Mucosal HIV Transmission Research Articles

Highly Sensitive Analysis of Cervical Mucosal HIV-1 Infection Using Reporter Viruses Expressing Secreted Nanoluciferase.

Ex vivo cervical tissue explant models offer a physiologically relevant approach for studying virus-host interactions that underlie mucosal HIV-1 transmission to women. However, the utility of cervical explant tissue (CET) models has been limited for both practical and technical reasons. These include assay variation, inadequate sensitivity for assessing HIV-1 infection and replication in tissue, and constraints imposed by the requirement for using multiple replica samples of CET to test each experimental variable and assay parameter. Here, we describe an experimental approach that employs secreted nanoluciferase (sNLuc) and current HIV-1 reporter virus technologies to overcome certain limitations of earlier ex vivo CET models. This method augments application of the CET model for investigating important questions involving mucosal HIV-1 transmission.

S11.1: Understanding the effect of microenvironment in the female genital tract on mucosal HIV transmission

S11.1: Understanding the effect of microenvironment in the female genital tract on mucosal HIV transmission

CD34T+ Humanized Mouse Model to Study Mucosal HIV-1 Transmission and Prevention.

Humanized mice are critical for HIV-1 research, but humanized mice generated from cord blood are inefficient at mucosal HIV-1 transmission. Most mucosal HIV-1 transmission studies in mice require fetal tissue-engraftment, the use of which is highly restricted or prohibited. We present a fetal tissue-independent model called CD34T+ with enhanced human leukocyte levels in the blood and improved T cell homing to the gut-associated lymphoid tissue. CD34T+ mice are highly permissive to intra-rectal HIV-1 infection and also show normal env diversification in vivo despite high viral replication. Moreover, mucosal infection in CD34T+ mice can be prevented by infusion of broadly neutralizing antibodies. CD34T+ mice can be rapidly and easily generated using only cord blood cells and do not require any complicated surgical procedures for the humanization process. Therefore, CD34T+ mice provide a novel platform for mucosal HIV-1 transmission studies as well as rapid in vivo testing of novel prevention molecules against HIV-1.

Innate Immune Response Against HIV-1.

The innate immune system is comprised of both cellular and humoral players that recognise and eradicate invading pathogens. Therefore, the interplay between retroviruses and innate immunity has emerged as an important component of viral pathogenesis. HIV-1 infection in humans that results in hematologic abnormalities and immune suppression is well represented by changes in the CD4/CD8 T cell ratio and consequent cell death causing CD4 lymphopenia. The innate immune responses by mucosal barriers such as complement, DCs, macrophages, and NK cells as well as cytokine/chemokine profiles attain great importance in acute HIV-1 infection, and thus, prevent mucosal capture and transmission of HIV-1. Conversely, HIV-1 has evolved to overcome innate immune responses through RNA-mediated rapid mutations, pathogen-associated molecular patterns (PAMPs) modification, down-regulation of NK cell activity and complement receptors, resulting in increased secretion of inflammatory factors. Consequently, epithelial tissues lining up female reproductive tract express innate immune sensors including anti-microbial peptides responsible for forming primary barriers and have displayed an effective potent anti-HIV activity during phase I/II clinical trials.

Innate immune mechanisms to oral pathogens in oral mucosa of HIV-infected individuals.

A crucial aspect of mucosal HIV transmission is the interaction between HIV, the local environmental milieu and immune cells. The oral mucosa comprises many host cell types including epithelial cells, CD4 + T cells, dendritic cells and monocytes/macrophages, as well as a diverse microbiome predominantly comprising bacterial species. While the oral epithelium is one of the first sites exposed to HIV through oral-genital contact and nursing infants, it is largely thought to be resistant to HIV transmission via mechanisms that are still unclear. HIV-1 infection is also associated with predisposition to secondary infections, such as tuberculosis, and other diseases including cancer. This review addresses the following questions that were discussed at the 8th World Workshop on Oral Health and Disease in AIDS held in Bali, Indonesia, 13 September –15 September 2019: (a) How does HIV infection affect epithelial cell signalling? (b) How does HIV infection affect the production of cytokines and other innate antimicrobial factors, (c) How is the mucosal distribution and function of immune cells altered in HIV infection? (d) How do T cells affect HIV (oral) pathogenesis and cancer? (e) How does HIV infection lead to susceptibility to TB infections?

