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 efforts to identify therapeutic approaches that will eliminate or reduce the size of this latent HIV-1 reservoir. In this regard, a sensitive assay that 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 into 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.

Epithelial cells in human cervical and colonic mucosa do not express HIV receptor. However, HIV transmission occurs across the unbreached epithelia by an unknown mechanism. In this study, the effect of HIV exposure on tight junction (TJ) and cytokine production in ectocervical and colon mucosal epithelia in tissue biopsies was investigated in an organ culture model. After HIV exposure, the distribution patterns and quantities of epithelial TJ and adherens proteins were evaluated by immunofluorescence staining followed by confocal microscopy. Cytokine mRNA in the mucosal epithelia was also evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR). HIV transmission was evaluated by measuring p24 production in culture supernatant. Our results showed there were no significant changes in the distribution and quantities of epithelial TJ/adherens junction (AJ) proteins after exposure to HIV. However, higher levels of CXCL10 and CXCL11 mRNA expression were detected in HIV-exposed ectocervical epithelia. In case of colon mucosa, higher levels of CXCL10 and IL-6 mRNA expression were detected in HIV-exposed colon mucosa. Our study suggests that HIV induces cytokine production in epithelial cells, which may facilitate HIV transmission by recruiting HIV target cells in the submucosal region. Furthermore, HIV transmission may not occur through epithelial TJ/AJ disruption.

ABSTRACT Curing HIV-1 infection will require elimination of persistent cellular reservoirs that harbor latent virus in the face of combination antiretroviral therapy (cART). Proposed immunotherapeutic strategies to cure HIV-1 infection include enhancing lysis of these infected cells by cytotoxic T lymphocytes (CTL). A major challenge in this strategy is overcoming viral immune escape variants that have evaded host immune control. Here we report that naive CD8+ T cells from chronic HIV-1-infected participants on long-term cART can be primed by dendritic cells (DC). These DC must be mature, produce high levels of interleukin 12p70 (IL-12p70), be responsive to CD40 ligand (CD40L), and be loaded with inactivated, autologous HIV-1. These DC-primed CD8+ T cell responders produced high levels of gamma interferon (IFN-γ) in response to a broad range of both conserved and variable regions of Gag and effectively killed CD4+ T cell targets that were either infected with the autologous latent reservoir-associated virus or loaded with autologous Gag peptides. In contrast, HIV-1-specific memory CD8+ T cells stimulated with autologous HIV-1-loaded DC produced IFN-γ in response to a narrow range of conserved and variable Gag peptides compared to the primed T cells and most notably, displayed significantly lower cytolytic function. Our findings highlight the need to selectively induce new HIV-1-specific CTL from naive precursors while avoiding activation of existing, dysfunctional memory T cells in potential curative immunotherapeutic strategies for HIV-1 infection. IMPORTANCE Current immunotherapeutic approaches aim to enhance antiviral immunity against the HIV-1 reservoir; however, it has yet to be shown whether T cells from persons on cART can recognize and eliminate virus-infected cells. We show that in persons on cART a personalized medicine approach using their dendritic cells to stimulate their naive T cells induces potent effector CTL in vitro that recognize and eradicate HIV-1-infected CD4+ T cells. Additionally, we show that the same stimulation of existing memory T cells results in cytokine secretion but limited effector function. Our study demonstrates that the naive T cell repertoire can recognize persistent HIV-1 during cART and supports immunotherapy strategies for an HIV-1 cure that targets naive T cells, rather than existing, dysfunctional, memory T cells.

