HIV Cell-to-Cell Spread Results in Earlier Onset of Viral Gene Expression by Multiple Infections per Cell.

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Mikaël Boullé

2016-11-01

Full Text Available Cell-to-cell spread of HIV, a directed mode of viral transmission, has been observed to be more rapid than cell-free infection. However, a mechanism for earlier onset of viral gene expression in cell-to-cell spread was previously uncharacterized. Here we used time-lapse microscopy combined with automated image analysis to quantify the timing of the onset of HIV gene expression in a fluorescent reporter cell line, as well as single cell staining for infection over time in primary cells. We compared cell-to-cell spread of HIV to cell-free infection, and limited both types of transmission to a two-hour window to minimize differences due to virus transit time to the cell. The mean time to detectable onset of viral gene expression in cell-to-cell spread was accelerated by 19% in the reporter cell line and by 35% in peripheral blood mononuclear cells relative to cell-free HIV infection. Neither factors secreted by infected cells, nor contact with infected cells in the absence of transmission, detectably changed onset. We recapitulated the earlier onset by infecting with multiple cell-free viruses per cell. Surprisingly, the acceleration in onset of viral gene expression was not explained by cooperativity between infecting virions. Instead, more rapid onset was consistent with a model where the fastest expressing virus out of the infecting virus pool sets the time for infection independently of the other co-infecting viruses.

Antigen-presenting cells represent targets for R5 HIV-1 infection in the first trimester pregnancy uterine mucosa.

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Romain Marlin

Full Text Available BACKGROUND: During the first trimester of pregnancy, HIV-1 mother-to-child transmission is relatively rare despite the permissivity of placental cells to cell-to-cell HIV-1 infection. The placenta interacts directly with maternal uterine cells (decidual cells but the physiological role of the decidua in the control of HIV-1 transmission and whether decidua could be a source of infected cells is unknown. METHODOLOGY/PRINCIPAL FINDINGS: To answer to this question, decidual mononuclear cells were exposed to HIV-1 in vitro. Decidual cells were shown to be more susceptible to infection by an R5 HIV-1, as compared to an X4 HIV-1. Infected cells were identified by flow cytometry analysis. The results showed that CD14(+ cells were the main targets of HIV-1 infection in the decidua. These infected CD14(+ cells expressed DC-SIGN, CD11b, CD11c, the Fc gamma receptor CD16, CD32 and CD64, classical MHC class-I and class-II and maturation and activation molecules CD83, CD80 and CD86. The permissivity of decidual tissue was also evaluated by histoculture. Decidual tissue was not infected by X4 HIV-1 but was permissive to R5 HIV-1. Different profiles of infection were observed depending on tissue localization. CONCLUSIONS/SIGNIFICANCE: The presence of HIV-1 target cells in the decidua in vitro and the low rate of in utero mother-to-child transmission during the first trimester of pregnancy suggest that a natural control occurs in vivo limiting cell-to-cell infection of the placenta and consequently infection of the fetus.

Cell-cell transmission of VSV-G pseudotyped lentivector particles.

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Amy M Skinner

Full Text Available Many replicating viruses, including HIV-1 and HTLV-1, are efficiently transmitted from the cell surface of actively infected cells upon contact with bystander cells. In a previous study, we reported the prolonged cell surface retention of VSV-G replication-deficient pseudotyped lentivector prior to endocytic entry. However, the competing kinetics of cell surface versus dissociation, neutralization or direct transfer to other cells have received comparatively little attention. Here we demonstrate that the relative efficiency of cell-cell surface transmission can outpace "cell-free" transduction at limiting vector input. This coincides with the prolonged half-life of cell bound vector but occurs, unlike HTLV-1, without evidence for particle aggregation. These studies suggest that cell-surface attachment stabilizes particles and alters neutralization kinetics. Our experiments provide novel insight into the underexplored cell-cell transmission of pseudotyped particles.

Vaginal microbiota and sexually transmitted infections that may influence transmission of cell-associated HIV.

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Cone, Richard A

2014-12-15

Vaginal microbiota and sexually transmitted infections (STIs) are likely to influence the transmission of cell-associated human immunodeficiency virus (HIV). Lactic acid produced by Lactobacillus-dominated microbiota (Nugent score 0-3) will likely inhibit transmission, especially female-to-male transmission. In contrast, polymicrobial microbiota (Nugent score 4-10), community state types IV-A and IV-B, and STIs will likely increase transmission of cell-associated HIV. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Dendritic cells exposed to MVA-based HIV-1 vaccine induce highly functional HIV-1-specific CD8(+ T cell responses in HIV-1-infected individuals.

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Núria Climent

Full Text Available Currently, MVA virus vectors carrying HIV-1 genes are being developed as HIV-1/AIDS prophylactic/therapeutic vaccines. Nevertheless, little is known about the impact of these vectors on human dendritic cells (DC and their capacity to present HIV-1 antigens to human HIV-specific T cells. This study aimed to characterize the interaction of MVA and MVA expressing the HIV-1 genes Env-Gag-Pol-Nef of clade B (referred to as MVA-B in human monocyte-derived dendritic cells (MDDC and the subsequent processes of HIV-1 antigen presentation and activation of memory HIV-1-specific T lymphocytes. For these purposes, we performed ex vivo assays with MDDC and autologous lymphocytes from asymptomatic HIV-infected patients. Infection of MDDC with MVA-B or MVA, at the optimal dose of 0.3 PFU/MDDC, induced by itself a moderate degree of maturation of MDDC, involving secretion of cytokines and chemokines (IL1-ra, IL-7, TNF-α, IL-6, IL-12, IL-15, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, IP-10, MIG, and IFN-α. MDDC infected with MVA or MVA-B and following a period of 48 h or 72 h of maturation were able to migrate toward CCL19 or CCL21 chemokine gradients. MVA-B infection induced apoptosis of the infected cells and the resulting apoptotic bodies were engulfed by the uninfected MDDC, which cross-presented HIV-1 antigens to autologous CD8(+ T lymphocytes. MVA-B-infected MDDC co-cultured with autologous T lymphocytes induced a highly functional HIV-specific CD8(+ T cell response including proliferation, secretion of IFN-γ, IL-2, TNF-α, MIP-1β, MIP-1α, RANTES and IL-6, and strong cytotoxic activity against autologous HIV-1-infected CD4(+ T lymphocytes. These results evidence the adjuvant role of the vector itself (MVA and support the clinical development of prophylactic and therapeutic anti-HIV vaccines based on MVA-B.

SAMHD1 controls cell cycle status, apoptosis and HIV-1 infection in monocytic THP-1 cells

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Bonifati, Serena; Daly, Michele B.; St Gelais, Corine; Kim, Sun Hee; Hollenbaugh, Joseph A.; Shepard, Caitlin; Kennedy, Edward M.; Kim, Dong-Hyun; Schinazi, Raymond F.; Kim, Baek; Wu, Li

2016-01-01

SAMHD1 limits HIV-1 infection in non-dividing myeloid cells by decreasing intracellular dNTP pools. HIV-1 restriction by SAMHD1 in these cells likely prevents activation of antiviral immune responses and modulates viral pathogenesis, thus highlighting a critical role of SAMHD1 in HIV-1 physiopathology. Here, we explored the function of SAMHD1 in regulating cell proliferation, cell cycle progression and apoptosis in monocytic THP-1 cells. Using the CRISPR/Cas9 technology, we generated THP-1 cells with stable SAMHD1 knockout. We found that silencing of SAMHD1 in cycling cells stimulates cell proliferation, redistributes cell cycle population in the G_1/G_0 phase and reduces apoptosis. These alterations correlated with increased dNTP levels and more efficient HIV-1 infection in dividing SAMHD1 knockout cells relative to control. Our results suggest that SAMHD1, through its dNTPase activity, affects cell proliferation, cell cycle distribution and apoptosis, and emphasize a key role of SAMHD1 in the interplay between cell cycle regulation and HIV-1 infection.

