Vaginal Lactobacillus fatty acid response mechanisms reveal a metabolite-targeted strategy for bacterial vaginosis treatment.

Bacterial vaginosis (BV), a common syndrome characterized by Lactobacillus-deficient vaginal microbiota, is associated with adverse health outcomes. BV often recurs after standard antibiotic therapy in part because antibiotics promote microbiota dominance by Lactobacillus iners instead of Lactobacillus crispatus, which has more beneficial health associations. Strategies to promote L. crispatus and inhibit L. iners are thus needed. We show that oleic acid (OA) and similar long-chain fatty acids simultaneously inhibit L. iners and enhance L. crispatus growth. These phenotypes require OA-inducible genes conserved in L. crispatus and related lactobacilli, including an oleate hydratase (ohyA) and putative fatty acid efflux pump (farE). FarE mediates OA resistance, while OhyA is robustly active in the vaginal microbiota and enhances bacterial fitness by biochemically sequestering OA in a derivative form only ohyA-harboring organisms can exploit. OA promotes L. crispatus dominance more effectively than antibiotics in an in vitro BV model, suggesting a metabolite-based treatment approach.

Lactobacillus-Deficient Cervicovaginal Bacterial Communities Are Associated with Increased HIV Acquisition in Young South African Women.

Elevated inflammation in the female genital tract is associated with increased HIV risk. Cervicovaginal bacteria modulate genital inflammation; however, their role in HIV susceptibility has not been elucidated. In a prospective cohort of young, healthy South African women, we found that individuals with diverse genital bacterial communities dominated by anaerobes other than Gardnerella were at over 4-fold higher risk of acquiring HIV and had increased numbers of activated mucosal CD4+ T cells compared to those with Lactobacillus crispatus-dominant communities. We identified specific bacterial taxa linked with reduced (L. crispatus) or elevated (Prevotella, Sneathia, and other anaerobes) inflammation and HIV infection and found that high-risk bacteria increased numbers of activated genital CD4+ T cells in a murine model. Our results suggest that highly prevalent genital bacteria increase HIV risk by inducing mucosal HIV target cells. These findings might be leveraged to reduce HIV acquisition in women living in sub-Saharan Africa.

Genetic variation of the HIV-1 subtype C transmitted/founder viruses long terminal repeat elements and the impact on transcription activation potential and clinical disease outcomes.

A genetic bottleneck is a hallmark of HIV-1 transmission such that only very few viral strains, termed transmitted/founder (T/F) variants establish infection in a newly infected host. Phenotypic characteristics of these variants may determine the subsequent course of disease. The HIV-1 5' long terminal repeat (LTR) promoter drives viral gene transcription and is genetically identical to the 3' LTR. We hypothesized that HIV-1 subtype C (HIV-1C) T/F virus LTR genetic variation is a determinant of transcriptional activation potential and clinical disease outcome. The 3'LTR was amplified from plasma samples of 41 study participants acutely infected with HIV-1C (Fiebig stages I and V/VI). Paired longitudinal samples were also available at one year post-infection for 31 of the 41 participants. 3' LTR amplicons were cloned into a pGL3-basic luciferase expression vector, and transfected alone or together with Transactivator of transcription (tat) into Jurkat cells in the absence or presence of cell activators (TNF-α, PMA, Prostratin and SAHA). Inter-patient T/F LTR sequence diversity was 5.7% (Renge: 2-12) with subsequent intrahost viral evolution observed in 48.4% of the participants analyzed at 12 months post-infection. T/F LTR variants exhibited differential basal transcriptional activity, with significantly higher Tat-mediated transcriptional activity compared to basal (p<0.001). Basal and Tat-mediated T/F LTR transcriptional activity showed significant positive correlation with contemporaneous viral loads and negative correlation with CD4 T cell counts (p<0.05) during acute infection respectively. Furthermore, Tat-mediated T/F LTR transcriptional activity significanly correlated positively with viral load set point and viral load; and negatively with CD4 T cell counts at one year post infection (all p<0.05). Lastly, PMA, Prostratin, TNF-α and SAHA cell stimulation resulted in enhanced yet heterologous transcriptional activation of different T/F LTR variants. Our data suggest that T/F LTR variants may influence viral transcriptional activity, disease outcomes and sensitivity to cell activation, with potential implications for therapeutic interventions.

