The virologic and cellular factors that are involved in transmission of human immunodeficiency virus type 1 (HIV-1) across the female genital tissue are poorly understood. We have recently developed a human cervical tissue-derived organ culture model to study heterosexual transmission of HIV-1 that mimics the in vivo situation. Using this model we investigated the role of phenotypic characteristics of HIV-1 and identified the cell types that are first infected during transmission. Our data indicate that the cell-free R5 HIV-1 was more efficiently transmitted than cell-free X4 HIV-1. Cell-free and cell-associated HIV-1 had comparable transmission efficiency regardless of whether the virus was of R5 or X4 type. We have demonstrated that memory CD4 Heterosexual transmission accounts for the majority of worldwide human immunodeficiency virus type 1 (HIV-1) infections in women (17,20,21). Very little is known about the role of biologic and molecular characteristics of HIV-1 during sexual transmission. Since semen has been shown to contain free infectious HIV-1 and HIV-1-infected cells (19,26,(32)(33)(34)(35), transmission could potentially occur via cell-free and cell-associated HIV-1. Studies on the viral envelope sequence in men during primary infection suggest that virus transmitted during sexual contact was mostly homogeneous and had macrophagetropic and non-syncytium-inducing phenotypes (35, 37). However, recent data of Long et al. (16) indicate that heterogeneous HIV-1 was found in women, whereas homogeneous virus was detected in men soon after sexual contact, indicating that the mechanism of sexual transmission of HIV-1 in women could be different than that in men.There are no in vivo data to indicate the cell types that first become infected in the reproductive tract of women. Although in chronically HIV-1-infected women T cells, macrophages, and Langerhans cells in cervical tissue are infected with HIV-1 (25, 26), analysis of lymph nodes in HIV-infected men during the acute phase of infection indicates that actively virus replication occurs in activated and resting CD4 ϩ T cells (36). A number of studies have demonstrated that productively HIV-1-infected cells in vivo are memory T cells (CD4 ϩ CD45RO ϩ ) that respond to antiretroviral therapy (22,24). There is controversy in the simian immunodeficiency virus (SIV)-rhesus monkey model system about the types of cells that become first infected during sexual transmission. Spira et al. (28) found that cells which initially become infected in the female genital tract are Langerhans cells present in the lamina propria, but the identity of these cells could not be determined by specific immunohistochemical staining on contiguous tissues. Moreover, this study could not determine whether these HIV-1 DNA-containing cells were productively infected. On the other hand, using simultaneous in situ hybridization and immunohistochemical staining, Zhang et al. (36) recently found that CD4 ϩ T (both activated and resting) cells and not dendritic cells were predominantly the...

Although antiretroviral therapy can suppress HIV-1 infection to undetectable levels of plasma viremia, integrated latent HIV-1 genomes that encode replication competent virus persist in resting CD4+ T cells. This latent HIV-1 reservoir represents a major barrier to a cure. Currently, there are substantial ongoing efforts to identify therapeutic approaches that will eliminate or reduce the size of this latent HIV-1 reservoir. In this regard, a sensitive assay which can accurately and rapidly quantify inducible replication competent latent HIV-1 from resting CD4+ T cells is essential for HIV-1 eradication studies. Here we describe a reporter cell-based assay to quantify inducible replication competent latent HIV-1. This assay has several advantages over existing technology in that it: (i) is sensitive; (ii) requires only a small blood volume; (iii) is faster, less labor intensive, and less expensive, and (iv) can be readily adapted to a high-throughput format. Using this assay we show that the size of the inducible latent HIV-1 reservoir in aviremic participants on therapy is approximately 70-fold larger than previous estimates.