AIDS Research and Human RetrovirusesVol. 30, No. S1 Mucosal Immune Activation and InflammationAn Approach to Unravel Cellular Mechanisms Responsible for Enhanced Neisseria Gonorrhea Induced HIV Acquisition and its Effect on MicrobicidesAnwesha Sanyal, Phalguni Gupta, Ming Ding, and Deena RatnerAnwesha SanyalSearch for more papers by this author, Phalguni GuptaSearch for more papers by this author, Ming DingSearch for more papers by this author, and Deena RatnerSearch for more papers by this authorPublished Online:30 Oct 2014https://doi.org/10.1089/aid.2014.5525.abstractAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"An Approach to Unravel Cellular Mechanisms Responsible for Enhanced Neisseria Gonorrhea Induced HIV Acquisition and its Effect on Microbicides." AIDS Research and Human Retroviruses, 30(S1), p. A238FiguresReferencesRelatedDetails Volume 30Issue S1Oct 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Anwesha Sanyal, Phalguni Gupta, Ming Ding, and Deena Ratner.An Approach to Unravel Cellular Mechanisms Responsible for Enhanced Neisseria Gonorrhea Induced HIV Acquisition and its Effect on Microbicides.AIDS Research and Human Retroviruses.Oct 2014.A238-A238.http://doi.org/10.1089/aid.2014.5525.abstractPublished in Volume: 30 Issue S1: October 30, 2014PDF download

In the absence of universally available antiretroviral (ARV) drugs or a vaccine against HIV-1, microbicides may offer the most immediate hope for controlling the AIDS pandemic. The most advanced and clinically effective microbicides are based on ARV agents that interfere with the earliest stages of HIV-1 replication. Our objective was to identify and characterize novel ARV-like inhibitors, as well as demonstrate their efficacy at blocking HIV-1 transmission. Abasic phosphorothioate 2' deoxyribose backbone (PDB) oligomers were evaluated in a variety of mechanistic assays and for their ability to inhibit HIV-1 infection and virus transmission through primary human cervical mucosa. Cellular and biochemical assays were used to elucidate the antiviral mechanisms of action of PDB oligomers against both lab-adapted and primary CCR5- and CXCR4-utilizing HIV-1 strains, including a multidrug-resistant isolate. A polarized cervical organ culture was used to test the ability of PDB compounds to block HIV-1 transmission to primary immune cell populations across ectocervical tissue. The antiviral activity and mechanisms of action of PDB-based compounds were dependent on oligomer size, with smaller molecules preventing reverse transcription and larger oligomers blocking viral entry. Importantly, irrespective of molecular size, PDBs potently inhibited virus infection and transmission within genital tissue samples. Furthermore, the PDB inhibitors exhibited excellent toxicity and stability profiles and were found to be safe for vaginal application in vivo. These results, coupled with the previously reported intrinsic anti-inflammatory properties of PDBs, support further investigations in the development of PDB-based topical microbicides for preventing the global spread of HIV-1.

Abstract Topical microbicides (vaginal gels) are one mechanism to prevent sexual transmission of human immunodeficiency virus type 1 (HIV-1). We show here that a new class of microbicides, abasic phosphorothioate 2’ deoxyribose backbone (PDB) oligomers, has significant anti-HIV-1 activities in human PBMC cell cultures when either the virus or cells are pre-incubated with the compound. HIV infection is also inhibited in an explant model of human cervical tissue. A second function of PDB other than its anti-HIV activity is its capacity to inhibit HIV-triggered TLR7/9 activation. PDB suppresses the release of HIV-induced pro-inflammatory cytokines/chemokines in cultures of human PBMC and ectocervical tissues. We also demonstrated that PDB is safe and non-toxic in in vitro cell cultures and in an in vivo murine vaginal toxicity model. The effect of PDB is stable as this compound retains activity at pH 4.4 and after transition to a neutral pH, following incubation with biological fluids (seminal plasma, vaginal fluids) and a normal vaginal flora Lactobacillus strain. PDB does not affect the growth of Lactobacillus strains. Phosphorothioate 2’ deoxyribose oligomers are novel microbicide candidates with dual-acting anti-HIV and anti-inflammatory activities and an excellent safety and stability profile.

Microbicides have been evaluated mostly against cell-free HIV-1. Because semen contains both cell-free and cell-associated HIV-1, HIV-1 transmission could occur via either or both sources. Therefore, it is important to examine the antiviral activity of microbicides against cell-associated HIV-1. The cyclic antimicrobial peptide retrocyclin RC-101 has been shown previously to have antiviral activity against cell-free HIV-1, with no associated cellular toxicity. In this article we have examined the antiviral activity of RC-101 against cell-associated HIV-1. The results demonstrate potent antiviral activity of RC-101 against cell-cell HIV-1 transmission in both CD4-dependent and CD4-independent assays against CCR5- and CXCR4-tropic HIV-1, with no cellular toxicity. Furthermore, this antiviral activity was retained in the presence of human seminal plasma. The potent antiviral activity of RC-101 against cell-associated HIV-1 reported here, and the previously reported antiviral activity in cervical tissues, suggest that RC-101 is an excellent and promising microbicide candidate against HIV-1.