Early Initiation of Antiretroviral Therapy Preserves the Metabolic Function of CD4+ T Cells in Subtype C Human Immunodeficiency Virus 1 Infection.

BACKGROUND: Immune dysfunction often persists in people living with human immunodeficiency virus (HIV) who are on antiretroviral therapy (ART), clinically manifesting as HIV-1-associated comorbid conditions. Early ART initiation may reduce incidence of HIV-1-associated immune dysfunction and comorbid conditions. Immunometabolism is a critical determinant of functional immunity. We investigated the effect of HIV-1 infection and timing of ART initiation on CD4+ T cell metabolism and function. METHODS: Longitudinal blood samples from people living with HIV who initiated ART during hyperacute HIV-1 infection (HHI; before peak viremia) or chronic HIV-1 infection (CHI) were assessed for the metabolic and immune functions of CD4+ T cells. Metabolite uptake and mitochondrial mass were measured using fluorescent analogues and MitoTracker Green accumulation, respectively, and were correlated with CD4+ T cell effector functions. RESULTS: Initiation of ART during HHI prevented dysregulation of glucose uptake by CD4+ T cells, but glucose uptake was reduced before and after ART initiation in CHI. Glucose uptake positively correlated with interleukin-2 and tumor necrosis factor-α production by CD4+ T cells. CHI was associated with elevated mitochondrial mass in effector memory CD4+ T cells that persisted after ART and correlated with PD-1 expression. CONCLUSIONS: ART initiation in HHI largely prevented metabolic impairment of CD4+ T cells. ART initiation in CHI was associated with persistently dysregulated immunometabolism of CD4+ T cells, which was associated with impaired cellular functions and exhaustion.

Cervicovaginal bacteria are a major modulator of host inflammatory responses in the female genital tract.

Colonization by Lactobacillus in the female genital tract is thought to be critical for maintaining genital health. However, little is known about how genital microbiota influence host immune function and modulate disease susceptibility. We studied a cohort of asymptomatic young South African women and found that the majority of participants had genital communities with low Lactobacillus abundance and high ecological diversity. High-diversity communities strongly correlated with genital pro-inflammatory cytokine concentrations in both cross-sectional and longitudinal analyses. Transcriptional profiling suggested that genital antigen-presenting cells sense gram-negative bacterial products in situ via Toll-like receptor 4 signaling, contributing to genital inflammation through activation of the NF-κB signaling pathway and recruitment of lymphocytes by chemokine production. Our study proposes a mechanism by which cervicovaginal microbiota impact genital inflammation and thereby might affect a woman's reproductive health, including her risk of acquiring HIV.

Subtle Longitudinal Alterations in Env Sequence Potentiate Differences in Sensitivity to Broadly Neutralizing Antibodies following Acute HIV-1 Subtype C Infection.