SAMHD1 controls cell cycle status, apoptosis and HIV-1 infection in monocytic THP-1 cells

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Bonifati, Serena [Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, OH (United States); Daly, Michele B. [Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA (United States); St Gelais, Corine; Kim, Sun Hee [Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, OH (United States); Hollenbaugh, Joseph A.; Shepard, Caitlin [Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA (United States); Kennedy, Edward M. [Department of Molecular Genetics and Microbiology, Duke University, Durham, NC (United States); Kim, Dong-Hyun [Department of Pharmacy, School of Pharmacy, Kyung-Hee University, Seoul (Korea, Republic of); Schinazi, Raymond F. [Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA (United States); Kim, Baek, E-mail: baek.kim@emory.edu [Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA (United States); Department of Pharmacy, School of Pharmacy, Kyung-Hee University, Seoul (Korea, Republic of); Wu, Li, E-mail: wu.840@osu.edu [Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, OH (United States)

2016-08-15

SAMHD1 limits HIV-1 infection in non-dividing myeloid cells by decreasing intracellular dNTP pools. HIV-1 restriction by SAMHD1 in these cells likely prevents activation of antiviral immune responses and modulates viral pathogenesis, thus highlighting a critical role of SAMHD1 in HIV-1 physiopathology. Here, we explored the function of SAMHD1 in regulating cell proliferation, cell cycle progression and apoptosis in monocytic THP-1 cells. Using the CRISPR/Cas9 technology, we generated THP-1 cells with stable SAMHD1 knockout. We found that silencing of SAMHD1 in cycling cells stimulates cell proliferation, redistributes cell cycle population in the G{sub 1}/G{sub 0} phase and reduces apoptosis. These alterations correlated with increased dNTP levels and more efficient HIV-1 infection in dividing SAMHD1 knockout cells relative to control. Our results suggest that SAMHD1, through its dNTPase activity, affects cell proliferation, cell cycle distribution and apoptosis, and emphasize a key role of SAMHD1 in the interplay between cell cycle regulation and HIV-1 infection.

HIV-1 gp120 induces NFAT nuclear translocation in resting CD4+ T-cells

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Cicala, Claudia; Arthos, James; Censoplano, Nina; Cruz, Catherine; Chung, Eva; Martinelli, Elena; Lempicki, Richard A.; Natarajan, Ven; VanRyk, Donald; Daucher, Marybeth; Fauci, Anthony S.

2006-01-01

The replication of human immunodeficiency virus (HIV) in CD4+ T-cells is strongly dependent upon the state of activation of infected cells. Infection of sub-optimally activated cells is believed to play a critical role in both the transmission of virus and the persistence of CD4+ T-cell reservoirs. There is accumulating evidence that HIV can modulate signal-transduction pathways in a manner that may facilitate replication in such cells. We previously demonstrated that HIV gp120 induces virus replication in resting CD4+ T cells isolated from HIV-infected individuals. Here, we show that in resting CD4+ T-cells, gp120 activates NFATs and induces their translocation into the nucleus. The HIV LTR encodes NFAT recognition sites, and NFATs may play a critical role in promoting viral replication in sub-optimally activated cells. These observations provide insight into a potential mechanism by which HIV is able to establish infection in resting cells, which may have implications for both transmission of HIV and the persistence of viral reservoirs

DJ1 Expression Downregulates in Neuroblastoma Cells (SK-N-MC Chronically Exposed to HIV-1 and Cocaine.

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Upal eRoy

2015-07-01

Full Text Available Background: HIV-associated neurological disorder (HAND has long been recognized as a consequence of Human Immunodeficiency Virus (HIV infection in the brain. The pathology of HAND gets more complicated with the recreational drug use such as cocaine. Recent studies have suggested multiple genetic influences involved in the pathology of addiction and HAND but only a fraction of the entire genetic risk has been investigated so far. In this regard, role of DJ1 protein (a gene linked to autosomal recessive early-onset Parkinson’s disease in regulating dopamine transmission and reactive oxygen species (ROS production in neuronal cells will be worth investigating in HIV-1 and cocaine exposed microenvironment. Being a very abundant protein in the brain, DJ1 could serve as a potential marker for early detection of HIV-1 and/or cocaine related neurological disorder.Methods: In vitro analysis was done to observe the effect of HIV-1 and/or cocaine on DJ1 protein expression in neuroblastoma cells (SK-N-MC. Gene expression and protein analysis of DJ1 was done on the HIV infected and/or cocaine treated SK-N-MC and compared to untreated cells using real time PCR, Western Blot and flow cytometry.Results: Gene expression and protein analysis indicated that there was a significant decrease in DJ1 expression in SK-N-MC chronically exposed to HIV-1 and/or cocaine.Conclusion: This is the first study to establish that DJ1 expression level in the neuronal cells significantly decreased in presence of HIV-1and/or cocaine indicating oxidative stress level of dopamine neurons.

HIV-1 Vif's Capacity To Manipulate the Cell Cycle Is Species Specific.

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Evans, Edward L; Becker, Jordan T; Fricke, Stephanie L; Patel, Kishan; Sherer, Nathan M

2018-04-01

Cells derived from mice and other rodents exhibit profound blocks to HIV-1 virion production, reflecting species-specific incompatibilities between viral Tat and Rev proteins and essential host factors cyclin T1 (CCNT1) and exportin-1 (XPO1, also known as CRM1), respectively. To determine if mouse cell blocks other than CCNT1 and XPO1 affect HIV's postintegration stages, we studied HIV-1 NL4-3 gene expression in mouse NIH 3T3 cells modified to constitutively express HIV-1-compatible versions of CCNT1 and XPO1 (3T3.CX cells). 3T3.CX cells supported both Rev-independent and Rev-dependent viral gene expression and produced relatively robust levels of virus particles, confirming that CCNT1 and XPO1 represent the predominant blocks to these stages. Unexpectedly, however, 3T3.CX cells were remarkably resistant to virus-induced cytopathic effects observed in human cell lines, which we mapped to the viral protein Vif and its apparent species-specific capacity to induce G 2 /M cell cycle arrest. Vif was able to mediate rapid degradation of human APOBEC3G and the PPP2R5D regulatory B56 subunit of the PP2A phosphatase holoenzyme in mouse cells, thus demonstrating that Vif NL4-3 's modulation of the cell cycle can be functionally uncoupled from some of its other defined roles in CUL5-dependent protein degradation. Vif was also unable to induce G 2 /M cell cycle arrest in other nonhuman cell types, including cells derived from nonhuman primates, leading us to propose that one or more human-specific cofactors underpin Vif's ability to modulate the cell cycle. IMPORTANCE Cells derived from mice and other rodents exhibit profound blocks to HIV-1 replication, thus hindering the development of a low-cost small-animal model for studying HIV/AIDS. Here, we engineered otherwise-nonpermissive mouse cells to express HIV-1-compatible versions of two species-specific host dependency factors, cyclin T1 (CCNT1) and exportin-1 (XPO1) (3T3.CX cells). We show that 3T3.CX cells rescue HIV-1

Anti-gp120 minibody gene transfer to female genital epithelial cells protects against HIV-1 virus challenge in vitro.

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Ussama M Abdel-Motal

Full Text Available Although cervico-vaginal epithelial cells of the female lower genital tract provide the initial defense system against HIV-1 infection, the protection is sometimes incomplete. Thus, enhancing anti-HIV-1 humoral immunity at the mucosal cell surface by local expression of anti-HIV-1 broadly neutralizing antibodies (BnAb that block HIV-1 entry would provide an important new intervention that could slow the spread of HIV/AIDS.This study tested the hypothesis that adeno-associated virus (AAV-BnAb gene transfer to cervico-vaginal epithelial cells will lead to protection against HIV-1. Accordingly, a recombinant AAV vector that encodes human b12 anti-HIV gp120 BnAb as a single-chain variable fragment Fc fusion (scFvFc, or "minibody" was constructed. The secreted b12 minibody was shown to be biologically functional in binding to virus envelope protein, neutralizing HIV-1 and importantly, blocking transfer and infectivity of HIV-1(bal in an organotypic human vaginal epithelial cell (VEC model. Furthermore, cervico-vaginal epithelial stem cells were found to be efficiently transduced by the optimal AAV serotype mediated expression of GFP.This study provides the foundation for a novel microbicide strategy to protect against sexual transmission of HIV-1 by AAV transfer of broadly neutralizing antibody genes to cervico-vaginal epithelial stem cells that could replenish b12 BnAb secreting cells through multiple menstrual cycles.

Polyclonal B cell differentiation and loss of gastrointestinal tract germinal centers in the earliest stages of HIV-1 infection.