Background: Cervical tissue-based organ cultures have been used successfully to evaluate microbicides for toxicity and antiviral activity. The antimicrobial peptide retrocyclin RC-101 has been shown to have potent anti-HIV activity in cell culture.Objective: To evaluate RC-101 in organ culture for toxicity and its ability to block HIV-1 transmission across cervical mucosa.Methods: A cervical tissue-based organ culture was used to measure antiviral activity of RC-101. Cytotoxicity in tissues was determined by immunostaining of cellular proteins and by measuring inflammatory cytokines using real-time reverse transcriptase-polymerase chain reaction and Luminex technology.Results: RC-101 blocked transmission of both R5 and X4 HIV-1 across cervical mucosa in this organ culture model. Furthermore, film-formulated RC-101 exhibited potent antiviral activity in organ culture. Such antiviral activity of RC-101 was retained in the presence of semen and vaginal fluid. RC-101 showed no cytotoxicity in cervical tissue. Furthermore, RC-101 did not induce proinflammatory cytokine response in tissues. RC-101 also did not have any effect on natural killer cell activity and proliferation of CD4 and CD8 cells and did not show chemotactic activity.Conclusions: Therefore, because of strong antiviral activity and low cytotoxicity in cervical tissues, RC-101 should be considered as an excellent microbicide candidate against HIV-1.

Background: Although there are different strains of HIV-1 in a chronically infected individual, only one or limited virus strains are successfully transmitted to a new individual. The reason for this "transmission bottleneck'' is as yet unknown.Methodology/Principal Findings: A human cervical explant model was used to measure HIV-1 transmission efficiency of viral strains from chronic infections, and transmitter/founder variants. We also evaluated the genetic characteristics of HIV-1 variants in the inoculums compared to those transmitted across the cervical mucosa. Eight different HIV-1 isolates were used in this study, six chronic isolates and two transmitter/founder viruses. The transmission efficiency of the chronic and transmitter/founder virus isolates and the viral diversity of chronic isolates before and after viral transmission were assessed. The results indicate that transmitter/founder viruses did not display higher transmission efficiency than chronic HIV-1 isolates. Furthermore, no evidence for a difference in diversity was found between the inoculums and transmitted virus strains. Phylogenetic analysis indicated that the sequences of variants in the inoculums and those present in transmitted virus intermingled irrespective of co-receptor usage. In addition, the inoculum and transmitted variants had a similar pairwise distance distribution.Conclusion: There was no selection of a single or limited number of viral variants during HIV-1 transmission across the cervical mucosa in the organ culture model, indicating that the cervical mucosa alone may not produce the transmission bottleneck of HIV-1 infection observed in vivo.

SIV model indicates that upon traversing the cervicovaginal mucosa, SIV/SIV‐infected cells migrate to regional lymph nodes where active replication occurs prior to systemic virus dissemination. The purpose of the study is to develop a model to study early HIV‐1 transmission events that occur after crossing the cervical mucosa into regional lymph nodes.

An urgent need exists for HIV-1 microbicides. Here, we describe the in vivo testing of lactic acid bacteria bioengineered to secrete cyanovirin-N. We fed pigtail macaques a yogurt formulation that used bioengineered strains as a starter culture. Cyanovirin-N expression could be detected in the rectal vault during and immediately after feeding. Ex vivo viral challenge of rectal tissue biopsies revealed that peak viral burden was significantly lower in tissue obtained from experimental animals compared with control animals. Formulation of candidate compounds in lactic acid bacteria and their oral administration seems to be a feasible strategy for mucosal delivery of microbicides.

Background RC-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 Findings RC-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/Significance Formulated 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.