Short Communication:Transgender Women on Feminizing Hormone Therapy Demonstrate a Distinct Rectal Mucosal Transcriptome from Cisgender Men.

Among transgender women (TGW), the effects of feminizing hormone therapy use on rectal mucosal (RM) HIV transmission are largely unknown. In this small pilot study, we compared the RM transcriptome in TGW utilizing feminizing hormone therapy with a group of cisgender men who have sex with men (MSM) engaging in condomless receptive anal intercourse (AI) and to a group of cisgender men who had never engaged in AI. There were 498 differentially expressed genes (DEGs) in TGW compared with men who had never engaged in AI, and 154 DEGs compared with the MSM. Among TGW, a unique RM transcriptome was identified that implicated pathways critical for mucosal immune responses, including upregulation of genes that mediate immune cell activation and the production of cytokines and other immune signaling molecules. Furthermore, gene set enrichment analyses identified immune signatures that implicated enrichment of proinflammatory immunological pathways in TGW, specifically involving interferon-α, interleukin-6, and tumor necrosis factor-α signaling, whereas metabolic pathways were shown to be enriched among the cisgender male groups. These findings suggest that TGW have a distinct RM immune environment influenced by the use of feminizing hormones, and consequently, there is an urgent need for further investigation into the immunological effects of gender-affirming hormone therapy and its potential impact on HIV mucosal transmission risk for transgender individuals.

Use of Monoclonal Antibodies to Prevent the Sexual Transmission of Human Immunodeficiency Virus Type 1

<P>Passive immunization has been used since the late 1800’s to prevent and treat human infectious diseases. Administration of animal immune sera and human immunoglobulin has given way to the use of monoclonal antibodies (mAbs) for passive immunization, and highly potent broadly neutralizing anti-HIV antibodies (bNAbs) are now being considered for HIV therapy and prophylaxis. Recent studies have shown that systemic and topical administration of bNAbs can effectively inhibit HIV/SHIV mucosal transmission in macaques and in humanized mice, and selected bNAbs are currently being tested in clinical trials for safety and efficacy in humans. In this review, we outline strategies for the selection, engineering and manufacture of human bNAbs to prevent the sexual transmission of HIV, describe the proof-of-concept animal studies that have demonstrated mAb-mediated protection against mucosal HIV transmission, and review clinical trials currently underway to test the safety and efficacy of mAb-based HIV prevention in humans.

Virologic Aspects of Mucosal Transmission

The transmission of HIV is generally inefficient. Despite the development of a diverse viral quasispecies in a chronically infected individual, a severe genetic bottleneck is observed during transmission, leading to only one or a few genetic variants establishing infection. This genetic bottleneck is the result of both stochastic events and selection pressures, such that viruses with specific traits are favored during transmission. This chapter discusses current models of HIV mucosal transmission, evidence for selection of specific viral traits during this process, and the biological characterization of transmitted founder viruses based on monkey models and human cohorts. The impact of transmitted viral phenotypes on disease progression is also described. Understanding in greater depth the key viral features required for transmission will be essential to the development of effective interventions for HIV prevention.

Determinants of Tenascin-C and HIV-1 envelope binding and neutralization.

Interactions between innate antiviral factors at mucosal surfaces and HIV-1 virions contribute to the natural inefficiency of HIV-1 transmission and are a platform to inform the development of vaccine and nonvaccine strategies to block mucosal HIV-1 transmission. Tenascin-C (TNC) is a large, hexameric extracellular matrix glycoprotein identified in breast milk and genital fluids that broadly neutralizes HIV-1 via interaction with the HIV-1 Envelope (Env) variable 3 (V3) loop. In this report, we characterize the specific determinants of the interaction between TNC and the HIV-1 Env. We observed that TNC binding and neutralization of HIV-1 is dependent on the TNC fibrinogen-like globe (fbg) and fibronectin-type III (fn) domains, oligomerization, and its newly-mapped glycan structure. Moreover, we observed that TNC-mediated neutralization is also dependent on Env V3 residues 321/322 and 326/327, which surround the IGDIR motif of the V3 loop, as well the N332 glycan, which is critical to the broadly neutralizing activity of glycan-dependent V3-specific antibodies such as PGT128. Our results demonstrate a striking parallel between innate and adaptive immune mechanisms of broad HIV neutralization and provide further insight into the host protein-virus interactions responsible for the natural inefficiency of mucosal HIV-1 transmission.