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Marc C Levesque

2009-07-01

Full Text Available The antibody response to HIV-1 does not appear in the plasma until approximately 2-5 weeks after transmission, and neutralizing antibodies to autologous HIV-1 generally do not become detectable until 12 weeks or more after transmission. Moreover, levels of HIV-1-specific antibodies decline on antiretroviral treatment. The mechanisms of this delay in the appearance of anti-HIV-1 antibodies and of their subsequent rapid decline are not known. While the effect of HIV-1 on depletion of gut CD4(+ T cells in acute HIV-1 infection is well described, we studied blood and tissue B cells soon after infection to determine the effect of early HIV-1 on these cells.In human participants, we analyzed B cells in blood as early as 17 days after HIV-1 infection, and in terminal ileum inductive and effector microenvironments beginning at 47 days after infection. We found that HIV-1 infection rapidly induced polyclonal activation and terminal differentiation of B cells in blood and in gut-associated lymphoid tissue (GALT B cells. The specificities of antibodies produced by GALT memory B cells in acute HIV-1 infection (AHI included not only HIV-1-specific antibodies, but also influenza-specific and autoreactive antibodies, indicating very early onset of HIV-1-induced polyclonal B cell activation. Follicular damage or germinal center loss in terminal ileum Peyer's patches was seen with 88% of follicles exhibiting B or T cell apoptosis and follicular lysis.Early induction of polyclonal B cell differentiation, coupled with follicular damage and germinal center loss soon after HIV-1 infection, may explain both the high rate of decline in HIV-1-induced antibody responses and the delay in plasma antibody responses to HIV-1. Please see later in the article for Editors' Summary.

New approaches to design HIV-1 T-cell vaccines.

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Perrin, Hélène; Canderan, Glenda; Sékaly, Rafick-Pierre; Trautmann, Lydie

2010-09-01

Following the evidence that T-cell responses are crucial in the control of HIV-1 infection, vaccines targeting T-cell responses were tested in recent clinical trials. However, these vaccines showed a lack of efficacy. This review attempts to define the qualitative and quantitative features that are desirable for T-cell-induced responses by vaccines. We also describe strategies that could lead to achievement of this goal. Using the yellow fever vaccine as a benchmark of an efficient vaccine, recent studies identified factors of immune protection and more importantly innate immune pathways needed for the establishment of long-term protective adaptive immunity. To prevent or control HIV-1 infection, a vaccine must induce efficient and persistent antigen-specific T cells endowed with mucosal homing capacity. Such cells should have the capability to counteract HIV-1 diversity and its rapid spread from the initial site of infection. To achieve this goal, the activation of a diversified innate immune response is critical. New systems biology approaches will provide more precise correlates of immune protection that will pave the way for new approaches in T-cell-based vaccines.

Characteristics of HIV-1 discordant couples enrolled in a trial of HSV-2 suppression to reduce HIV-1 transmission: the partners study.

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Jairam R Lingappa

Full Text Available The Partners HSV-2/HIV-1 Transmission Study (Partners Study is a phase III, placebo-controlled trial of daily acyclovir for genital herpes (HSV-2 suppression among HIV-1/HSV-2 co-infected persons to reduce HIV-1 transmission to their HIV-1 susceptible partners, which requires recruitment of HIV-1 serodiscordant heterosexual couples. We describe the baseline characteristics of this cohort.HIV-1 serodiscordant heterosexual couples, in which the HIV-1 infected partner was HSV-2 seropositive, had a CD4 count >or=250 cells/mcL and was not on antiretroviral therapy, were enrolled at 14 sites in East and Southern Africa. Demographic, behavioral, clinical and laboratory characteristics were assessed.Of the 3408 HIV-1 serodiscordant couples enrolled, 67% of the HIV-1 infected partners were women. Couples had cohabitated for a median of 5 years (range 2-9 with 28% reporting unprotected sex in the month prior to enrollment. Among HIV-1 susceptible participants, 86% of women and 59% of men were HSV-2 seropositive. Other laboratory-diagnosed sexually transmitted infections were uncommon (500 relative to <350, respectively, p<0.001.The Partners Study successfully enrolled a cohort of 3408 heterosexual HIV-1 serodiscordant couples in Africa at high risk for HIV-1 transmission. Follow-up of this cohort will evaluate the efficacy of acyclovir for HSV-2 suppression in preventing HIV-1 transmission and provide insights into biological and behavioral factors determining heterosexual HIV-1 transmission.ClinicalTrials.gov NCT00194519.

Where in the Cell Are You? Probing HIV-1 Host Interactions through Advanced Imaging Techniques

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Brennan S. Dirk

2016-10-01

Full Text Available Viruses must continuously evolve to hijack the host cell machinery in order to successfully replicate and orchestrate key interactions that support their persistence. The type-1 human immunodeficiency virus (HIV-1 is a prime example of viral persistence within the host, having plagued the human population for decades. In recent years, advances in cellular imaging and molecular biology have aided the elucidation of key steps mediating the HIV-1 lifecycle and viral pathogenesis. Super-resolution imaging techniques such as stimulated emission depletion (STED and photoactivation and localization microscopy (PALM have been instrumental in studying viral assembly and release through both cell–cell transmission and cell–free viral transmission. Moreover, powerful methods such as Forster resonance energy transfer (FRET and bimolecular fluorescence complementation (BiFC have shed light on the protein-protein interactions HIV-1 engages within the host to hijack the cellular machinery. Specific advancements in live cell imaging in combination with the use of multicolor viral particles have become indispensable to unravelling the dynamic nature of these virus-host interactions. In the current review, we outline novel imaging methods that have been used to study the HIV-1 lifecycle and highlight advancements in the cell culture models developed to enhance our understanding of the HIV-1 lifecycle.

HIV-1 transgenic rats develop T cell abnormalities

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Reid, William; Abdelwahab, Sayed; Sadowska, Mariola; Huso, David; Neal, Ashley; Ahearn, Aaron; Bryant, Joseph; Gallo, Robert C.; Lewis, George K.; Reitz, Marvin

2004-01-01

HIV-1 infection leads to impaired antigen-specific T cell proliferation, increased susceptibility of T cells to apoptosis, progressive impairment of T-helper 1 (Th1) responses, and altered maturation of HIV-1-specific memory cells. We have identified similar impairments in HIV-1 transgenic (Tg) rats. Tg rats developed an absolute reduction in CD4 + and CD8 + T cells able to produce IFN-γ following activation and an increased susceptibility of T cells to activation-induced apoptosis. CD4 + and CD8 + effector/memory (CD45RC - CD62L - ) pools were significantly smaller in Tg rats compared to non-Tg controls, although the converse was true for the naieve (CD45RC + CD62L + ) T cell pool. Our interpretation is that the HIV transgene causes defects in the development of T cell effector function and generation of specific effector/memory T cell subsets, and that activation-induced apoptosis may be an essential factor in this process

Low mother-to-child-transmission rate of Hepatitis C virus in cART treated HIV-1 infected mothers.

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Snijdewind, I J M; Smit, C; Schutten, M; Nellen, F J B; Kroon, F P; Reiss, P; van der Ende, M E

2015-07-01

Maternal transmission is the most common cause of HCV infection in children. HIV co-infection and high levels of plasma HCV-RNA have been associated with increased HCV transmission rates. We assessed the vertical HCV transmission rate in the HIV-HCV co-infected group of pregnant women on cART. We conducted a retrospective study in a Dutch cohort of HIV-positive pregnant women and their children. We identified co-infected mothers. Results of the HCV tests of the children were obtained. All 21 women were on cART at the time of delivery. We analyzed data of the 24 live-born children at risk for mother-to-child transmission (MTCT) of HCV between 1996 and 2009. HIV-RNA was cell count was 419 cells/μl (290-768). There was no transmission of HIV. The median plasma HCV-RNA in our cohort of 23 non-transmitting deliveries in 21 women was 3.5×10E5 viral eq/ml (IQR 9.6×104-1.5×106veq/mL). One of 24 live-born children was found to be infected with HCV genotype 1. At the time of delivery the maternal plasma HIV-RNA was cell count was 160 cells/μl and maternal plasma HCV-RNA was 4.6×10E6 veq/ml. This amounted to a prevalence of HCV-MTCT of 4%. In this well-defined cohort of HIV-HCV co-infected pregnant women, all treated with cART during pregnancy, a modest rate of vertical HCV transmission was observed. Copyright © 2015 Elsevier B.V. All rights reserved.

Role of Semen on Vaginal HIV-1 Transmission and Maraviroc Protection

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Council, Olivia D.; Swanson, Michael D.; Spagnuolo, Rae Ann

2015-01-01

We used bone marrow/liver/thymus (BLT) humanized mice to establish the effect of semen on vaginal HIV infection and on the efficacy of topically applied maraviroc. Our results demonstrate that vaginal transmission of cell-free HIV occurs efficiently in the presence of semen and that topically applied maraviroc efficiently prevents HIV transmission in the presence of semen. We also show that semen has no significant effect on the transmission of transmitted/founder viruses or cell-associated viruses. PMID:26392489

Identification of Nevirapine-Resistant HIV-1 in the Latent Reservoir after Single-Dose Nevirapine to Prevent Mother-to-Child Transmission of HIV-1

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Wind-Rotolo, Megan; Durand, Christine; Cranmer, Lisa; Reid, Alison; Martinson, Neil; Doherty, Meg; Jilek, Benjamin L.; Kagaayi, Joseph; Kizza, Allan; Pillay, Visva; Laeyendecker, Oliver; Reynolds, Steven J.; Eshleman, Susan H.; Lau, Bryan; Ray, Stuart C.; Siliciano, Janet D.; Quinn, Thomas C.; Siliciano, Robert F.