Broadly neutralizing antibodies (bNAbs) for HIV-1 prevention or cure strategies must inhibit transmitted/founder and reservoir viruses. Establishing sensitivity of circulating viruses to bNAbs and genetic patterns affecting neutralization variability may guide rational bNAbs selection for clinical development. We analyzed 326 single env genomes from nine individuals followed longitudinally following acute HIV-1 infection, with samples collected at ~1 week after the first detection of plasma viremia; 300 to 1,709 days postinfection but prior to initiating antiretroviral therapy (ART) (median = 724 days); and ~1 year post ART initiation. Sequences were assessed for phylogenetic relatedness, potential N- and O-linked glycosylation, and variable loop lengths (V1 to V5). A total of 43 env amplicons (median = 3 per patient per time point) were cloned into an expression vector and the TZM-bl assay was used to assess the neutralization profiles of 15 bNAbs targeting the CD4 binding site, V1/V2 region, V3 supersite, MPER, gp120/gp41 interface, and fusion peptide. At 1 µg/mL, the neutralization breadths were as follows: VRC07-LS and N6.LS (100%), VRC01 (86%), PGT151 (81%), 10-1074 and PGT121 (80%), and less than 70% for 10E8, 3BNC117, CAP256.VRC26, 4E10, PGDM1400, and N123-VRC34.01. Features associated with low sensitivity to V1/V2 and V3 bNAbs were higher potential glycosylation sites and/or relatively longer V1 and V4 domains, including known "signature" mutations. The study shows significant variability in the breadth and potency of bNAbs against circulating HIV-1 subtype C envelopes. VRC07-LS, N6.LS, VRC01, PGT151, 10-1074, and PGT121 display broad activity against subtype C variants, and major determinants of sensitivity to most bNAbs were within the V1/V4 domains. IMPORTANCE Broadly neutralizing antibodies (bNAbs) have potential clinical utility in HIV-1 prevention and cure strategies. However, bNAbs target diverse epitopes on the HIV-1 envelope and the virus may evolve to evade immune responses. It is therefore important to identify antibodies with broad activity in high prevalence settings, as well as the genetic patterns that may lead to neutralization escape. We investigated 15 bNAbs with diverse biophysical properties that target six epitopes of the HIV-1 Env glycoprotein for their ability to inhibit viruses that initiated infection, viruses circulating in plasma at chronic infection before antiretroviral treatment (ART), or viruses that were archived in the reservoir during ART in subtype C infected individuals in South Africa, a high burden country. We identify the antibodies most likely to be effective for clinical use in this setting and describe mutational patterns associated with neutralization escape from these antibodies.

Population-specific diversity of the immunoglobulin constant heavy G chain (IGHG) genes.

Human immunoglobulin G (IgG) molecules, IgG1, IgG2 and IgG3, exhibit substantial inter-individual variation in their constant heavy chain regions, as discovered by serological methods. This polymorphism is encoded by the IGHG1, IGHG2, and IGHG3 genes and may influence antibody function. We sequenced the coding fragments of these genes in 95 European Americans, 94 African Americans, and 94 Black South Africans. Striking differences were observed between the population groups, including extremely low amino acid sequence variation in IGHG1 among South Africans, and higher IGHG2 and IGHG3 diversity in individuals of African descent compared to individuals of European descent. Molecular definition of the loci illustrates a greater level of allelic polymorphism than previously described, including the presence of common IGHG2 and IGHG3 variants that were indistinguishable serologically. Comparison of our data with the 1000 Genome Project sequences indicates overall agreement between the datasets, although some inaccuracies in the 1000 Genomes Project are likely. These data represent the most comprehensive analysis of IGHG polymorphisms across major populations, which can now be applied to deciphering their functional impact.

Association between the cytokine storm, immune cell dynamics, and viral replicative capacity in hyperacute HIV infection.

INTRODUCTION: Immunological damage in acute HIV infection (AHI) may predispose to detrimental clinical sequela. However, studies on the earliest HIV-induced immunological changes are limited, particularly in sub-Saharan Africa. We assessed the plasma cytokines kinetics, and their associations with virological and immunological parameters, in a well-characterized AHI cohort where participants were diagnosed before peak viremia. METHODS: Blood cytokine levels were measured using Luminex and ELISA assays pre-infection, during the hyperacute infection phase (before or at peak viremia, 1-11 days after the first detection of viremia), after peak viremia (24-32 days), and during the early chronic phase (77-263 days). Gag-protease-driven replicative capacities of the transmitted/founder viruses were determined using a green fluorescent reporter T cell assay. Complete blood counts were determined before and immediately following AHI detection before ART initiation. RESULTS: Untreated AHI was associated with a cytokine storm of 12 out of the 33 cytokines analyzed. Initiation of ART during Fiebig stages I-II abrogated the cytokine storm. In untreated AHI, virus replicative capacity correlated positively with IP-10 (rho = 0.84, P < 0.001) and IFN-alpha (rho = 0.59, P = 0.045) and inversely with nadir CD4+ T cell counts (rho = - 0.58, P = 0.048). Hyperacute HIV infection before the initiation of ART was associated with a transient increase in monocytes (P < 0.001), decreased lymphocytes (P = 0.011) and eosinophils (P = 0.003) at Fiebig stages I-II, and decreased eosinophils (P < 0.001) and basophils (P = 0.007) at Fiebig stages III-V. Levels of CXCL13 during the untreated hyperacute phase correlated inversely with blood eosinophils (rho = - 0.89, P < 0.001), basophils (rho = - 0.87, P = 0.001) and lymphocytes (rho = - 0.81, P = 0.005), suggesting their trafficking into tissues. In early treated individuals, time to viral load suppression correlated positively with plasma CXCL13 at the early chronic phase (rho = 0.83, P = 0.042). CONCLUSION: While commencement of ART during Fiebig stages I-II of AHI abrogated the HIV-induced cytokine storm, significant depletions of eosinophils, basophils, and lymphocytes, as well as transient expansions of monocytes, were still observed in these individuals in the hyperacute phase before the initiation of ART, suggesting that even ART initiated during the onset of viremia does not abrogate all HIV-induced immune changes.