Mucosa: Key Interactions Determining Sexual Transmission of the HIV Infection.

In the context of HIV sexual transmission at the genital mucosa, initial interactions between the virus and the mucosal immunity determine the outcome of the exposure. Hence, these interactions have been deeply explored in attempts to undercover potential targets for developing preventative strategies. The knowledge gained has led to propose a hypothetical model for mucosal HIV transmission. Subsequent research studies on this topic further revealed new mechanisms and identified new host-HIV interactions. This review aims at integrating these findings to inform better and update the current model of HIV transmission. At the earliest stage of virus exposure, the epithelial integrity and the presence of antiviral factors are critical in preventing viral entry to the submucosa. However, the virus has been shown to enter to the submucosa in the presence of physical abrasion or via epithelial transmigration using paracellular passage or transcytosis mechanisms. The efficiency of these processes is greater with cell-associated viral inoculums and can be influenced by the presence of viral and immune factors, and by the structure of the exposed epithelium. Once the virus reaches the submucosa, dendritic cells and fibroblasts, as recently described, have been shown in vitro of being capable of facilitating the transfer of viral particles to susceptible cells, leading to viral dissemination, most likely in a trans-infection manner. The presence of activated CD4+ T cells in submucosa increases the probability of infection, where the predominant microbiota could be implicated through the modulation of an inflammatory microenvironment. Other factors such as genital fluids and hormones could also play an essential role in HIV transmission. Here, we review the most recent evidence described for mucosal HIV-transmission contributing with the understanding of this phenomenon.

Short Communication: Decreased Plasma Calcitonin Gene-Related Peptide as a Novel Biomarker for HIV-1 Disease Progression

HIV-1 mucosal transmission in genital epithelia occurs through infection of Langerhans cells and subsequent transinfection of CD4+ T cells. We previously reported that the vasodilator neuropeptide calcitonin gene-related peptide (CGRP), secreted upon activation of sensory peripheral neurons that innervate all mucosal epithelia, significantly inhibits transinfection. To investigate the association between CGRP and HIV-1 during infection, we evaluated circulating CGRP levels in HIV-1-infected patients. Plasma was obtained from combination antiretroviral therapy (cART)-naive or cART-treated patients with primary/acute (PHI) or chronic (CHI) HIV-1 infection, as well as from individuals who naturally control HIV-1 infection, namely exposed seronegatives (ESNs), elite controllers (ECs), and long-term nonprogressors (LTNPs). CGRP plasma levels were measured using an enzyme immunoassay. Compared with healthy HIV-1-negative controls, CGRP plasma levels significantly decreased in PHI patients and even further in CHI patients, but remained unchanged in ESNs, ECs, and LTNPs. Moreover, CGRP plasma levels were restored to baseline upon cART in both PHI and CHI. Finally, CGRP plasma levels directly correlated with CD4+ T cell counts and inversely with viral loads. Altogether, CGRP could serve as a novel diagnostic plasma biomarker for progression of HIV-1 infection. Moreover, administration of CGRP to cART-naive HIV-1-infected patients, to compensate for CGRP decline, could help controlling on-going HIV-1 infection.

Anti-HIV IgM protects against mucosal SHIV transmission.