2009-01-01

Background Intrapartum single-dose nevirapine decreases mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1) but promotes nevirapine resistance. Although resistant viruses fade to undetectable levels in plasma, they may persist as stably integrated proviruses within the latent reservoir in resting CD4+ T cells, potentially complicating future treatment. Methods Blood samples were collected from 60 women from South Africa and Uganda >6 months after they had received single-dose nevirapine. To selectively analyze the stable latent form of HIV-1, resting CD4+ T cells were isolated and activated in the presence of reverse-transcriptase inhibitors and integrase inhibitors, which allows for the specific isolation of viruses produced by cells with stably integrated proviral DNA. These viruses were then analyzed for nevirapine resistance. Results Although only a small number of latently infected cells were present in each blood sample (mean, 162 cells), nevirapine resistance mutations (K103N and G190A) were detected in the latent reservoir of 4 (8%) of 50 evaluable women. Conclusions A single dose of nevirapine can establish antiretroviral resistance within the latent reservoir. This results in a potentially lifelong risk of reemergence of nevirapine-resistant virus and highlights the need for strategies to prevent transmission that do not compromise successful future treatment. PMID:19338474

Alemtuzumab-induced elimination of HIV-1-infected immune cells.

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Ruxrungtham, Kiat; Sirivichayakul, Sunee; Buranapraditkun, Supranee; Krause, Werner

2016-01-01

Currently, there is no drug known that is able to eradicate either HIV or HIV-infected host cells. The effectiveness of all available treatments is based on the prevention of viral replication. We investigated whether the monoclonal, CD52 receptor-targeting antibody, alemtuzumab, which is currently approved for the treatment of multiple sclerosis, is able to eliminate HIV-infected immune cells. In blood samples from healthy donors and from HIV-1-infected subjects who were either treatment-naïve or resistant to HAART, we studied whether the CD52 expression on T cells and their subsets (CD3, CD4, CD8), B cells (CD19), dendritic cells (CD123) and monocytes (CD11c) is retained in HIV-1 infection and whether alemtuzumab is able to eradicate infected cells, using four-colour flow cytometry. We found that CD52 expression on immune cells is retained in HIV-1 infection regardless of CD4 cell count, viral load and treatment status, and is amenable to alemtuzumab-induced depletion. For the first time it could be shown in vitro that HIV-1-infected immune cells can be eliminated by using the monoclonal antibody alemtuzumab.

The impact of pregnancy on the HIV-1-specific T cell function in infected pregnant women.

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Hygino, Joana; Vieira, Morgana M; Kasahara, Taissa M; Xavier, Luciana F; Blanco, Bernardo; Guillermo, Landi V C; Filho, Renato G S; Saramago, Carmen S M; Lima-Silva, Agostinho A; Oliveira, Ariane L; Guimarães, Vander; Andrade, Arnaldo F B; Bento, Cleonice A M

2012-12-01

Evidences indicate that pregnancy can alter the Ag-specific T-cell responses. This work aims to evaluate the impact of pregnancy on the in vitro HIV-1-specific immune response. As compared with non-pregnant patients, lower T-cell proliferation and higher IL-10 production were observed in T-cell cultures from pregnant patients following addition of either mitogens or HIV-1 antigens. In our system, the main T lymphocyte subset involved in producing IL-10 was CD4(+)FoxP3(-). Depletion of CD4(+) cells elevated TNF-α and IFN-γ production. Interestingly, the in vitro HIV-1 replication was lower in cell cultures from pregnant patients, and it was inversely related to IL-10 production. In these cultures, the neutralization of IL-10 by anti-IL-10 mAb elevated TNF-α release and HIV-1 replication. In conclusion, our results reveal that pregnancy-related events should favor the expansion of HIV-1-specific IL-10-secreting CD4(+) T-cells in HIV-1-infected women, which should, in the scenario of pregnancy, help to reduce the risk of vertical HIV-1 transmission. Copyright © 2012 Elsevier Inc. All rights reserved.

Acyclovir and Transmission of HIV-1 from Persons Infected with HIV-1 and HSV-2

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Celum, Connie; Wald, Anna; Lingappa, Jairam R.; Magaret, Amalia S.; Wang, Richard S.; Mugo, Nelly; Mujugira, Andrew; Baeten, Jared M.; Mullins, James I.; Hughes, James P.; Bukusi, Elizabeth A.; Cohen, Craig R.; Katabira, Elly; Ronald, Allan; Kiarie, James; Farquhar, Carey; Stewart, Grace John; Makhema, Joseph; Essex, Myron; Were, Edwin; Fife, Kenneth H.; de Bruyn, Guy; Gray, Glenda E.; McIntyre, James A.; Manongi, Rachel; Kapiga, Saidi; Coetzee, David; Allen, Susan; Inambao, Mubiana; Kayitenkore, Kayitesi; Karita, Etienne; Kanweka, William; Delany, Sinead; Rees, Helen; Vwalika, Bellington; Stevens, Wendy; Campbell, Mary S.; Thomas, Katherine K.; Coombs, Robert W.; Morrow, Rhoda; Whittington, William L.H.; McElrath, M. Juliana; Barnes, Linda; Ridzon, Renee; Corey, Lawrence

2010-01-01

BACKGROUND Most persons who are infected with human immunodeficiency virus type 1 (HIV-1) are also infected with herpes simplex virus type 2 (HSV-2), which is frequently reactivated and is associated with increased plasma and genital levels of HIV-1. Therapy to suppress HSV-2 reduces the frequency of reactivation of HSV-2 as well as HIV-1 levels, suggesting that suppression of HSV-2 may reduce the risk of transmission of HIV-1. METHODS We conducted a randomized, placebo-controlled trial of suppressive therapy for HSV-2 (acyclovir at a dose of 400 mg orally twice daily) in couples in which only one of the partners was seropositive for HIV-1 (CD4 count, ≥250 cells per cubic millimeter) and that partner was also infected with HSV-2 and was not taking antiretroviral therapy at the time of enrollment. The primary end point was transmission of HIV-1 to the partner who was not initially infected with HIV-1; linkage of transmissions was assessed by means of genetic sequencing of viruses. RESULTS A total of 3408 couples were enrolled at 14 sites in Africa. Of the partners who were infected with HIV-1, 68% were women, and the baseline median CD4 count was 462 cells per cubic millimeter. Of 132 HIV-1 seroconversions that occurred after randomization (an incidence of 2.7 per 100 person-years), 84 were linked within couples by viral sequencing: 41 in the acyclovir group and 43 in the placebo group (hazard ratio with acyclovir, 0.92, 95% confidence interval [CI], 0.60 to 1.41; P = 0.69). Suppression with acyclovir reduced the mean plasma concentration of HIV-1 by 0.25 log10 copies per milliliter (95% CI, 0.22 to 0.29; P<0.001) and the occurrence of HSV-2–positive genital ulcers by 73% (risk ratio, 0.27; 95% CI, 0.20 to 0.36; P<0.001). A total of 92% of the partners infected with HIV-1 and 84% of the partners not infected with HIV-1 remained in the study for 24 months. The level of adherence to the dispensed study drug was 96%. No serious adverse events related to acyclovir

Antiretroviral pre-exposure prophylaxis prevents vaginal transmission of HIV-1 in humanized BLT mice.

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Paul W Denton

2008-01-01

Full Text Available Worldwide, vaginal transmission now accounts for more than half of newly acquired HIV-1 infections. Despite the urgency to develop and implement novel approaches capable of preventing HIV transmission, this process has been hindered by the lack of adequate small animal models for preclinical efficacy and safety testing. Given the importance of this route of transmission, we investigated the susceptibility of humanized mice to intravaginal HIV-1 infection.We show that the female reproductive tract of humanized bone marrow-liver-thymus (BLT mice is reconstituted with human CD4+ T and other relevant human cells, rendering these humanized mice susceptible to intravaginal infection by HIV-1. Effects of HIV-1 infection include CD4+ T cell depletion in gut-associated lymphoid tissue (GALT that closely mimics what is observed in HIV-1-infected humans. We also show that pre-exposure prophylaxis with antiretroviral drugs is a highly effective method for preventing vaginal HIV-1 transmission. Whereas 88% (7/8 of BLT mice inoculated vaginally with HIV-1 became infected, none of the animals (0/5 given pre-exposure prophylaxis of emtricitabine (FTC/tenofovir disoproxil fumarate (TDF showed evidence of infection (Chi square = 7.5, df = 1, p = 0.006.The fact that humanized BLT mice are susceptible to intravaginal infection makes this system an excellent candidate for preclinical evaluation of both microbicides and pre-exposure prophylactic regimens. The utility of humanized mice to study intravaginal HIV-1 transmission is particularly highlighted by the demonstration that pre-exposure prophylaxis can prevent intravaginal HIV-1 transmission in the BLT mouse model.