Metagenomic Sequencing of HIV-1 in the Blood and Female Genital Tract Reveals Little Quasispecies Diversity during Acute Infection.

Heterosexual transmission of human immunodeficiency virus type 1 (HIV-1) is associated with a significant bottleneck in the viral quasispecies population, yet the timing of that bottleneck is poorly understood. We characterized HIV-1 diversity in the blood and female genital tract (FGT) within 2 weeks after detection of infection in three women enrolled in a unique prospective cohort in South Africa. We assembled full-length HIV-1 genomes from matched cervicovaginal lavage (CVL) samples and plasma. Deep sequencing allowed us to identify intrahost single-nucleotide variants (iSNVs) and to characterize within-sample HIV-1 diversity. Our results demonstrated very little HIV-1 diversity in the FGT and plasma by the time viremia was detectable. Within each subject, the consensus HIV-1 sequences were identical in plasma and CVL fluid. No iSNV was present at >6% frequency. One subject had 77 low-frequency iSNVs across both CVL fluid and plasma, another subject had 14 iSNVs in only CVL fluid from the earliest time point, and the third subject had no iSNVs in CVL fluid or plasma. Overall, the small amount of diversity that we detected was greater in the FGT than in plasma and declined over the first 2 weeks after viremia was detectable, compatible with a very early HIV-1 transmission bottleneck. To our knowledge, our study represents the earliest genomic analysis of HIV-1 in the FGT after transmission. Further, the use of metagenomic sequencing allowed us to characterize other organisms in the FGT, including commensal bacteria and sexually transmitted infections, highlighting the utility of the method to sequence both HIV-1 and its metagenomic environment.IMPORTANCE Due to error-prone replication, HIV-1 generates a diverse population of viruses within a chronically infected individual. When HIV-1 is transmitted to a new individual, one or a few viruses establish the new infection, leading to a genetic bottleneck in the virus population. Understanding the timing and nature of this bottleneck may provide insight into HIV-1 vaccine design and other preventative strategies. We examined the HIV-1 population in three women enrolled in a unique prospective cohort in South Africa who were followed closely during the earliest stages of HIV-1 infection. We found very little HIV-1 diversity in the blood and female genital tract during the first 2 weeks after virus was detected in the bloodstream. These results are compatible with a very early HIV-1 population bottleneck, suggesting the need to study the HIV-1 population in the female genital tract before virus is detectable in the bloodstream.

Differences in HIV-1 reservoir size, landscape characteristics and decay dynamics in acute and chronic treated HIV-1 Clade C infection.

