Studies in SIV-infected macaques show that the virus reservoir is particularly refractory to conventional suppressive antiretroviral therapy (ART). We posit that optimized ART regimens designed to have robust penetration in tissue reservoirs and long-lasting antiviral activity may be advantageous for HIV or SIV remission. Here we treat macaques infected with RT-SHIV with oral emtricitabine/tenofovir alafenamide and long-acting cabotegravir/rilpivirine without (n = 4) or with (n = 4) the immune activator vesatolimod after the initial onset of viremia. We document full suppression in all animals during treatment (4-12 months) and no virus rebound after treatment discontinuation (1.5-2 years of follow up) despite CD8 + T cell depletion. We show efficient multidrug penetration in virus reservoirs and persisting rilpivirine in plasma for 2 years after the last dose. Our results document a type of virus remission that is achieved through early treatment initiation and provision of ultra long-lasting antiviral activity that persists after treatment cessation.

Despite their importance for immunity against sexually transmitted infections (STIs), the composition of the female reproductive tract (FRT) memory CD4 T cell population in response to changes in the local tissue environment during the menstrual cycle remains poorly defined. Here we show that across humans, non-human primates (NHP), and mice, FRT CD4 T cells comprise distinct subsets corresponding to migratory memory (TMM) and resident memory (TRM) cells. TMM display tissue-itinerant trafficking characteristics, restricted FRT tissue distribution, with distinct transcriptional properties and effector responses to infection. CD4 T cell subset fluctuations synchronized with cycle-driven proinflammatory changes within the local tissue environment and oral administration of a CCR5 antagonist inhibited cycle phase-specific migratory T cell surveillance. This study provides novel insights into the dynamic nature of FRT memory CD4 T cells and identifies the menstrual cycle as a key regulator of memory T cell defense at the site of STI exposure. Copyright The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

BACKGROUND: The menstrual cycle influences HIV infection-risk in women, although the timing and underlying mechanism are unclear. Here we investigated the contribution of the menstrual cycle to HIV susceptibility through evaluating immune behavior with infection-risk over time. METHODS: Blood and vaginal lavage samples were collected from 18 pig-tailed macaques to evaluate immune changes over reproductive cycles, and from 5 additional animals undergoing repeated vaginal exposures to simian HIV (SHIV). Peripheral blood mononuclear cell (PBMC) samples from healthy women (n = 10) were prospectively collected over the course of a menstrual cycle to profile T cell populations. Immune properties from PBMC and vaginal lavage samples were measured by flow cytometry. Plasma progesterone was measured by enzyme immunoassay. The oscillation frequency of progesterone concentration and CCR5 expression on CD4 T cells was calculated using the Lomb-Scargle periodogram. SHIV infection was monitored in plasma by RT-PCR. Immune measures were compared using generalized estimating equations (GEE). FINDINGS: Macaques cycle-phases were associated with fluctuations in systemic immune properties and a type-1 inflammatory T cell response with corresponding CCR5+ memory CD4 T cell (HIV target cell) infiltration into the vaginal lumen at the late luteal phase. Power spectral analysis identified CCR5 oscillation frequencies synchronized with reproductive cycles. In a repetitive low-dose vaginal challenge model, productive SHIV(163P3) infection only occurred during intervals of mounting type-1 T cell responses (n = 5/5). Finally, we identify similar type-1 inflammatory T cell responses over the menstrual cycle are occurring in healthy women. INTERPRETATION: These data demonstrate that periodic shifts in the immune landscape under menstrual cycle regulation drives bystander CCR5+ CD4 T cell recruitment and HIV susceptibility in the female reproductive tract. FUNDING: This study was supported by the U.S. Centers for Disease Control and Prevention, Atlanta, GA 30329 and NIH grants to Emory University (K23AI114407 to A.N.S., the Emory University Center for AIDS research [P30AI050409], and Atlanta Clinical and Translational Sciences Institute [KLR2TR000455, UL1TR000454]). DISCLAIMER: The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the U.S. Centers for Disease Control and Prevention or the Department of Health and Human Services.

We examined the relationship between CSF immune cells and neurocognition and neuronal damage in HIV+ individuals before and after initiating antiretroviral therapy. Multivariate analysis at baseline indicated that greater CD4+ T cell abundance was associated with better cognition (p = .017), while higher CSF HIV RNA was associated with increased neuronal damage (p = .014). Following 24 weeks of antiretroviral therapy, CD8+ T cells, HLA-DR expressing CD4+ and CD8+ T cells, B cells, NK cells, and non-classical monocyte percentage decreased in CSF. Female gender was negatively associated with cognitive performance over time, as was higher percentage of HLA-DR expressing CD8+ T cells at baseline.