Sexual transmission and propagation of SIV and HIV in resting and activated CD4+ T cells

NARCIS (Netherlands)

Zhang, Z.; Schuler, T.; Zupancic, M.; Wietgrefe, S.; Staskus, K. A.; Reimann, K. A.; Reinhart, T. A.; Rogan, M.; Cavert, W.; Miller, C. J.; Veazey, R. S.; Notermans, D.; Little, S.; Danner, S. A.; Richman, D. D.; Havlir, D.; Wong, J.; Jordan, H. L.; Schacker, T. W.; Racz, P.; Tenner-Racz, K.; Letvin, N. L.; Wolinsky, S.; Haase, A. T.

1999-01-01

In sexual transmission of simian immunodeficiency virus, and early and later stages of human immunodeficiency virus-type 1 (HIV-1) infection, both viruses were found to replicate predominantly in CD4(+) T cells at the portal of entry and in lymphoid tissues. Infection was propagated not only in

Calcitonin Gene-Related Peptide Induces HIV-1 Proteasomal Degradation in Mucosal Langerhans Cells.

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Bomsel, Morgane; Ganor, Yonatan

2017-12-01

The neuroimmune dialogue between peripheral neurons and Langerhans cells (LCs) within mucosal epithelia protects against incoming pathogens. LCs rapidly internalize human immunodeficiency virus type 1 (HIV-1) upon its sexual transmission and then trans -infect CD4 + T cells. We recently found that the neuropeptide calcitonin gene-related peptide (CGRP), secreted mucosally from peripheral neurons, inhibits LC-mediated HIV-1 trans -infection. In this study, we investigated the mechanism of CGRP-induced inhibition, focusing on HIV-1 degradation in LCs and its interplay with trans -infection. We first show that HIV-1 degradation occurs in endolysosomes in untreated LCs, and functionally blocking such degradation with lysosomotropic agents results in increased trans -infection. We demonstrate that CGRP acts via its cognate receptor and at a viral postentry step to induce faster HIV-1 degradation, but without affecting the kinetics of endolysosomal degradation. We reveal that unexpectedly, CGRP shifts HIV-1 degradation from endolysosomes toward the proteasome, providing the first evidence for functional HIV-1 proteasomal degradation in LCs. Such efficient proteasomal degradation significantly inhibits the first phase of trans -infection, and proteasomal, but not endolysosomal, inhibitors abrogate CGRP-induced inhibition. Together, our results establish that CGRP controls the HIV-1 degradation mode in LCs. The presence of endogenous CGRP within innervated mucosal tissues, especially during the sexual response, to which CGRP contributes, suggests that HIV-1 proteasomal degradation predominates in vivo Hence, proteasomal, rather than endolysosomal, HIV-1 degradation in LCs should be enhanced clinically to effectively restrict HIV-1 trans -infection. IMPORTANCE During sexual transmission, HIV-1 is internalized and degraded in LCs, the resident antigen-presenting cells in mucosal epithelia. Yet during trans -infection, infectious virions escaping degradation are transferred

Human embryonic stem cell (hES derived dendritic cells are functionally normal and are susceptible to HIV-1 infection

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Bandi Sriram

2008-01-01

Full Text Available Abstract Background Human embryonic stem (hES cells hold considerable promise for cell replacement and gene therapies. Their remarkable properties of pluripotency, self-renewal, and tractability for genetic modification potentially allows for the production of sizeable quantities of therapeutic cells of the hematopoietic lineage. Dendritic cells (DC arise from CD34+ hematopoietic progenitor cells (HPCs and are important in many innate and adaptive immune functions. With respect to HIV-1 infection, DCs play an important role in the efficient capture and transfer of the virus to susceptible cells. With an aim of generating DCs from a renewable source for HIV-1 studies, here we evaluated the capacity of hES cell derived CD34+ cells to give rise to DCs which can support HIV-1 infection. Results Undifferentiated hES cells were cultured on S17 mouse bone marrow stromal cell layers to derive CD34+ HPCs which were subsequently grown in specific cytokine differentiation media to promote the development of DCs. The hES derived DCs (hES-DC were subjected to phenotypic and functional analyses and compared with DCs derived from fetal liver CD34+ HPC (FL-DC. The mature hES-DCs displayed typical DC morphology consisting of veiled stellate cells. The hES-DCs also displayed characteristic phenotypic surface markers CD1a, HLA-DR, B7.1, B7.2, and DC-SIGN. The hES-DCs were found to be capable of antigen uptake and stimulating naïve allogeneic CD4+ T cells in a mixed leukocyte reaction assay. Furthermore, the hES-DCs supported productive HIV-1 viral infection akin to standard DCs. Conclusion Phenotypically normal and functionally competent DCs that support HIV-1 infection can be derived from hES cells. hES-DCs can now be exploited in applied immunology and HIV-1 infection studies. Using gene therapy approaches, it is now possible to generate HIV-1 resistant DCs from anti-HIV gene transduced hES-CD34+ hematopoietic progenitor cells.

The transcriptome of HIV-1 infected intestinal CD4+ T cells exposed to enteric bacteria.

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Alyson C Yoder

2017-02-01

Full Text Available Global transcriptome studies can help pinpoint key cellular pathways exploited by viruses to replicate and cause pathogenesis. Previous data showed that laboratory-adapted HIV-1 triggers significant gene expression changes in CD4+ T cell lines and mitogen-activated CD4+ T cells from peripheral blood. However, HIV-1 primarily targets mucosal compartments during acute infection in vivo. Moreover, early HIV-1 infection causes extensive depletion of CD4+ T cells in the gastrointestinal tract that herald persistent inflammation due to the translocation of enteric microbes to the systemic circulation. Here, we profiled the transcriptome of primary intestinal CD4+ T cells infected ex vivo with transmitted/founder (TF HIV-1. Infections were performed in the presence or absence of Prevotella stercorea, a gut microbe enriched in the mucosa of HIV-1-infected individuals that enhanced both TF HIV-1 replication and CD4+ T cell death ex vivo. In the absence of bacteria, HIV-1 triggered a cellular shutdown response involving the downregulation of HIV-1 reactome genes, while perturbing genes linked to OX40, PPAR and FOXO3 signaling. However, in the presence of bacteria, HIV-1 did not perturb these gene sets or pathways. Instead, HIV-1 enhanced granzyme expression and Th17 cell function, inhibited G1/S cell cycle checkpoint genes and triggered downstream cell death pathways in microbe-exposed gut CD4+ T cells. To gain insights on these differential effects, we profiled the gene expression landscape of HIV-1-uninfected gut CD4+ T cells exposed to bacteria. Microbial exposure upregulated genes involved in cellular proliferation, MAPK activation, Th17 cell differentiation and type I interferon signaling. Our findings reveal that microbial exposure influenced how HIV-1 altered the gut CD4+ T cell transcriptome, with potential consequences for HIV-1 susceptibility, cell survival and inflammation. The HIV-1- and microbe-altered pathways unraveled here may serve as a

Long-term nonprogression and broad HIV-1-specific proliferative T-cell responses

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Nesrina eImami

2013-03-01

Full Text Available Complex mechanisms underlying the maintenance of fully functional, proliferative, HIV-1-specific T-cell responses involve processes from early T-cell development through to the final stages of T-cell differentiation and antigen recognition. Virus-specific proliferative CD4 and CD8 T-cell responses, important for the control of infection, are observed in some HIV-1+ patients during early stages of disease, and are maintained in long-term nonprogressing subjects. In the vast majority of HIV-1+ patients, full immune functionality is lost when proliferative HIV-1-specific T-cell responses undergo a variable progressive decline throughout the course of chronic infection. This appears irreparable despite administration of potent combination antiretroviral therapy, which to date is non-curative, necessitating life-long administration and the development of effective, novel, therapeutic interventions. While a sterilising cure, involving clearance of virus from the host, remains a primary aim, a functional cure may be a more feasible goal with considerable impact on worldwide HIV-1 infection. Such an approach would enable long-term co-existence of host and virus in the absence of toxic and costly drugs. Effective immune homeostasis coupled with a balanced response appropriately targeting conserved viral antigens, in a manner that avoids hyperactivation and exhaustion, may prove to be the strongest correlate of durable viral control. This review describes novel concepts underlying full immune functionality in the context of HIV-1 infection, which may be utilised in future strategies designed to improve upon existing therapy. The aim will be to induce long-term nonprogressor or elite controller status in every infected host, through immune-mediated control of viraemia and reduction of viral reservoirs, leading to lower HIV-1 transmission rates.