Background: Persisting HIV reservoir viruses in resting CD4 T cells and other cellular subsets are the main barrier to cure efforts. Antiretroviral therapy (ART) intensification by early initiation has been shown to enable post-treatment viral control in some cases but the underlying mechanisms are not fully understood. We hypothesized that ART initiated during the hyperacute phase of infection before peak will affect the size, decay dynamics and landscape characteristics of HIV-1 subtype C viral reservoirs. Methods: We studied 35 women at high risk of infection from Durban, South Africa identified with hyperacute HIV infection by twice weekly testing for plasma HIV-1 RNA. Study participants included 11 who started ART at a median of 456 (297-1203) days post onset of viremia (DPOV), and 24 who started ART at a median of 1 (1-3) DPOV. We used peripheral blood mononuclear cells (PBMC) to measure total HIV-1 DNA by ddPCR and to sequence reservoir viral genomes by full length individual proviral sequencing (FLIP-seq) from onset of detection of HIV up to 1 year post treatment initiation. Results: Whereas ART in hyperacute infection blunted peak viremia compared to untreated individuals (p<0.0001), there was no difference in total HIV-1 DNA measured contemporaneously (p=0.104). There was a steady decline of total HIV DNA in early treated persons over 1 year of ART (p=0.0004), with no significant change observed in the late treated group. Total HIV-1 DNA after one year of treatment was lower in the early treated compared to the late treated group (p=0.02). Generation of 697 single viral genome sequences revealed a difference in the longitudinal proviral genetic landscape over one year between untreated, late treated, and early treated infection: the relative contribution of intact genomes to the total pool of HIV-1 DNA after 1 year was higher in untreated infection (31%) compared to late treated (14%) and early treated infection (0%). Treatment initiated in both late and early infection resulted in a more rapid decay of intact (13% and 51% per month) versus defective (2% and 35% per month) viral genomes. However, intact genomes were still observed one year post chronic treatment initiation in contrast to early treatment where intact genomes were no longer detectable. Moreover, early ART reduced phylogenetic diversity of intact genomes and limited the seeding and persistence of cytotoxic T lymphocyte immune escape variants in the reservoir. Conclusions: Overall, our results show that whereas ART initiated in hyperacute HIV-1 subtype C infection did not impact reservoir seeding, it was nevertheless associated with more rapid decay of intact viral genomes, decreased genetic complexity and immune escape in reservoirs, which could accelerate reservoir clearance when combined with other interventional strategies.

Generation and characterization of infectious molecular clones of transmitted/founder HIV-1 subtype C viruses.

The genetic diversity of HIV impedes vaccine development. Identifying the viral properties of transmitted/founder (T/F) variants may provide a common vaccine target. To study the biological nature of T/F viruses, we constructed full-length clones from women detected during Fiebig stage I acute HIV-1 infection (AHI) from heterosexual male-to-female (MTF) transmission; and clones after one year of infection using In-Fusion-based cloning. Eighteen full-length T/F clones were generated from 9 women and six chronic infection clones were from 2 individuals. All clones but one were non-recombinant subtype C. Three of the 5 T/F clones and 3 chronic clones tested replicated efficiently in PBMCs and utilised CCR5 coreceptor for cell entry. Transmitted/founder and chronic infection clones displayed heterogenous in vitro replicative capacity and resistance to type I interferon. T/F viruses had shorter Env glycoproteins and fewer N-linked glycosylation sites in Env. Our findings suggest MTF transmission may select viruses with compact envelopes.

Increasing the meaningful involvement of women in HIV cure-related research: a qualitative interview study in the United States.

BACKGROUND: Cisgender women represent over half of people living with HIV globally. However, current research efforts toward a cure for HIV focus predominantly on cisgender men. The under-representation of women in HIV cure clinical studies is particularly problematic given data suggesting that sex-dependent phenotypes limit scientific discovery. OBJECTIVE: We aimed to generate considerations to increase the meaningful involvement of women in HIV cure-related research. MATERIALS AND METHODS: We conducted in-depth interviews with biomedical researchers and community members to better understand factors that could increase the meaningful involvement of women in HIV cure clinical trials. Participants were affiliated with academia, industry, community advisory boards, and community-based organizations, and were identified using listings from the AIDS Clinical Trials Group and the Martin Delaney Collaboratories. We used conventional content analysis to analyze the qualitative data. RESULTS: We recruited 27 participants, of whom 11 were biomedical researchers and 16 were community members. Participants included 25 cisgender women, 1 transgender woman, and 1 cisgender man. Key considerations emerged, including the need to ensure that HIV cure studies reflect HIV epidemiologic trends and having accurate representation by sex and gender in HIV cure research. To increase the meaningful involvement of women, recommendations included instituting intentional enrollment goals, frequent and mandatory reporting on enrollment, and incentives for sites to enroll women. Additional themes included the need for agency and self-determination, attention to lived experiences, trauma and healing, and adequate support for women (e.g. logistical, psychosocial, mental, emotional, and physical). Participants noted that women would be willing to participate in HIV cure trials, related procedures (e.g. biopsies), and analytical treatment interruptions. They also expressed a desired for women-centered and holistic clinical trial designs that account for intersectionality. CONCLUSIONS: Our empirical inquiry extends recent calls to action to increase diversity of people involved in HIV cure research. Redressing the under-inclusion of women in HIV cure research is an urgent imperative. The entire field must mobilize and reform to achieve this goal. Meaningfully involving women across the gender spectrum in HIV cure research is needed to ensure that interventions are safe, effective, scalable, and acceptable for all people with HIV.

