Ann M. Carias

Defining the Interaction of HIV-1 with the Mucosal Barriers of the Female Reproductive Tract

ABSTRACT Worldwide, HIV-1 infects millions of people annually, the majority of whom are women. To establish infection in the female reproductive tract (FRT), HIV-1 in male ejaculate must overcome numerous innate and adaptive immune factors, traverse the genital epithelium, and establish infection in underlying CD4+ target cells. How the virus achieves this remains poorly defined. By utilizing a new technique, we define how HIV-1 interacts with different tissues of the FRT using human cervical explants and in vivo exposure in the rhesus macaque vaginal transmission model. Despite previous claims of the squamous epithelium being an efficient barrier to virus entry, we reveal that HIV-1 can penetrate both intact columnar and squamous epithelial barriers to depths where the virus can encounter potential target cells. In the squamous epithelium, we identify virus entry occurring through diffusive percolation, penetrating areas where cell junctions are absent. In the columnar epithelium, we illustrate that virus does not transverse barriers as well as previously thought due to mucus impediment. We also show a statistically significant correlation between the viral load of inocula and the ability of HIV-1 to pervade the squamous barrier. Overall, our results suggest a diffusive percolation mechanism for the initial events of HIV-1 entry. With these data, we also mathematically extrapolate the number of HIV-1 particles that penetrate the mucosa per coital act, providing a biological description of the mechanism for HIV-1 transmission during the acute and chronic stages of infection.

Prevention of SHIV transmission by topical IFN-β treatment.

Understanding vaginal and rectal HIV transmission and protective cellular and molecular mechanisms is critical for designing new prevention strategies, including those required for an effective vaccine. The determinants of protection against HIV infection are, however, poorly understood. Increasing evidence suggest that innate immune defenses may help protect mucosal surfaces from HIV transmission in highly exposed, uninfected subjects. More recent studies suggest that systemically administered type 1 interferon protects against simian immunodeficiency virus infection of macaques. Here we hypothesized that topically applied type 1 interferons might stimulate vaginal innate responses that could protect against HIV transmission. We therefore applied a recombinant human type 1 interferon (IFN-β) to the vagina of rhesus macaques and vaginally challenged them with pathogenic simian/human immunodeficiency virus (SHIV). Vaginal administration of IFN-β resulted in marked local changes in immune cell phenotype, increasing immune activation and HIV co-receptor expression, yet provided significant protection from SHIV acquisition as interferon response genes were also upregulated. These data suggest that protection from vaginal HIV acquisition may be achieved by activating innate mucosal defenses.

Characterization of the Influence of Semen-Derived Enhancer of Virus Infection on the Interaction of HIV-1 with Female Reproductive Tract Tissues

ABSTRACT The majority of human immunodeficiency virus type 1 (HIV-1) transmission events occur in women when semen harboring infectious virus is deposited onto the mucosal barriers of the vaginal, ectocervical, and endocervical epithelia. Seminal factors such as semen-derived enhancer of virus infection (SEVI) fibrils were previously shown to greatly enhance the infectivity of HIV-1 in cell culture systems. However, when SEVI is intravaginally applied to living animals, there is no effect on vaginal transmission. To define how SEVI might function in the context of sexual transmission, we applied HIV-1 and SEVI to intact human and rhesus macaque reproductive tract tissues to determine how it influences virus interactions with these barriers. We show that SEVI binds HIV-1 and sequesters most virions to the luminal surface of the stratified squamous epithelium, significantly reducing the number of virions that penetrated the tissue. In the simple columnar epithelium, SEVI was no longer fibrillar in structure and was detached from virions but allowed significantly deeper epithelial virus penetration. These observations reveal that the action of SEVI in intact tissues is very different in the anatomical context of sexual transmission and begin to explain the lack of stimulation of infection observed in the highly relevant mucosal transmission model. IMPORTANCE The most common mode of HIV-1 transmission in women occurs via genital exposure to the semen of HIV-infected men. A productive infection requires the virus to penetrate female reproductive tract epithelial barriers to infect underlying target cells. Certain factors identified within semen, termed semen-derived enhancers of virus infection (SEVI), have been shown to significantly enhance HIV-1 infectivity in cell culture. However, when applied to the genital tracts of living female macaques, SEVI did not enhance virus transmission. Here we show that SEVI functions very differently in the context of intact mucosal tissues. SEVI decreases HIV-1 penetration of squamous epithelial barriers in humans and macaques. At the mucus-coated columnar epithelial barrier, the HIV-1/SEVI interaction is disrupted. These observations suggest that SEVI may not play a significant stimulatory role in the efficiency of male-to-female sexual transmission of HIV.