HIV-1 tat promotes integrin-mediated HIV transmission to dendritic cells by binding Env spikes and competes neutralization by anti-HIV antibodies.

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Paolo Monini

Full Text Available Use of Env in HIV vaccine development has been disappointing. Here we show that, in the presence of a biologically active Tat subunit vaccine, a trimeric Env protein prevents in monkeys virus spread from the portal of entry to regional lymph nodes. This appears to be due to specific interactions between Tat and Env spikes that form a novel virus entry complex favoring R5 or X4 virus entry and productive infection of dendritic cells (DCs via an integrin-mediated pathway. These Tat effects do not require Tat-transactivation activity and are blocked by anti-integrin antibodies (Abs. Productive DC infection promoted by Tat is associated with a highly efficient virus transmission to T cells. In the Tat/Env complex the cysteine-rich region of Tat engages the Env V3 loop, whereas the Tat RGD sequence remains free and directs the virus to integrins present on DCs. V2 loop deletion, which unshields the CCR5 binding region of Env, increases Tat/Env complex stability. Of note, binding of Tat to Env abolishes neutralization of Env entry or infection of DCs by anti-HIV sera lacking anti-Tat Abs, which are seldom present in natural infection. This is reversed, and neutralization further enhanced, by HIV sera containing anti-Tat Abs such as those from asymptomatic or Tat-vaccinated patients, or by sera from the Tat/Env vaccinated monkeys. Thus, both anti-Tat and anti-Env Abs are required for efficient HIV neutralization. These data suggest that the Tat/Env interaction increases HIV acquisition and spreading, as a mechanism evolved by the virus to escape anti-Env neutralizing Abs. This may explain the low effectiveness of Env-based vaccines, which are also unlikely to elicit Abs against new Env epitopes exposed by the Tat/Env interaction. As Tat also binds Envs from different clades, new vaccine strategies should exploit the Tat/Env interaction for both preventative and therapeutic interventions.

Oral keratinocytes support non-replicative infection and transfer of harbored HIV-1 to permissive cells.

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Vacharaksa, Anjalee; Asrani, Anil C; Gebhard, Kristin H; Fasching, Claudine E; Giacaman, Rodrigo A; Janoff, Edward N; Ross, Karen F; Herzberg, Mark C

2008-07-17

Oral keratinocytes on the mucosal surface are frequently exposed to HIV-1 through contact with infected sexual partners or nursing mothers. To determine the plausibility that oral keratinocytes are primary targets of HIV-1, we tested the hypothesis that HIV-1 infects oral keratinocytes in a restricted manner. To study the fate of HIV-1, immortalized oral keratinocytes (OKF6/TERT-2; TERT-2 cells) were characterized for the fate of HIV-specific RNA and DNA. At 6 h post inoculation with X4 or R5-tropic HIV-1, HIV-1gag RNA was detected maximally within TERT-2 cells. Reverse transcriptase activity in TERT-2 cells was confirmed by VSV-G-mediated infection with HIV-NL4-3Deltaenv-EGFP. AZT inhibited EGFP expression in a dose-dependent manner, suggesting that viral replication can be supported if receptors are bypassed. Within 3 h post inoculation, integrated HIV-1 DNA was detected in TERT-2 cell nuclei and persisted after subculture. Multiply spliced and unspliced HIV-1 mRNAs were not detectable up to 72 h post inoculation, suggesting that HIV replication may abort and that infection is non-productive. Within 48 h post inoculation, however, virus harbored by CD4 negative TERT-2 cells trans infected co-cultured peripheral blood mononuclear cells (PBMCs) or MOLT4 cells (CD4+ CCR5+) by direct cell-to-cell transfer or by releasing low levels of infectious virions. Primary tonsil epithelial cells also trans infected HIV-1 to permissive cells in a donor-specific manner. Oral keratinocytes appear, therefore, to support stable non-replicative integration, while harboring and transmitting infectious X4- or R5-tropic HIV-1 to permissive cells for up to 48 h.

Oral keratinocytes support non-replicative infection and transfer of harbored HIV-1 to permissive cells

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Giacaman Rodrigo A

2008-07-01

Full Text Available Abstract Background Oral keratinocytes on the mucosal surface are frequently exposed to HIV-1 through contact with infected sexual partners or nursing mothers. To determine the plausibility that oral keratinocytes are primary targets of HIV-1, we tested the hypothesis that HIV-1 infects oral keratinocytes in a restricted manner. Results To study the fate of HIV-1, immortalized oral keratinocytes (OKF6/TERT-2; TERT-2 cells were characterized for the fate of HIV-specific RNA and DNA. At 6 h post inoculation with X4 or R5-tropic HIV-1, HIV-1gag RNA was detected maximally within TERT-2 cells. Reverse transcriptase activity in TERT-2 cells was confirmed by VSV-G-mediated infection with HIV-NL4-3Δenv-EGFP. AZT inhibited EGFP expression in a dose-dependent manner, suggesting that viral replication can be supported if receptors are bypassed. Within 3 h post inoculation, integrated HIV-1 DNA was detected in TERT-2 cell nuclei and persisted after subculture. Multiply spliced and unspliced HIV-1 mRNAs were not detectable up to 72 h post inoculation, suggesting that HIV replication may abort and that infection is non-productive. Within 48 h post inoculation, however, virus harbored by CD4 negative TERT-2 cells trans infected co-cultured peripheral blood mononuclear cells (PBMCs or MOLT4 cells (CD4+ CCR5+ by direct cell-to-cell transfer or by releasing low levels of infectious virions. Primary tonsil epithelial cells also trans infected HIV-1 to permissive cells in a donor-specific manner. Conclusion Oral keratinocytes appear, therefore, to support stable non-replicative integration, while harboring and transmitting infectious X4- or R5-tropic HIV-1 to permissive cells for up to 48 h.

HIV-1 adaptation to NK cell-mediated immune pressure

DEFF Research Database (Denmark)

Elemans, Marjet; Boelen, Lies; Rasmussen, Michael

2017-01-01

The observation, by Alter et al., of the enrichment of NK cell “escape” variants in individuals carrying certain Killer-cell Immunoglobulin-like Receptor (KIR) genes is compelling evidence that natural killer (NK) cells exert selection pressure on HIV-1. Alter et al hypothesise that variant pepti...

Visualization of HIV-1 interactions with penile and foreskin epithelia: clues for female-to-male HIV transmission.

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Minh H Dinh

2015-03-01

Full Text Available To gain insight into female-to-male HIV sexual transmission and how male circumcision protects against this mode of transmission, we visualized HIV-1 interactions with foreskin and penile tissues in ex vivo tissue culture and in vivo rhesus macaque models utilizing epifluorescent microscopy. 12 foreskin and 14 cadaveric penile specimens were cultured with R5-tropic photoactivatable (PA-GFP HIV-1 for 4 or 24 hours. Tissue cryosections were immunofluorescently imaged for epithelial and immune cell markers. Images were analyzed for total virions, proportion of penetrators, depth of virion penetration, as well as immune cell counts and depths in the tissue. We visualized individual PA virions breaching penile epithelial surfaces in the explant and macaque model. Using kernel density estimated probabilities of localizing a virion or immune cell at certain tissue depths revealed that interactions between virions and cells were more likely to occur in the inner foreskin or glans penis (from local or cadaveric donors, respectively. Using statistical models to account for repeated measures and zero-inflated datasets, we found no difference in total virions visualized at 4 hours between inner and outer foreskins from local donors. At 24 hours, there were more virions in inner as compared to outer foreskin (0.0495 +/- 0.0154 and 0.0171 +/- 0.0038 virions/image, p = 0.001. In the cadaveric specimens, we observed more virions in inner foreskin (0.0507 +/- 0.0079 virions/image than glans tissue (0.0167 +/- 0.0033 virions/image, p<0.001, but a greater proportion was seen penetrating uncircumcised glans tissue (0.0458 +/- 0.0188 vs. 0.0151 +/- 0.0100 virions/image, p = 0.099 and to significantly greater mean depths (29.162 +/- 3.908 vs. 12.466 +/- 2.985 μm. Our in vivo macaque model confirmed that virions can breach penile squamous epithelia in a living model. In summary, these results suggest that the inner foreskin and glans epithelia may be important sites