Unsanitary blood/plasma collecting activities in central China during the 1990's caused a high prevalence of blood-borne HIV-1 infection. Although the genetic characterization of the proviral DNA of HIV-1 circulating in the infected former blood donors (FBDs) has been reported, there is little information about the biological characteristics of virus isolates in these FBDs. In this study, we have examined the biologic properties of HIV-1 isolates from AIDS patients and long-term non-progressors (LTNP) of FBDs. Our results indicate that the growth properties, co-receptor usage and syncitium inducing capabilities of the HIV-1 isolates are associated with the disease status of patients. The virus isolates from LTNPs replicated slower, used the CCR5 co-receptor and were of non-syncytium inducing phenotype. In contrast, HIV-1 isolates from AIDS patients showed high replication kinetics, used both CCR5 and CXCR4 co-receptors and induced syncytium formation. A higher level of cytopathicity was also detected in syncytium inducing virus compared to non-syncytium inducing isolates irrespective of patients' disease statuses. Although there was no significant differences in the binding and penetration of the target cells between the isolates from LTNPs and those from AIDS patients, viral DNA synthesis of viral isolates from LTNPs was much slower than the DNA synthesized by the isolates from AIDS patients, indicating a restriction at a post-entry step. Analysis of deduced amino acid sequences in C2-V5 regions of these isolates has provided a molecular basis for further identification of viral phenotypes of HIV-1 subtype B'. This study has provided valuable information on the biological properties of circulating HIV-1 strains among Chinese FBDs to better understand their viral characteristics and design more appropriate vaccine candidates for FBDs.

Background: Plasma viral load has been shown to be a meaningful prognostic marker for disease progression in untreated, HIV-1 subtype B-infected subjects in United States and Western Europe and therefore used as a prognostic marker for disease progression. Because of high expenses of commercially available viral load assays, the role of viral load in disease progression has not been evaluated in HIV-1 subtype C-infected patients in India. Methods: We developed an inexpensive real-time reverse transcriptase-polymerase chain reaction assay to quantify viral load in plasma of HIV-1 subtype C-infected subjects from India and used it in a longitudinal analysis of viral load and CD4 cell number in HIV-infected subjects from Calcutta, India. Results: The real-time reverse transcriptase-polymerase chain reaction assay can quantify plasma viral load with a linear range of detection from 102 to 109 HIV-1 RNA copies per input. Longitudinal analysis of viral load in a cohort of 39 subjects over an average period of approximately 3 years indicates that 1-log increase in HIV-1 RNA level was associated with a decline of 67 CD4 cell count. Furthermore, HIV-1 RNA level between 500 and 50,000 copies per milliliter would predict a 12.9% decrease in CD4 cell count per year, whereas HIV-1 RNA levels above 50,000 copies HIV-1 RNA per milliliter would predict a 25.3% decrease in CD4 cells per year. In addition, we estimated that the mean incubation period of disease development, as defined by the loss of CD4 below 200, is 8.2 years. Conclusion: Our report on the level of viral load on predicting CD4 decline in Indian subjects with HIV-1 provides an additional important tool to the physicians for treating and planning a therapeutic strategy to control HIV-1 infection in India.

HIV-1 subtype C has been the predominant subtype throughout the course of the HIV-1 epidemic in India regardless of the geographic region of the country. In an effort to understand the mechanism of subtype C predominance in this country, we have investigated the in vitro replication fitness and transmission efficiency of HIV-1 subtypes A and C from India. Using a dual infection growth competition assay, we found that primary HIV-1 subtype C isolates had higher overall relative fitness in PBMC than subtype A primary isolates. Moreover, in an ex vivo cervical tissue derived organ culture, subtype C isolates displayed higher transmission efficiency across cervical mucosa than subtype A isolates. We found that higher fitness of subtype C was not due to a trans effect exerted by subtype C infected PBMC. A half genome A/C recombinant clone in which the 3′ half of the viral genome of subtype A was replaced with the corresponding subtype C3′ half, had similar replicative fitness as the parental subtype A. These results suggest that the higher replication fitness and transmission efficiency of subtype C virus compared to subtype A virus from India is most probably not due to the envelope gene alone and may be due to genes present within the 5′ half of the viral genome or to a more complex interaction between the genes located within the two halves of the viral genome. These data provide a model to explain the asymmetric distribution of subtype C over other subtypes in India.

ABSTRACT 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.