Vaginal Lactobacillus fatty acid response mechanisms reveal a novel strategy for bacterial vaginosis treatment.

Bacterial vaginosis (BV), a common syndrome characterized by Lactobacillus-deficient vaginal microbiota, is associated with adverse health outcomes. BV often recurs after standard antibiotic therapy in part because antibiotics promote microbiota dominance by Lactobacillus iners instead of Lactobacillus crispatus, which has more beneficial health associations. Strategies to promote L. crispatus and inhibit L. iners are thus needed. We show that oleic acid (OA) and similar long-chain fatty acids simultaneously inhibit L. iners and enhance L. crispatus growth. These phenotypes require OA-inducible genes conserved in L. crispatus and related species, including an oleate hydratase (ohyA) and putative fatty acid efflux pump (farE). FarE mediates OA resistance, while OhyA is robustly active in the human vaginal microbiota and sequesters OA in a derivative form that only ohyA-harboring organisms can exploit. Finally, OA promotes L. crispatus dominance more effectively than antibiotics in an in vitro model of BV, suggesting a novel approach for treatment.

CD8 lymphocytes mitigate HIV-1 persistence in lymph node follicular helper T cells during hyperacute-treated infection.

HIV persistence in tissue sites despite ART is a major barrier to HIV cure. Detailed studies of HIV-infected cells and immune responses in native lymph node tissue environment is critical for gaining insight into immune mechanisms impacting HIV persistence and clearance in tissue sanctuary sites. We compared HIV persistence and HIV-specific T cell responses in lymph node biopsies obtained from 14 individuals who initiated therapy in Fiebig stages I/II, 5 persons treated in Fiebig stages III-V and 17 late treated individuals who initiated ART in Fiebig VI and beyond. Using multicolor immunofluorescence staining and in situ hybridization, we detect HIV RNA and/or protein in 12 of 14 Fiebig I/II treated persons on suppressive therapy for 1 to 55 months, and in late treated persons with persistent antigens. CXCR3+ T follicular helper cells harbor the greatest amounts of gag mRNA transcripts. Notably, HIV-specific CD8+ T cells responses are associated with lower HIV antigen burden, suggesting that these responses may contribute to HIV suppression in lymph nodes during therapy. These results reveal HIV persistence despite the initiation of ART in hyperacute infection and highlight the contribution of virus-specific responses to HIV suppression in tissue sanctuaries during suppressive ART.

Hypermethylation at the CXCR5 gene locus limits trafficking potential of CD8+ T cells into B-cell follicles during HIV-1 infection.

CD8+ T cells play an important role in HIV control. However, in human lymph nodes (LNs), only a small subset of CD8+ T cells express CXCR5, the chemokine receptor required for cell migration into B-cell follicles, which are major sanctuaries for HIV persistence in individuals on therapy. Here, we investigate the impact of HIV infection on follicular CD8+ T cell (fCD8) frequencies, trafficking patterns, and CXCR5 regulation. We show that, although HIV infection results in a marginal increase in fCD8s in LNs, the majority of HIV-specific CD8+ T cells are CXCR5- (non-fCD8s) (P < .003). Mechanistic investigations using Assay for Transposase-Accessible Chromatin using sequencing showed that non-fCD8s have closed chromatin at the CXCR5 transcriptional start site (TSS). DNA bisulfite sequencing identified DNA hypermethylation at the CXCR5 TSS as the most probable cause of closed chromatin. Transcriptional factor footprint analysis revealed enrichment of transforming growth factors (TGFs) at the TSS of fCD8s. In vitro stimulation of non-fCD8s with recombinant TGF-ß resulted in a significant increase in CXCR5 expression (fCD8s). Thus, this study identifies TGF-ß signaling as a viable strategy for increasing fCD8 frequencies in follicular areas of the LN where they are needed to eliminate HIV-infected cells, with implications for HIV cure strategies.