Selective elimination of HIV-1-infected cells by Env-directed, HIV-1-based virus-like particles

International Nuclear Information System (INIS)

Peretti, Silvia; Schiavoni, Ilaria; Pugliese, Katherina; Federico, Maurizio

2006-01-01

We recently showed that both replicating and resting cells cultivated with ganciclovir (GCV) were killed when challenged with vesicular stomatitis virus G glycoprotein pseudotyped HIV-1-based virus-like particles (VLPs) carrying the Nef7 (i.e., an HIV-1 Nef mutant incorporating in virions at high levels)/herpes simplex virus-1 thymidine kinase (HSV-TK) fusion product. On this basis, a novel anti-HIV therapeutic approach based on Nef7/TK VLPs expressing X4 or R5 HIV cell receptor complexes has been attempted. We here report that (CD4-CXCR4) and (CD4-CCR5) Nef7-based VLPs efficiently enter cells infected by X4- or R5-tropic HIV-1 strains, respectively. Importantly, the delivery of the VLP-associated Nef7/TK led to cell death upon GCV treatment. Of interest, VLPs were effective also against non-replicating, HIV-1-infected primary human monocyte-derived macrophages. HIV-targeted VLPs represent a promising candidate for the treatment of persistently HIV-1-infected cells that are part of virus reservoirs resistant to HAART therapies

HIV type 1 chemokine receptor usage in mother-to-child transmission.

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Salvatori, F; Scarlatti, G

2001-07-01

To investigate the role of the HIV-1 phenotype in mother-to-child HIV-1 transmission, we evaluated coreceptor usage and replication kinetics in chemokine receptor-expressing U87MG.CD4 cells of primary isolates from 32 HIV-1-infected mothers of Italian origin, none under preventive antiretroviral therapy, and from their infected infants. Five of 15 mothers of infected children and 2 of 17 mothers of uninfected children harbored viruses able to use CXCR4 as coreceptor. However, all isolates used CCR5, alone or in association with CXCR4. The replicative capacity in coreceptor-expressing cells of the viral isolates did not differ between the two groups of mothers. All mothers with an R5 virus transmitted a virus with the same coreceptor usage, whereas those four with a multitropic virus transmitted such a virus in one case. Although the presence of a mixed viral population was documented in the mothers, we did not observe transmission solely of X4 viruses. Interestingly, the only child infected with a multitropic virus carried a defective CCR5 allele. Analysis of the env V3 region of the provirus from this child revealed infection with multiple viral variants with a predominance of R5-type over X4-type sequences. These findings show that CCR5 usage of a viral isolate is not a discriminating risk factor for vertical transmission. Furthermore, X4 viruses can be transmitted to the newborn, although less frequently. In particular, we document the transmission of multiple viral variants with different coreceptor usage in a Delta32 CCR5 heterozygous child, and demonstrate that the heterozygous genotype per se does not contribute to the restriction of R5-type virus spread.

Potent Inhibition of HIV-1 Replication in Resting CD4 T Cells by Resveratrol and Pterostilbene.

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Chan, Chi N; Trinité, Benjamin; Levy, David N

2017-09-01

HIV-1 infection of resting CD4 T cells plays a crucial and numerically dominant role during virus transmission at mucosal sites and during subsequent acute replication and T cell depletion. Resveratrol and pterostilbene are plant stilbenoids associated with several health-promoting benefits. Resveratrol has been shown to inhibit the replication of several viruses, including herpes simplex viruses 1 and 2, papillomaviruses, severe acute respiratory syndrome virus, and influenza virus. Alone, resveratrol does not inhibit HIV-1 infection of activated T cells, but it does synergize with nucleoside reverse transcriptase inhibitors in these cells to inhibit reverse transcription. Here, we demonstrate that resveratrol and pterostilbene completely block HIV-1 infection at a low micromolar dose in resting CD4 T cells, primarily at the reverse transcription step. The anti-HIV effect was fully reversed by exogenous deoxynucleosides and Vpx, an HIV-1 and simian immunodeficiency virus protein that increases deoxynucleoside triphosphate (dNTP) levels. These findings are consistent with the reported ability of resveratrol to inhibit ribonucleotide reductase and to lower dNTP levels in cells. This study supports the potential use of resveratrol, pterostilbene, or related compounds as adjuvants in anti-HIV preexposure prophylaxis (PrEP) formulations. Copyright © 2017 American Society for Microbiology.

HIV-1 replication in cell lines harboring INI1/hSNF5 mutations

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Wu Xuhong

2006-08-01

Full Text Available Abstract Background INI1/hSNF5 is a cellular protein that directly interacts with HIV-1 integrase (IN. It is specifically incorporated into HIV-1 virions. A dominant negative mutant derived from INI1 inhibits HIV-1 replication. Recent studies indicate that INI1 is associated with pre-integration and reverse transcription complexes that are formed upon viral entry into the target cells. INI1 also is a tumor suppressor, biallelically deleted/mutated in malignant rhabdoid tumors. We have utilized cell lines derived from the rhabdoid tumors, MON and STA-WT1, that harbor either null or truncating mutations of INI1 respectively, to assess the effect of INI1 on HIV-1 replication. Results We found that while HIV-1 virions produced in 293T cells efficiently transduced MON and STA-WT1 cells, HIV-1 particle production was severely reduced in both of these cells. Reintroduction of INI1 into MON and STA-WT1 significantly enhanced the particle production in both cell lines. HIV-1 particles produced in MON cells were reduced for infectivity, while those produced in STA-WT1 were not. Further analysis indicated the presence of INI1 in those virions produced from STA-WT1 but not from those produced from MON cells. HIV-1 produced in MON cells were defective for synthesis of early and late reverse transcription products in the target cells. Furthermore, virions produced in MON cells were defective for exogenous reverse transcriptase activity carried out using exogenous template, primer and substrate. Conclusion Our results suggest that INI1-deficient cells exhibit reduced particle production that can be partly enhanced by re-introduction of INI1. Infectivity of HIV-1 produced in some but not all INI1 defective cells, is affected and this defect may correlate to the lack of INI1 and/or some other proteins in these virions. The block in early events of virion produced from MON cells appears to be at the stage of reverse transcription. These studies suggest that

HIV-1 replication in cell lines harboring INI1/hSNF5 mutations.

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Sorin, Masha; Yung, Eric; Wu, Xuhong; Kalpana, Ganjam V

2006-08-31

INI1/hSNF5 is a cellular protein that directly interacts with HIV-1 integrase (IN). It is specifically incorporated into HIV-1 virions. A dominant negative mutant derived from INI1 inhibits HIV-1 replication. Recent studies indicate that INI1 is associated with pre-integration and reverse transcription complexes that are formed upon viral entry into the target cells. INI1 also is a tumor suppressor, biallelically deleted/mutated in malignant rhabdoid tumors. We have utilized cell lines derived from the rhabdoid tumors, MON and STA-WT1, that harbor either null or truncating mutations of INI1 respectively, to assess the effect of INI1 on HIV-1 replication. We found that while HIV-1 virions produced in 293T cells efficiently transduced MON and STA-WT1 cells, HIV-1 particle production was severely reduced in both of these cells. Reintroduction of INI1 into MON and STA-WT1 significantly enhanced the particle production in both cell lines. HIV-1 particles produced in MON cells were reduced for infectivity, while those produced in STA-WT1 were not. Further analysis indicated the presence of INI1 in those virions produced from STA-WT1 but not from those produced from MON cells. HIV-1 produced in MON cells were defective for synthesis of early and late reverse transcription products in the target cells. Furthermore, virions produced in MON cells were defective for exogenous reverse transcriptase activity carried out using exogenous template, primer and substrate. Our results suggest that INI1-deficient cells exhibit reduced particle production that can be partly enhanced by re-introduction of INI1. Infectivity of HIV-1 produced in some but not all INI1 defective cells, is affected and this defect may correlate to the lack of INI1 and/or some other proteins in these virions. The block in early events of virion produced from MON cells appears to be at the stage of reverse transcription. These studies suggest that presence of INI1 or some other host factor in virions and

Ectopic expression of anti-HIV-1 shRNAs protects CD8+ T cells modified with CD4ζ CAR from HIV-1 infection and alleviates impairment of cell proliferation

International Nuclear Information System (INIS)

Kamata, Masakazu; Kim, Patrick Y.; Ng, Hwee L.; Ringpis, Gene-Errol E.; Kranz, Emiko; Chan, Joshua; O'Connor, Sean; Yang, Otto O.; Chen, Irvin S.Y.