Worldwide, most new HIV infections occur through mucosal exposure. Immunoglobulin M (IgM) is the first antibody class generated in response to infectious agents; IgM is present in the systemic circulation and in mucosal fluids as secretory IgM. We sought to investigate for the first time the role of IgM in preventing AIDS virus acquisition in vivo. Recombinant polymeric monoclonal IgM was generated from the neutralizing monoclonal IgG1 antibody 33C6-IgG1, tested in vitro, and given by passive intrarectal immunization to rhesus macaques 30 min before intrarectal challenge with simian-human immunodeficiency virus (SHIV) that carries an HIV-1 envelope gene. In vitro, 33C6-IgM captured virions more efficiently and neutralized the challenge SHIV with a 50% inhibitory molar concentration (IC50) that was 1 log lower than that for 33C6-IgG1. The IgM form also exhibited significantly higher affinity and avidity compared with 33C6-IgG1. After intrarectal administration, 33C6-IgM prevented viremia in four out of six rhesus macaques after high-dose intrarectal SHIV challenge. Five out of six rhesus macaques given 33C6-IgG1 were protected at a five times higher molar concentration compared with the IgM form; all untreated controls became highly viremic. Rhesus macaques passively immunized with 33C6-IgM with breakthrough infection had notably early development of autologous neutralizing antibody responses. Our primate model data provide the first proof-of-concept that mucosal IgM can prevent mucosal HIV transmission and have implications for HIV prevention and vaccine development.

HIV transmission from infected CD4+ T cells to allogenic T and dendritic cells is inhibited by broadly neutralizing antibodies.

In the semen, both free virus and infected cells are able to establish HIV infection during sexual intercourse. An efficient vaccine should therefore inhibit both infectious states. The aim of this study was to analyze the capacity of broadly neutralizing antibodies (bNAbs) to inhibit HIV transmission by the infected cells. We developed an in-vitro model aiming to mimic mucosal HIV transmission via infected cells. PHA-activated CD4+ T cells stained with PKH26 from donor A were infected and co-cultured with CD4+ T cells and dendritic cells from donor B in the presence of bNAbs. We showed that dendritic cells were the preferential HIV target cells at early time points in this co-culture model. In the context of this co-culture model where infection and transmission occurred simultaneously, bNAbs efficiently inhibited HIV replication as well as HIV transmission from infected cells to allogenic dendritic cells and CD4+ T cells. Overall, our results indicate that dendritic cells, in addition to CD4+ T cells, are key cells that are efficiently infected by HIV and bNAbs are potent inhibitors of infection of both target cells. Future HIV prophylactic vaccine design should develop immune strategies able to prevent the infection of dendritic cells, in addition to the inhibition of CD4+ T-cell infection.

Role of Porphyromonas gingivalis outer membrane vesicles in oral mucosal transmission of HIV

The association between mucosal microbiota and HIV-1 infection has garnered great attention in the field of HIV-1 research. Previously, we reported a receptor-independent HIV-1 entry into epithelial cells mediated by a Gram-negative invasive bacterium, Porphyromonas gingivalis. Here, we present evidence showing that P. gingivalis outer membrane vesicles (OMVs) promote mucosal transmission of HIV-1. We demonstrated, using the Dynabeads technology, a specific interaction between HIV-1 and P. gingivalis OMVs which led to an OMV-dependent viral entry into oral epithelial cells. HIV-1 was detected in human oral keratinocytes (HOKs) after a 20 minute exposure to the HIV-vesicle complexes. After entry, most of the complexes appeared to dissociate, HIV-1 was reverse-transcribed, and viral DNA was integrated into the genome of HOKs. Meanwhile, some of the complexes exited the original host and re-entered neighboring HOKs and permissive cells of HIV-1. Moreover, P. gingivalis vesicles enhanced HIV-1 infection of MT4 cells at low infecting doses that are not able to establish an efficient infection alone. These findings suggest that invasive bacteria and their OMVs with ability to interact with HIV-1 may serve as a vehicle to translocate HIV through the mucosa, establish mucosal transmission of HIV-1, and enhance HIV-1 infectivity.

Depo-Provera alters cervical immune responses and increases HIV infectivity ex vivo: a longitudinal study.