Expression profile of human Fc receptors in mucosal tissue: Implications for antibody-dependent cellular effector functions targeting HIV-1 transmission

The majority of new Human Immunodeficiency Virus (HIV)-1 infections are acquired via sexual transmission at mucosal surfaces. Partial efficacy (31.2%) of the Thai RV144 HIV-1 vaccine trial has been correlated with Antibody-dependent Cellular Cytotoxicity (ADCC) mediated by non-neutralizing antibodies targeting the V1V2 region of the HIV-1 envelope. This has led to speculation that ADCC and other antibody-dependent cellular effector functions might provide an important defense against mucosal acquisition of HIV-1 infection. However, the ability of antibody-dependent cellular effector mechanisms to impact on early mucosal transmission events will depend on a variety of parameters including effector cell type, frequency, the class of Fc-Receptor (FcR) expressed, the number of FcR per cell and the glycoslyation pattern of the induced antibodies. In this study, we characterize and compare the frequency and phenotype of IgG (CD16 [FcγRIII], CD32 [FcγRII] and CD64 [FcγRI]) and IgA (CD89 [FcαR]) receptor expression on effector cells within male and female genital mucosal tissue, colorectal tissue and red blood cell-lysed whole blood. The frequency of FcR expression on CD14+ monocytic cells, myeloid dendritic cells and natural killer cells were similar across the three mucosal tissue compartments, but significantly lower when compared to the FcR expression profile of effector cells isolated from whole blood, with many cells negative for all FcRs. Of the three tissues tested, penile tissue had the highest percentage of FcR positive effector cells. Immunofluorescent staining was used to determine the location of CD14+, CD11c+ and CD56+ cells within the three mucosal tissues. We show that the majority of effector cells across the different mucosal locations reside within the subepithelial lamina propria. The potential implication of the observed FcR expression patterns on the effectiveness of FcR-dependent cellular effector functions to impact on the initial events in mucosal transmission and dissemination warrants further mechanistic studies.

Progesterone-Based Intrauterine Device Use Is Associated with a Thinner Apical Layer of the Human Ectocervical Epithelium and a Lower ZO-1 mRNA Expression

ABSTRACT Currently, whether hormonal contraceptives affect male to female human immunodeficiency virus (HIV) transmission is being debated. In this study, we investigated whether the use of progesterone-based intrauterine devices (pIUDs) is associated with a thinning effect on the ectocervical squamous epithelium, down-regulation of epithelial junction proteins, and/or alteration of HIV target cell distribution in the human ectocervix. Ectocervical tissue biopsies from healthy premenopausal volunteers using pIUDs were collected and compared to biopsies obtained from two control groups, namely women using combined oral contraceptives (COCs) or who do not use hormonal contraceptives. In situ staining and image analysis were used to measure epithelial thickness and the presence of HIV receptors in tissue biopsies. Messenger RNA levels of epithelial junction markers were measured by quantitative PCR. The epithelial thickness displayed by women in the pIUD group was similar to those in the COC group, but significantly thinner as compared to women in the no hormonal contraceptive group. The thinner epithelial layer of the pIUD group was specific to the apical layer of the ectocervix. Furthermore, the pIUD group expressed significantly lower levels of the tight junction marker ZO-1 within the epithelium as compared to the COC group. Similar expression levels of HIV receptors and coreceptors CD4, CCR5, DC-SIGN, and Langerin were observed in the three study groups. Thus, women using pIUD displayed a thinner apical layer of the ectocervical epithelium and reduced ZO-1 expression as compared to control groups. These data suggest that pIUD use may weaken the ectocervical epithelial barrier against invading pathogens, including HIV.

Phagocytosis: Cell biology view of antiviral function

Purpose of reviewRecently, studies have suggested a role for Fc-mediated effector functions in viremic control of HIV infection and blocking HIV acquisition. Although progress has been made in identifying the mechanisms responsible for regulating various innate functions, minimal research has been performed concerning macrophage-specific phagocytosis and antiviral effects. Recent findingsOf what research has been performed, phagocytosis has been identified as a possible key player in antiviral functions during initial infection, offering protection at the HIV mucosal entry sites. Recent research has also highlighted the importance of various antibody characteristics, such as polymorphism, immunoglobulin subclass, and glycan structure on those effector functions modulated. Lastly, despite recent failures in HIV vaccine trials, the RV144 Thai trial illustrated 31.2% efficacy against heterosexual infection. When these protective results were looked at in depth, vaccine-induced antibodies were increased when infection rates decreased, suggesting that HIV might be neutralized through receptor-mediated effector mechanisms, including phagocytosis. Importantly, these data instilled the awareness that the identification of protective immune correlates is imperative to successfully develop vaccine strategies. SummaryIn this review, we address the antiviral mechanisms of phagocytosis, focusing on complement-mediated phagocytosis and Fc-receptor-mediated antibody-dependent cellular phagocytosis in relation to HIV transmission and infection.

Increases in Endogenous or Exogenous Progestins Promote Virus-Target Cell Interactions within the Non-human Primate Female Reproductive Tract.

