with increased risk in the vagina and penis are not identical. Just as importantly, the microbiota associated with comparatively less inflammation and HIV risk-i.e., the optimal microbiota-are very different at the two sites. In the vagina, Lactobacillus spp. are immunoregulatory and may protect against HIV acquisition, whereas on the penis, "skin type" flora such as Corynebacterium are associated with reduced inflammation. Compared to its vaginal counterpart, much less is known about the dynamics of the penile microbiome, the ability of clinical interventions to alter the penile microbiome, or the impact of natural/induced microbiome alterations on penile immunology and HIV risk.

Quantitative Immunofluorescent Imaging of Immune Cells in Mucosal Tissues.

Understanding the interplay between commensals, pathogens, and immune cells in the skin and mucosal tissues is critical to improve prevention and treatment of a myriad of diseases. While high-parameter flow cytometry is the current gold standard for immune cell characterization in blood, it is less suitable for mucosal tissues, where structural and spatial information is lost during tissue disaggregation. Immunofluorescence overcomes this limitation, serving as an excellent alternative for studying immune cells in mucosal tissues. However, the use of immunofluorescent microscopy for analyzing clinical samples is hampered by a lack of high-throughput quantitative analysis techniques. In this chapter, we describe methods for sectioning, staining, and imaging whole sections of human foreskin tissue. We also describe methods to automate immune cell quantification from immunofluorescent images, including image preprocessing and methods to quantify both circular and irregularly shaped immune cells using open-source software.

Immune milieu and microbiome of the distal urethra in Ugandan men: impact of penile circumcision and implications for HIV susceptibility.

BACKGROUND: Coronal sulcus (CS) anaerobe abundance and IL-8 levels are linked to HIV acquisition, and are dramatically reduced after penile circumcision (PC). The distal urethra may be the site of some HIV acquisition before PC, and presumably most acquisition post PC. We describe the immune milieu and microbiome of the distal urethra in uncircumcised Ugandan men, and define the impact of PC. Participants consisted of HIV-negative, genital symptom-free adult Ugandan men undergoing PC (n = 51). Urethral and coronal sulcus swabs were collected at baseline and at 6- and 12-months post-PC. Soluble immune factors were quantified by multiplex ELISA, and bacterial abundance assessed by 16S rRNA qPCR and sequencing. RESULTS: At baseline, the urethra was enriched compared to the CS for most cytokines (including IL-8 and MIP-1ß) and soluble E-cadherin (sE-cadherin, an epithelial disruption marker), although CS levels of IL-1α and IL-1ß were higher. Baseline total bacterial abundance was ≥ 20-fold higher in the CS than the urethra (median 27,100 vs. 1200 gene copies/swab, p = 0.001), and anaerobes comprised 58% of CS bacteria vs. 42% of urethral bacteria. PC did not alter urethral IL-8 (median 806 at baseline vs. 1130 pg/ml at 12 months; p = 0.062) and urethral sE-cadherin increased (113,223 vs. 158,385 pg/ml, p = 0.009), despite five- and sevenfold drops in total bacterial and anaerobe abundance after PC, respectively. However, PC dramatically reduced CS levels of sE-cadherin (15,843 vs. 837 pg/ml, p < 0.001) and most cytokines (IL-8; 34 vs. 3 pg/ml, p < 0.001), while reducing total bacterial and anaerobe abundance by 13-fold and 60-fold, respectively (both P ≤ 0.004). CONCLUSIONS: The urethra is immunologically rich with characteristics of an HIV-susceptible tissue site. However, PC had no impact on urethral immunology and may have reduced epithelial integrity, despite modest reductions in total bacteria and anaerobes, suggesting that HIV protection from PC is not mediated via immune or microbiome alterations in the urethra. Video abstract.

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.

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.

Penile bacteria associated with HIV seroconversion, inflammation, and immune cells.

The foreskin is a site of heterosexual acquisition of HIV-1 among uncircumcised men. However, some men remain HIV-negative despite repeated, unprotected vaginal intercourse with HIV-positive partners, while others become infected after few exposures. The foreskin microbiome includes a diverse group of anaerobic bacteria that have been linked to HIV acquisition. However, these anaerobes tend to coassociate, making it difficult to determine which species might increase HIV risk and which may be innocent bystanders. Here, we show that 6 specific anaerobic bacterial species, Peptostreptococcus anaerobius, Prevotella bivia, Prevotella disiens, Dialister propionicifaciens, Dialister micraerophilus, and a genetic near neighbor of Dialister succinatiphilus, significantly increased cytokine production, recruited HIV-susceptible CD4+ T cells to the inner foreskin, and were associated with HIV acquisition. This strongly suggests that the penile microbiome increases host susceptibility to HIV and that these species are potential targets for microbiome-based prevention strategies.