Abstract Depo-Provera (Depot medroxyprogesterone acetate, MPA) is associated with an increased risk of HIV acquisition; however, its impact on the cervicovaginal immune milieu and HIV infectivity ex vivo is not well characterized. In a longitudinal study, we investigated the impact of Depo-Provera use by healthy women on levels of cervical immune mediators, expression of immune markers for HIV preference (CCR5, integrin a4b7, CD38) on cervical CD4+ T cells, and HIV infectivity ex vivo at baseline (visit 1), one month (visit 2) and three months (visit 3) after Depo-Provera treatment. Forty-seven healthy women who met the eligibility criteria were enrolled (median age 26, IQR 22.5–30). Cervical cytobrush cells and secretions from vaginal, endocervical, and rectal swabs were collected. Flow cytometry analysis indicated a significant increase in the frequency of cervical CCR5+CD4+T cells at visit 3 compared to visit 1. There was also an increasing trend in the frequency of cervical CD4+ cells expressing integrin a4b7 in response to Depo-Provera. Levels of several cytokines including G-CSF, GM-CSF, IL-6, MIP-1b, and VEGF in vaginal and cervical secretions were significantly decreased at visits 2 and 3 compared to visit 1. At visit 3, cervical levels of immune mediators remained suppressed despite a decreased serum concentration of MPA, suggesting a prolonged effect of Depo-Provera on mucosal immune responses. Cervicovaginal secretions from women after receiving Depo-Provera (visits 2 and 3) promoted HIV infectivity ex vivo compared to those at visit 1. Further studies on the modulation of mucosal immune responses and HIV transmission by Depo-Provera will offer insights into developing better strategies for HIV prevention.

Human Semen or Seminal Plasma Does Not Enhance HIV-1BaL Ex Vivo Infection of Human Colonic Explants.

To determine whether human whole semen (WS) and seminal plasma (SP) either previously frozen or freshly acquired altered ex vivo infectibility of human colonic explants or was associated with histology or toxicity changes, which may influence mucosal HIV-1 transmission in vivo. Pooled human semen samples were freshly obtained from study volunteers (never frozen) and from commercial sources (frozen/thawed). Endoscopically acquired rectal biopsies were evaluated for toxicity following titered ex vivo WS/SP exposure by histological grading and by MTT assay. The ex vivo HIV-1 biopsy challenge model was used to evaluate effects of exposure to either previously frozen or freshly acquired WS/SP on HIVBaL infectibility at a range of viral inocula (104-100 TCID50). To evaluate the effects at lower viral inocula of HIV-1 (10-2-102), experiments in the presence or absence of WS/SP were also performed utilizing TZM-bl cells. MTT assays and histological scoring demonstrated no tissue degradation of biopsies when exposed for 2 h to concentrations of 10% or 100% of either fresh or previously frozen WS/SP. Ex vivo biopsy HIV-1 challenge experiments showed no differences in the presence of freshly acquired or previously frozen/thawed WS/SP compared with control; no differences were seen with lower infectious titers on TZM-bl cells. Within the limits of assay sensitivity and variability, these data show no toxicity or significant enhancement of HIV-1 infectibility of human rectal mucosa using the colorectal explant model with either pooled fresh or frozen/thawed nonautologous human semen.

HIV internalization into oral and genital epithelial cells by endocytosis and macropinocytosis leads to viral sequestration in the vesicles

Recently, we showed that HIV-1 is sequestered, i.e., trapped, in the intracellular vesicles of oral and genital epithelial cells. Here, we investigated the mechanisms of HIV-1 sequestration in vesicles of polarized tonsil, foreskin and cervical epithelial cells. HIV-1 internalization into epithelial cells is initiated by multiple entry pathways, including clathrin-, caveolin/lipid raft-associated endocytosis and macropinocytosis. Inhibition of HIV-1 attachment to galactosylceramide and heparan sulfate proteoglycans, and virus endocytosis and macropinocytosis reduced HIV-1 sequestration by 30–40%. T-cell immunoglobulin and mucin domain 1 (TIM-1) were expressed on the apical surface of polarized tonsil, cervical and foreskin epithelial cells. However, TIM-1-associated HIV-1 macropinocytosis and sequestration were detected mostly in tonsil epithelial cells. Sequestered HIV-1 was resistant to trypsin, pronase, and soluble CD4, indicating that the sequestered virus was intracellular. Inhibition of HIV-1 intraepithelial sequestration and elimination of vesicles containing virus in the mucosal epithelium may help in the prevention of HIV-1 mucosal transmission.

Mucosal IgA Responses: Damaged in Established HIV Infection-Yet, Effective Weapon against HIV Transmission.