Cysteine dependence of Lactobacillus iners is a potential therapeutic target for vaginal microbiota modulation.

Vaginal microbiota composition affects many facets of reproductive health. Lactobacillus iners-dominated microbial communities are associated with poorer outcomes, including higher risk of bacterial vaginosis (BV), compared with vaginal microbiota rich in L. crispatus. Unfortunately, standard-of-care metronidazole therapy for BV typically results in dominance of L. iners, probably contributing to post-treatment relapse. Here we generate an L. iners isolate collection comprising 34 previously unreported isolates from 14 South African women with and without BV and 4 previously unreported isolates from 3 US women. We also report an associated genome catalogue comprising 1,218 vaginal Lactobacillus isolate genomes and metagenome-assembled genomes from >300 women across 4 continents. We show that, unlike L. crispatus, L. iners growth is dependent on L-cysteine in vitro and we trace this phenotype to the absence of canonical cysteine biosynthesis pathways and a restricted repertoire of cysteine-related transport mechanisms. We further show that cysteine concentrations in cervicovaginal lavage samples correlate with Lactobacillus abundance in vivo and that cystine uptake inhibitors selectively inhibit L. iners growth in vitro. Combining an inhibitor with metronidazole promotes L. crispatus dominance of defined BV-like communities in vitro by suppressing L. iners growth. Our findings enable a better understanding of L. iners biology and suggest candidate treatments to modulate the vaginal microbiota to improve reproductive health for women globally.

Antigen Presenting Cells Contribute to Persistent Immune Activation Despite Antiretroviral Therapy Initiation During Hyperacute HIV-1 Infection.

Human immunodeficiency virus (HIV)-induced changes in immune cells during the acute phase of infection can cause irreversible immunological damage and predict the rate of disease progression. Antiretroviral therapy (ART) remains the most effective strategy for successful immune restoration in immunocompromised people living with HIV and the earlier ART is initiated after infection, the better the long-term clinical outcomes. Here we explored the effect of ART on peripheral antigen presenting cell (APC) phenotype and function in women with HIV-1 subtype C infection who initiated ART in the hyperacute phase (before peak viremia) or during chronic infection. Peripheral blood mononuclear cells obtained longitudinally from study participants were used for immunophenotyping and functional analysis of monocytes and dendritic cells (DCs) using multiparametric flow cytometry and matched plasma was used for measurement of inflammatory markers IL-6 and soluble CD14 (sCD14) by enzyme-linked immunosorbent assay. HIV infection was associated with expansion of monocyte and plasmacytoid DC (pDC) frequencies and perturbation of monocyte subsets compared to uninfected persons despite antiretroviral treatment during hyperacute infection. Expression of activation marker CD69 on monocytes and pDCs in early treated HIV was similar to uninfected individuals. However, despite early ART, HIV infection was associated with elevation of plasma IL-6 and sCD14 levels which correlated with monocyte activation. Furthermore, HIV infection with or without early ART was associated with downmodulation of the co-stimulatory molecule CD86. Notably, early ART was associated with preserved toll-like receptor (TLR)-induced IFN-α responses of pDCs. Overall, this data provides evidence of the beneficial impact of ART initiated in hyperacute infection in preservation of APC functional cytokine production activity; but also highlights persistent inflammation facilitated by monocyte activation even after prolonged viral suppression and suggests the need for therapeutic interventions that target residual immune activation.

Epigenetic Regulation of BST-2 Expression Levels and the Effect on HIV-1 Pathogenesis.