BACKGROUND: While prior epidemiologic studies have suggested that injectable progestin-based contraceptive depot medroxyprogesterone acetate (DMPA) use may increase a woman's risk of acquiring HIV, recent data have suggested that DMPA users may be at a similar risk for HIV acquisition as users of the copper intrauterine device and levonorgestrel implant. Use of the etonogestrel Implant (Eng-Implant) is increasing but there are currently no studies evaluating its effect on HIV acquisition risk. OBJECTIVE: Evaluate the potential effect of the Eng-Implant use on HIV acquisition risk by analyzing HIV target cells and cytokine profiles in the lower genital tract and blood of adult premenopausal HIV-negative women using the Eng-Implant. METHODS: We prospectively obtained paired cervicovaginal lavage (CVL) and blood samples at 4 study visits over 16 weeks from women between ages 18-45, with normal menses (22-35 day intervals), HIV uninfected with no recent hormonal contraceptive or copper intrauterine device (IUD) use, no clinical signs of a sexually transmitted infection at enrollment and who were medically eligible to initiate Eng-Implant. Participants attended pre-Eng-Implant study visits (week -2, week 0) with the Eng-Implant inserted at the end of the week 0 study visit and returned for study visits at weeks 12 and 14. Genital tract leukocytes (enriched from CVL) and peripheral blood mononuclear cells (PBMC) from the study visits were evaluated for markers of activation (CD38, HLA-DR), retention (CD103) and trafficking (CCR7) on HIV target cells (CCR5+CD4+ T cells) using multicolor flow cytometry. Cytokines and chemokines in the CVL supernatant and blood plasma were measured in a Luminex assay. We estimated and compared study endpoints among the samples collected before and after contraception initiation with repeated-measures analyses using linear mixed models. RESULTS: Fifteen of 18 women who received an Eng-Implant completed all 4 study visits. The percentage of CD4+ T cells in CVL was not increased after implant placement but the percentage of CD4+ T cells expressing the HIV co-receptor CCR5 did increase after implant placement (p = 0.02). In addition, the percentage of central memory CD4+ T-cells (CCR7+) in CVL increased after implant placement (p = 0.004). The percentage of CVL CD4+, CCR5+ HIV target cells expressing activation markers after implant placement was either reduced (HLA-DR+, p = 0.01) or unchanged (CD38+, p = 0.45). Most CVL cytokine and chemokine concentrations were not significantly different after implant placement except for a higher level of the soluble lymphocyte activation marker (sCD40L; p = 0.04) and lower levels of IL12p70 (p = 0.02) and G-CSF (p<0.001). In systemic blood, none of the changes noted in CVL after implant placement occurred except for decreases in the percentage CD4 T-cells expressing HLA-DR+ T cells (p = 0.006) and G-CSF (p = 0.02). CONCLUSIONS: Eng-Implant use was associated with a moderate increase in the availability of HIV target cells in the genital tract, however the percentage of these cells that were activated did not increase and there were minimal shifts in the overall immune environment. Given the mixed nature of these findings, it is unclear if these implant-induced changes alter HIV risk.

Recently, the IL-36 cytokines were shown to be elevated in women with non-Lactobacillus-dominated vaginal microbiomes. Here, we evaluated IL36G expression in clinical samples from women with and without bacterial vaginosis (BV) and a human 3-D cervical epithelial cell model. IL36G expression was significantly elevated in cervicovaginal epithelial cells isolated from BV-positive women and corresponded with increased neutrophil counts relative to BV-negative women. Additionally, specific BV-associated bacterial species as well as a polymicrobial cocktail significantly induced IL36G expression in vitro. These findings suggest that IL-36gamma may exhibit an important function in the host response to BV and other sexually transmitted infections.

HSV-2 is a neurotropic virus that causes a persistent, lifelong infection that increases risk for other sexually transmitted infections. The vaginal epithelium is the first line of defense against HSV-2 and coordinates the immune response through the secretion of immune mediators, including the proinflammatory cytokine IL-36gamma. Previously, we showed that IL-36gamma treatment promoted transient polymorphonuclear cell infiltration to the vaginal cavity and protected against lethal HSV-2 challenge. In this report, we reveal that IL-36gamma specifically induces transient neutrophil infiltration but does not impact monocyte and macrophage recruitment. Using IL-36gamma(-/-) mice in a lethal HSV-2 challenge model, we show that neutrophil counts are significantly reduced at 1 and 2 d postinfection and that KC-mediated mature neutrophil recruitment is impaired in IL-36gamma(-/-) mice. Additionally, IL-36gamma(-/-) mice develop genital disease more rapidly, have significantly reduced survival time, and exhibit an increased incidence of hind limb paralysis that is linked to productive HSV-2 infection in the brain stem. IL-36gamma(-/-) mice also exhibit a significant delay in clearance of the virus from the vaginal epithelium and a more rapid spread of HSV-2 to the spinal cord, bladder, and colon. We further show that the decreased survival time and increased virus spread observed in IL-36gamma(-/-) mice are not neutrophil-dependent, suggesting that IL-36gamma may function to limit HSV-2 spread in the nervous system. Ultimately, we demonstrate that IL-36gamma is a key regulator of neutrophil recruitment in the vaginal microenvironment and may function to limit HSV-2 neuroinvasion.