Currently, there are mounting data suggesting that HIV-1 acquisition in women can be affected by the use of certain hormonal contraceptives. However, in non-human primate models, endogenous or exogenous progestin-dominant states are shown to increase acquisition. To gain mechanistic insights into this increased acquisition, we studied how mucosal barrier function and CD4+ T-cell and CD68+ macrophage density and localization changed in the presence of natural progestins or after injection with high-dose DMPA. The presence of natural or injected progestins increased virus penetration of the columnar epithelium and the infiltration of susceptible cells into a thinned squamous epithelium of the vaginal vault, increasing the likelihood of potential virus interactions with target cells. These data suggest that increasing either endogenous or exogenous progestin can alter female reproductive tract barrier properties and provide plausible mechanisms for increased HIV-1 acquisition risk in the presence of increased progestin levels.

Long-term direct visualization of passively transferred fluorophore-conjugated antibodies

The use of therapeutic antibodies, delivered by intravenous (IV) instillation, is a rapidly expanding area of biomedical treatment for a variety of conditions. However, little is known about how the antibodies are anatomically distributed following infusion and the underlying mechanism mediating therapeutic antibody distribution to specific anatomical sites remains to be elucidated. Current efforts utilize low resolution and sensitivity methods such as ELISA and indirect labeling imaging techniques, which often leads to high background and difficulty in assessing biodistribution. Here, using the in vivo non-human primate model, we demonstrate that it is possible to utilize the fluorophores Cy5 and Cy3 directly conjugated to antibodies for direct visualization and quantification of passively transferred antibodies in plasma, tissue, and in mucosal secretions. Antibodies were formulated with 1-2 fluorophores per antibody to minimally influence antibody function. Fluorophore conjugated Gamunex-C (pooled human IgG) were tested for binding to protein A, via surface plasmon resonance, and showed similar levels of binding when compared to unlabeled Gamunex-C. In order to assess the effect fluorophore labeling has on turnover and localization, rhesus macaques were IV infused with either labeled or unlabeled Gamunex-C. Plasma, vaginal Weck-Cel fluid, cervicovaginal mucus, and vaginal/rectal tissue biopsies were collected up to 8weeks. Similar turnover and biodistribution was observed between labeled and unlabeled antibodies, showing that the labeling process did not have an obvious deleterious effect on localization or turnover. Cy5 and Cy3 labeled antibodies were readily detected in the same pattern regardless of fluorophore. Tissue distribution was measured in macaque vaginal and rectal biopsies. The labeled antibody in macaque biopsies was found to have similar biodistribution pattern to endogenous antibodies in macaque and human tissues. In the vaginal and rectal mucosa, endogenous and infused antibodies were found primarily within the lamina propria. In the mucosal squamous epithelium of the vaginal vault, significant antibody was also observed in a striated pattern in the superficial, nonviable, stratum corneum. Endogenous antibody distribution in both human and macaque squamous tissues exhibited a similar pattern as seen with the labeled and unlabeled antibodies. This proof-of-principle study reveals that the labeled antibody is stable and physiologically similar relative to endogenous antibody setting the stage for future work to better understand the mechanisms of how antibodies reach unique anatomical sites. Direct visualization of fluorophore-conjugated antibodies following passive infusion can be utilized to assess the kinetics of biodistribution of infused antibodies and may be a useful approach to monitor and predict efficacy of therapeutic antibodies.

HIV interactions and the perils of epithelial thinning in the female reproductive tract

Background Currently, there is much debate on whether epithelial thinning from hormonal contraceptives can increase HIV acquisition. Previously, we illustrated that HIV can penetrate to depths in squamous epithelium where it can interact with target cells, such as CD4+ T-cells and macrophages. Using a similar approach, we show that epithelial thinning affects virus penetration, along with target cell and cellular junction distribution.

Barriers of Mucosal Entry of HIV/SIV

Most new HIV infections, over 80%, occur through sexual transmission. During sexual transmission, the virus must bypass specific female and male reproductive tract anatomical barriers to encounter viable target cells. Understanding the generally efficient ability of these barrier to exclude HIV and the precise mechanisms of HIV translocation beyond these genital barriers is essential for vaccine and novel therapeutic development. In this review, we explore the mucosal, barriers of cervico-vaginal and penile tissues that comprise the female and male reproductive tracts. The unique cellular assemblies f the squamous and columnar epithelium are illustrate highlighting their structure and function. Each anatomical tissue offers a unique barrier to virus entry in healthy individuals. Unfortunately barrier dysfunction can lead to HIV transmission. How these diverse mucosal barriers have the potential to fail is considered, highlighting those anatomical areas that are postulated to offer a weaker barrier and are; therefore, more susceptible to viral ingress. Risk factors, such as sexually transmitted infections, microbiome dysbiosis, and high progestin environments are also associated with increased acquisition of HIV. How these states may affect the integrity of mucosal barriers leading to HIV acquisition are discussed suggesting mechanisms of transmission and revealing potential targets for intervention.