HIV-1 must overcome host antiviral restriction factors for efficient replication. We hypothesized that elevated levels of bone marrow stromal cell antigen 2 (BST-2), a potent host restriction factor that interferes with HIV-1 particle release in some human cells and is antagonized by the viral protein Vpu, may associate with viral control. Using cryopreserved samples, from HIV-1 seronegative and seropositive Black women, we measured in vitro expression levels of BST-2 mRNA using a real-time PCR assay and protein levels were validated by Western blotting. The expression level of BST-2 showed an association with viral control within two independent cohorts of Black HIV infected females (r=-0.53, p=0.015, [n =21]; and r=-0.62, p=0.0006, [n=28]). DNA methylation was identified as a mechanism regulating BST-2 levels, where increased BST-2 methylation results in lower expression levels and associates with worse HIV disease outcome. We further demonstrate the ability to regulate BST-2 levels using a DNA hypomethylation drug. Our results suggest BST-2 as a factor for potential therapeutic intervention against HIV and other diseases known to involve BST-2.

Modeling the temporal dynamics of cervicovaginal microbiota identifies targets that may promote reproductive health.

BACKGROUND: Cervicovaginal bacterial communities composed of diverse anaerobes with low Lactobacillus abundance are associated with poor reproductive outcomes such as preterm birth, infertility, cervicitis, and risk of sexually transmitted infections (STIs), including human immunodeficiency virus (HIV). Women in sub-Saharan Africa have a higher prevalence of these high-risk bacterial communities when compared to Western populations. However, the transition of cervicovaginal communities between high- and low-risk community states over time is not well described in African populations. RESULTS: We profiled the bacterial composition of 316 cervicovaginal swabs collected at 3-month intervals from 88 healthy young Black South African women with a median follow-up of 9 months per participant and developed a Markov-based model of transition dynamics that accurately predicted bacterial composition within a broader cross-sectional cohort. We found that Lactobacillus iners-dominant, but not Lactobacillus crispatus-dominant, communities have a high probability of transitioning to high-risk states. Simulating clinical interventions by manipulating the underlying transition probabilities, our model predicts that the population prevalence of low-risk microbial communities could most effectively be increased by manipulating the movement between L. iners- and L. crispatus-dominant communities. CONCLUSIONS: The Markov model we present here indicates that L. iners-dominant communities have a high probability of transitioning to higher-risk states. We additionally identify transitions to target to increase the prevalence of L. crispatus-dominant communities. These findings may help guide future intervention strategies targeted at reducing bacteria-associated adverse reproductive outcomes among women living in sub-Saharan Africa. Video Abstract.

Antigen Presenting Cells Link the Female Genital Tract Microbiome to Mucosal Inflammation, With Hormonal Contraception as an Additional Modulator of Inflammatory Signatures.

The microbiome of the female genital tract (FGT) is closely linked to reproductive health outcomes. Diverse, anaerobe-dominated communities with low Lactobacillus abundance are associated with a number of adverse reproductive outcomes, such as preterm birth, cervical dysplasia, and sexually transmitted infections (STIs), including HIV. Vaginal dysbiosis is associated with local mucosal inflammation, which likely serves as a biological mediator of poor reproductive outcomes. Yet the precise mechanisms of this FGT inflammation remain unclear. Studies in humans have been complicated by confounding demographic, behavioral, and clinical variables. Specifically, hormonal contraception is associated both with changes in the vaginal microbiome and with mucosal inflammation. In this study, we examined the transcriptional landscape of cervical cell populations in a cohort of South African women with differing vaginal microbial community types. We also investigate effects of reproductive hormones on the transcriptional profiles of cervical cells, focusing on the contraceptive depot medroxyprogesterone acetate (DMPA), the most common form of contraception in sub-Saharan Africa. We found that antigen presenting cells (APCs) are key mediators of microbiome associated FGT inflammation. We also found that DMPA is associated with significant transcriptional changes across multiple cell lineages, with some shared and some distinct pathways compared to the inflammatory signature seen with dysbiosis. These results highlight the importance of an integrated, systems-level approach to understanding host-microbe interactions, with an appreciation for important variables, such as reproductive hormones, in the complex system of the FGT mucosa.