We explored the association between violence victimization and increased risk for acquiring sexually transmitted infections (STIs) in women by measuring cellular immune barrier properties from the female reproductive tract. STI-negative participants reporting repeated prior victimization occurrences through the lifetime trauma and victimization history (LTVH) instrument were more likely to exhibit alterations in barrier homeostasis and the composition of critical immune mediators irrespective of demographic parameters or presence of bacterial vaginosis. By combining cellular data with mixed-effect linear modeling, we uncovered differences in local T cells, MHCII+ antigen-presenting cells, and epithelial cells indicative of altered trafficking behavior, increased immunosuppressive function, and decreased barrier integrity at sites of STI exposure that correlate most strongly with LTVH score. These data evidence a biological link between a history of violence victimization and risk of STI acquisition through immune dysregulation in the female reproductive tract.

Successful clinical trials of antiretroviral pre-exposure prophylaxis (PrEP) to prevent HIV infection have been reported in heterosexual men and women, men who have sex with men (MSM), and injection drug users1. A daily oral drug regimen containing nucleoside reverse transcriptase inhibitors (NRTIs) tenofovir disoproxil fumarate (TDF) alone or in combination with emtricitabine (FTC) have been effective when participant adherence is high1. Analysis of PrEP dosing patterns estimate taking at least four TDF doses per week provides 96% protection from HIV infection and at least two doses per week provides 76% protection in MSM and transgender women2, though some estimate more frequent dosing in heterosexual women may be needed3. TDF and FTC require phosphorylation in HIV target cells to tenofovir-diphosphate (TFV-DP) and emtricitabine-triphosphate (FTC-TP) to provide PrEP protection as competitive analogs of naturally occurring deoxyadenosine triphosphate (dATP) and deoxycytidine triphosphate (dCTP), respectively. However, it is unclear if physiological conditions that increase dNTP concentrations can affect pharmacokinetics (PK) and pharmacodynamics (PD) of NRTIs. This may be particularly relevant when cellular deoxynucleoside triphosphate (dNTP) pools in HIV target cells increase in response to immune activation. Decreased ratios of phosphorylated NRTIs to their respective dNTPs have been associated with cell activation in vitro and in nonhuman primate studies4,5, yet this observation remains unexplored in PK and PD studies that measured intracellular drug6-8. The study presented here compared dATP and dCTP concentrations to TFV-DP and FTC-TP in peripheral blood mononuclear cell (PBMCs) of persons using daily oral Truvada™ during a successful HIV PrEP study. We further examined if variations among intracellular drug:dNTP ratios were related to lymphocyte activation.

The female genital tract (FGT) provides a means of entry to pathogens, including HIV, yet immune cell populations at this barrier between host and environment are not well defined. We initiated a study of healthy women to characterize resident T cell populations in the lower FGT from lavage and patient-matched peripheral blood to investigate potential mechanisms of HIV sexual transmission. Surprisingly, we observed FGT CD4 T cell populations were primarily CCR7hi, consistent with a central memory or recirculating memory T cell phenotype. In addition, roughly half of these CCR7hi CD4 T cells expressed CD69, consistent with resident memory T cells, whereas the remaining CCR7hi CD4 T cells lacked CD69 expression, consistent with recirculating memory CD4 T cells that traffic between peripheral tissues and lymphoid sites. HIV susceptibility markers CCR5 and CD38 were increased on FGT CCR7hi CD4 T cells compared with blood, yet migration to the lymphoid homing chemokines CCL19 and CCL21 was maintained. Infection with GFP-HIV showed that FGT CCR7hi memory CD4 T cells are susceptible HIV targets, and productive infection of CCR7hi memory T cells did not alter chemotaxis to CCL19 and CCL21. Variations of resident CCR7hi FGT CD4 T cell populations were detected during the luteal phase of the menstrual cycle, and longitudinal analysis showed the frequency of this population positively correlated to progesterone levels. These data provide evidence women may acquire HIV through local infection of migratory CCR7hi CD4 T cells, and progesterone levels predict opportunities for HIV to access these novel target cells.