Quantifying the clonality and dynamics of the within-host HIV-1 latent reservoir.

Among people living with human immunodeficiency virus type 1 (HIV-1), the long-term persistence of a population of cells carrying transcriptionally silent integrated viral DNA (provirus) remains the primary barrier to developing an effective cure. Ongoing cell division via proliferation is generally considered to be the driving force behind the persistence of this latent HIV-1 reservoir. The contribution of this mechanism (clonal expansion) is supported by the observation that proviral sequences sampled from the reservoir are often identical. This outcome is quantified as the 'clonality' of the sample population, e.g. the fraction of provirus sequences observed more than once. However, clonality as a quantitative measure is inconsistently defined and its statistical properties are not well understood. In this Reflections article, we use mathematical and phylogenetic frameworks to formally examine the inherent problems of using clonality to characterize the dynamics and proviral composition of the reservoir. We describe how clonality is not adequate for this task due to the inherent complexity of how infected cells are 'labeled' by proviral sequences-the outcome of a sampling process from the evolutionary history of active viral replication before treatment-as well as variation in cell birth and death rates among lineages and over time. Lastly, we outline potential directions in statistical and phylogenetic research to address these issues.

The Effect of Gender-Affirming Medical Care on the Vaginal and Neovaginal Microbiomes of Transgender and Gender-Diverse People.

Transgender and gender diverse individuals may seek gender-affirming medical care, such as hormone therapy or surgery, to produce primary and/or secondary sex characteristics that are more congruent with their gender. Gender-affirming medical care for transmasculine individuals can include testosterone therapy, which suppresses circulating estrogen and can lead to changes in the vaginal epithelium that are reminiscent of the post-menopausal period in cisgender females. Among transfeminine individuals, gender-affirming medical care can include vaginoplasty, which is the surgical creation of a vulva and neovaginal canal, commonly using penile and scrotal skin. The effect of gender-affirming medical care on the vagina of transmasculine individuals and on the neovagina of transfeminine individuals is poorly characterized. This review summarizes what is known of the epithelium and local microbiota of the testosterone-exposed vagina and the neovagina. We focus on potential pathogens and determinants of gynecological health and identify key knowledge gaps for future research.

Foreskin surface area is not associated with sub-preputial microbiome composition or penile cytokines.

OBJECTIVE: Male circumcision (MC) reduces acquisition of HIV-1 in heterosexual men by at least 60%, but the biological mechanism for this protection is incompletely understood. Previous studies have shown that a larger foreskin size, increased abundance of anaerobic bacteria in the sub-preputial space, and higher levels of pro-inflammatory cytokines on the penis are all prospectively associated with risk of HIV-1 acquisition. Since coverage of the glans on the non-erect penis is dependent on foreskin size, a larger foreskin could result in a less aerobic environment that might preferentially support anaerobic bacterial growth and induce inflammation. We therefore assessed the relationship between foreskin size, penile microbiome composition and local inflammation. METHODS: This is a retrospective, cross-sectional analysis of 82 HIV-uninfected men who participated in a randomized trial of MC for HIV-1 prevention in Rakai, Uganda between 2003-2006. Sub-preputial swabs were collected prior to MC and assessed for cytokines (multiplexed immunosorbent assay) and bacterial load (qPCR) and taxon abundance (sequencing). Foreskin size was measured immediately after MC. RESULTS: Foreskin surface area did not correlate with total bacterial load (rho = 0.05) nor the abundance of key taxa of bacteria previously associated with HIV-1 risk (rho = 0.04-0.25). Foreskin surface area also did not correlate with sub-preputial cytokine concentrations previously associated with HIV-1 risk (IL-8 rho = 0.05). CONCLUSIONS: Larger foreskin size is not associated with either increased penile anaerobes or pro-inflammatory cytokines. These data suggest that foreskin size does not increase HIV-1 risk through changes in penile microbiome composition or penile inflammation.