HIV infection not only destroys CD4+ T cells but also inflicts serious damage to the B-cell compartment, such as lymphadenopathy, destruction of normal B-cell follicle architecture, polyclonal hypergammaglobulinemia, increased apoptosis of B cells, and irreversible loss of memory B-cell responses with advanced HIV disease. Subepithelial B cells and plasma cells are also affected, which results in loss of mucosal IgG and IgA antibodies. This leaves the mucosal barrier vulnerable to bacterial translocation. The ensuing immune activation in mucosal tissues adds fuel to the fire of local HIV replication. We postulate that compromised mucosal antibody defenses also facilitate superinfection of HIV-positive individuals with new HIV strains. This in turn sets the stage for the generation of circulating recombinant forms of HIV. What can the mucosal B-cell compartment contribute to protect a healthy, uninfected host against mucosal HIV transmission? Here, we discuss proof-of-principle studies we have performed using passive mucosal immunization, i.e., topical administration of preformed anti-HIV monoclonal antibodies (mAbs) as IgG1, dimeric IgA1 (dIgA1), and dIgA2 isotypes, alone or in combination. Our data indicate that mucosally applied anti-HIV envelope mAbs can provide potent protection against mucosal transmission of simian-human immunodeficiency virus. Our review also discusses the induction of mucosal antibody defenses by active vaccination and potential strategies to interrupt the vicious cycle of bacterial translocation, immune activation, and stimulation of HIV replication in individuals with damaged mucosal barriers.

Highly Human Immunodeficiency Virus-Exposed Seronegative Men Have Lower Mucosal Innate Immune Reactivity.

Risk of HIV acquisition varies, and some individuals are highly HIV-1-exposed, yet, persistently seronegative (HESN). The immunologic mechanisms contributing to this phenomenon are an area of intense interest. As immune activation and inflammation facilitate disease progression in HIV-1-infected persons and gastrointestinal-associated lymphoid tissue is a highly susceptible site for transmission, we hypothesized that reduced gut mucosal immune reactivity may contribute to reduced HIV-1 susceptibility in HESN men with a history of numerous rectal sexual exposures. To test this, we used ex vivo mucosal explants from freshly acquired colorectal biopsies from healthy control and HESN subjects who were stimulated with specific innate immune ligands and inactivated whole pathogens. Immune reactivity was then assessed via cytokine arrays and proteomic analysis. Mucosal immune cell compositions were quantified via immunohistochemistry. We found that explants from HESN subjects produced less proinflammatory cytokines compared with controls following innate immune stimulation; while noninflammatory cytokines were similar between groups. Proteomic analysis identified several immune response proteins to be differentially expressed between HIV-1-stimulated HESN and control explants. Immunohistochemical examination of colorectal mucosa showed similar amounts of T cells, macrophages, and dendritic cells between groups. The results of this pilot study suggest that mucosal innate immune reactivity is dampened in HESN versus control groups, despite presence of similar densities of immune cells in the colorectal mucosa. This observed modulation of the rectal mucosal immune response may contribute to lower risk of mucosal HIV-1 transmission in these individuals.

Predicting HIV-1 transmission and antibody neutralization efficacy in vivo from stoichiometric parameters.

The potential of broadly neutralizing antibodies targeting the HIV-1 envelope trimer to prevent HIV-1 transmission has opened new avenues for therapies and vaccines. However, their implementation remains challenging and would profit from a deepened mechanistic understanding of HIV-antibody interactions and the mucosal transmission process. In this study we experimentally determined stoichiometric parameters of the HIV-1 trimer-antibody interaction, confirming that binding of one antibody is sufficient for trimer neutralization. This defines numerical requirements for HIV-1 virion neutralization and thereby enables mathematical modelling of in vitro and in vivo antibody neutralization efficacy. The model we developed accurately predicts antibody efficacy in animal passive immunization studies and provides estimates for protective mucosal antibody concentrations. Furthermore, we derive estimates of the probability for a single virion to start host infection and the risks of male-to-female HIV-1 transmission per sexual intercourse. Our work thereby delivers comprehensive quantitative insights into both the molecular principles governing HIV-antibody interactions and the initial steps of mucosal HIV-1 transmission. These insights, alongside the underlying, adaptable modelling framework presented here, will be valuable for supporting in silico pre-trial planning and post-hoc evaluation of HIV-1 vaccination or antibody treatment trials.