CD8؉ T cells display a noncytotoxic activity that suppresses transcription of human immunodeficiency virus type 1 (HIV-1) in an antigen-independent and major histocompatibility complex-unrestricted manner. To date, the precise cellular and molecular factors mediating this CD8 ؉ T-cell effector function remain unsolved. Despite evidence indicating the dependence of the activity on cell-cell contact, the possibility of a membranemediated activity that represses transcription from the viral promoter remains unexplored. We therefore investigated whether this inhibition of HIV-1 transcription might be elicited by a membrane-bound determinant. Using a CD8؉ T-cell line displaying potent noncytotoxic HIV-1 suppression activity, we have identified a membrane-localized HIV-1-suppressing activity that is concomitantly secreted as 30-to 100-nm endosomederived tetraspanin-rich vesicles known as exosomes. Purified exosomes from CD8 ؉ T-cell culture supernatant noncytotoxically suppressed CCR5-tropic (R5) and CXCR4-tropic (X4) replication of HIV-1 in vitro through a protein moiety. Similar antiviral activity was also found in exosomes isolated from two HIV-1-infected subjects. The antiviral exosomes specifically inhibited HIV-1 transcription in both acute and chronic models of infection. Our results, for the first time, indicate the existence of an antiviral membrane-bound factor consistent with the hallmarks defining noncytotoxic CD8 ؉ T-cell suppression of HIV-1.

Microbicide candidates with promising in vitro activity are often advanced for evaluations using human primary tissue explants relevant to the in vivo mucosal transmission of human immunodeficiency virus type 1 (HIV-1), such as tonsil, cervical, or rectal tissue. To compare virus growth or the anti-HIV-1 efficacies of candidate microbicides in tissue explants, a novel soft-endpoint method was evaluated to provide a single, objective measurement of virus growth. The applicability of the soft endpoint is shown across several different ex vivo tissue types, with the method performed in different laboratories, and for a candidate microbicide (PRO 2000). The soft-endpoint method was compared to several other endpoint methods, including (i) the growth of virus on specific days after infection, (ii) the area under the virus growth curve, and (iii) the slope of the virus growth curve. Virus growth at the assay soft endpoint was compared between laboratories, methods, and experimental conditions, using nonparametric statistical analyses. Intra-assay variability determinations using the coefficient of variation demonstrated higher variability for virus growth in rectal explants. Significant virus inhibition by PRO 2000 and significant differences in the growth of certain primary HIV-1 isolates were observed by the majority of laboratories. These studies indicate that different laboratories can provide consistent measurements of anti-HIV-1 microbicide efficacy when (i) the soft endpoint or another standardized endpoint is used, (ii) drugs and/or virus reagents are centrally sourced, and (iii) the same explant tissue type and method are used. Application of the soft-endpoint method reduces the inherent variability in comparisons of preclinical assays used for microbicide development.

BackgroundRC-101 is a congener of the antiretroviral peptide retrocyclin, which we and others have reported is active against clinical HIV-1 isolates from all major clades, does not hemagglutinate, and is non-toxic and non-inflammatory in cervicovaginal cell culture. Herein, film-formulated RC-101 was assessed for its antiviral activity in vitro, safety in vivo, retention in the cervix and vagina, and ability to remain active against HIV-1 and SHIV after intravaginal application in macaques.Methodology/Principal FindingsRC-101 was formulated as a quick-dissolving film (2000 µg/film), retained complete activity in vitro as compared to unformulated peptide, and was applied intravaginally in six pigtailed macaques daily for four days. At one and four days following the final application, the presence of RC-101 was assessed in peripheral blood, cervicovaginal lavage, cytobrushed cervicovaginal cells, and biopsied cervical and vaginal tissues by quantitative western blots. One day following the last film application, cervical biopsies from RC-101-exposed and placebo-controlled macaques were collected and were subjected to challenge with RT-SHIV in an ex vivo organ culture model. RC-101 peptide was detected primarily in the cytobrush and biopsied cervical and vaginal tissues, with little to no peptide detected in lavage samples, suggesting that the peptide was associated with the cervicovaginal epithelia. RC-101 remained in the tissues and cytobrush samples up to four days post-application, yet was not detected in any sera or plasma samples. RC-101, extracted from cytobrushes obtained one day post-application, remained active against HIV-1 BaL. Importantly, cervical biopsies from RC-101-treated animals reduced RT-SHIV replication in ex vivo organ culture as compared to placebo-treated animals.Conclusions/SignificanceFormulated RC-101 was stable in vivo and was retained in the mucosa. The presence of antivirally active RC-101 after five days in vivo suggests that RC-101 would be an important molecule to develop further as a topical microbicide to prevent HIV-1 transmission.