2015-01-01

Chimeric antigen receptors (CARs) are artificially engineered receptors that confer a desired specificity to immune effector T cells. As an HIV-1-specific CAR, CD4ζ CAR has been extensively tested in vitro as well as in clinical trials. T cells modified with this CAR mediated highly potent anti-HIV-1 activities in vitro and were well-tolerated in vivo, but exerted limited effects on viral load and reservoir size due to poor survival and/or functionality of the transduced cells in patients. We hypothesize that ectopic expression of CD4ζ on CD8 + T cells renders them susceptible to HIV-1 infection, resulting in poor survival of those cells. To test this possibility, highly purified CD8 + T cells were genetically modified with a CD4ζ-encoding lentiviral vector and infected with HIV-1. CD8 + T cells were vulnerable to HIV-1 infection upon expression of CD4ζ as evidenced by elevated levels of p24 Gag in cells and culture supernatants. Concurrently, the number of CD4ζ-modified CD8 + T cells was reduced relative to control cells upon HIV-1 infection. To protect these cells from HIV-1 infection, we co-expressed two anti-HIV-1 shRNAs previously developed by our group together with CD4ζ. This combination vector was able to suppress HIV-1 infection without impairing HIV-1-dependent effector activities of CD4ζ. In addition, the number of CD4ζ-modified CD8 + T cells maintained similar levels to that of the control even under HIV-1 infection. These results suggest that protecting CD4ζ-modified CD8 + T cells from HIV-1 infection is required for prolonged HIV-1-specific immune surveillance. - Highlights: • Ectopic expression of CD4ζ CAR in CD8 + T cells renders them susceptible to HIV-1 infection. • Co-expression of two anti-HIV-1 shRNAs protects CD4ζ CAR-modified CD8 + T cells from HIV-1 infection. • Protecting CD4ζ CAR-modified CD8 + T cells from HIV-1 infection suppresses its cytopathic effect

Inhibition of infection and transmission of HIV-1 and lack of significant impact on the vaginal commensal lactobacilli by carbohydrate-binding agents.

Science.gov (United States)

Petrova, Mariya I; Mathys, Leen; Lebeer, Sarah; Noppen, Sam; Van Damme, Els J M; Tanaka, Haruo; Igarashi, Yasuhiro; Vaneechoutte, Mario; Vanderleyden, Jos; Balzarini, Jan

2013-09-01

A selection of carbohydrate-binding agents (CBAs) with different glycan specificities were evaluated for their inhibitory effect against HIV infection and transmission, and their interaction with vaginal commensal bacteria. Several assays were used for the antiviral evaluation: (i) cell-free virus infection of human CD4+ T lymphocyte C8166 cells; (ii) syncytium formation in co-cultures of persistently HIV-1-infected HUT-78/HIV-1 and non-infected CD4+ SupT1 cells; (iii) DC-SIGN-directed capture of HIV-1 particles; and (iv) transmission of DC-SIGN-captured HIV-1 particles to uninfected CD4+ C8166 cells. CBAs were also examined for their interaction with vaginal commensal lactobacilli using several viability, proliferation and adhesion assays. The CBAs showed efficient inhibitory activity in the nanomolar to low-micromolar range against four events that play a crucial role in HIV-1 infection and transmission: cell-free virus infection, fusion between HIV-1-infected and non-infected cells, HIV-1 capture by DC-SIGN and transmission of DC-SIGN-captured virus to T cells. As candidate microbicides should not interfere with the normal human microbiota, we examined the effect of CBAs against Lactobacillus strains, including a variety of vaginal strains, a gastrointestinal strain and several non-human isolates. None of the CBAs included in our studies inhibited the growth of these bacteria in several media, affected their viability or had any significant impact on their adhesion to HeLa cell monolayers. The CBAs in this study were inhibitory to HIV-1 in several in vitro infection and transmission models, and may therefore qualify as potential microbicide candidates. The lack of significant impact on commensal vaginal lactobacilli is an important property of these CBAs in view of their potential microbicidal use.

Complement-Opsonized HIV-1 Alters Cross Talk Between Dendritic Cells and Natural Killer (NK Cells to Inhibit NK Killing and to Upregulate PD-1, CXCR3, and CCR4 on T Cells

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Rada Ellegård

2018-04-01

Full Text Available Dendritic cells (DCs, natural killer (NK cells, and T cells play critical roles during primary HIV-1 exposure at the mucosa, where the viral particles become coated with complement fragments and mucosa-associated antibodies. The microenvironment together with subsequent interactions between these cells and HIV at the mucosal site of infection will determine the quality of immune response that ensues adaptive activation. Here, we investigated how complement and immunoglobulin opsonization influences the responses triggered in DCs and NK cells, how this affects their cross talk, and what T cell phenotypes are induced to expand following the interaction. Our results showed that DCs exposed to complement-opsonized HIV (C-HIV were less mature and had a poor ability to trigger IFN-driven NK cell activation. In addition, when the DCs were exposed to C-HIV, the cytotolytic potentials of both NK cells and CD8 T cells were markedly suppressed. The expression of PD-1 as well as co-expression of negative immune checkpoints TIM-3 and LAG-3 on PD-1 positive cells were increased on both CD4 as well as CD8 T cells upon interaction with and priming by NK–DC cross talk cultures exposed to C-HIV. In addition, stimulation by NK–DC cross talk cultures exposed to C-HIV led to the upregulation of CD38, CXCR3, and CCR4 on T cells. Together, the immune modulation induced during the presence of complement on viral surfaces is likely to favor HIV establishment, dissemination, and viral pathogenesis.

Ectopic expression of anti-HIV-1 shRNAs protects CD8{sup +} T cells modified with CD4ζ CAR from HIV-1 infection and alleviates impairment of cell proliferation

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Kamata, Masakazu, E-mail: masa3k@ucla.edu [Division of Hematology-Oncology, David Geffen School of Medicine at UCLA, Los Angeles, CA (United States); Kim, Patrick Y. [Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA (United States); Ng, Hwee L. [Division of Infectious Diseases, David Geffen School of Medicine at UCLA, Los Angeles, CA (United States); Ringpis, Gene-Errol E.; Kranz, Emiko; Chan, Joshua; O' Connor, Sean [Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA (United States); Yang, Otto O. [Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA (United States); Division of Infectious Diseases, David Geffen School of Medicine at UCLA, Los Angeles, CA (United States); UCLA AIDS Institute, Los Angeles, CA (United States); AIDS Healthcare Foundation, Los Angeles, CA (United States); Chen, Irvin S.Y. [Division of Hematology-Oncology, David Geffen School of Medicine at UCLA, Los Angeles, CA (United States); Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA (United States); UCLA AIDS Institute, Los Angeles, CA (United States)

2015-07-31

Chimeric antigen receptors (CARs) are artificially engineered receptors that confer a desired specificity to immune effector T cells. As an HIV-1-specific CAR, CD4ζ CAR has been extensively tested in vitro as well as in clinical trials. T cells modified with this CAR mediated highly potent anti-HIV-1 activities in vitro and were well-tolerated in vivo, but exerted limited effects on viral load and reservoir size due to poor survival and/or functionality of the transduced cells in patients. We hypothesize that ectopic expression of CD4ζ on CD8{sup +} T cells renders them susceptible to HIV-1 infection, resulting in poor survival of those cells. To test this possibility, highly purified CD8{sup +} T cells were genetically modified with a CD4ζ-encoding lentiviral vector and infected with HIV-1. CD8{sup +} T cells were vulnerable to HIV-1 infection upon expression of CD4ζ as evidenced by elevated levels of p24{sup Gag} in cells and culture supernatants. Concurrently, the number of CD4ζ-modified CD8{sup +} T cells was reduced relative to control cells upon HIV-1 infection. To protect these cells from HIV-1 infection, we co-expressed two anti-HIV-1 shRNAs previously developed by our group together with CD4ζ. This combination vector was able to suppress HIV-1 infection without impairing HIV-1-dependent effector activities of CD4ζ. In addition, the number of CD4ζ-modified CD8{sup +} T cells maintained similar levels to that of the control even under HIV-1 infection. These results suggest that protecting CD4ζ-modified CD8{sup +} T cells from HIV-1 infection is required for prolonged HIV-1-specific immune surveillance. - Highlights: • Ectopic expression of CD4ζ CAR in CD8{sup +} T cells renders them susceptible to HIV-1 infection. • Co-expression of two anti-HIV-1 shRNAs protects CD4ζ CAR-modified CD8{sup +} T cells from HIV-1 infection. • Protecting CD4ζ CAR-modified CD8{sup +} T cells from HIV-1 infection suppresses its cytopathic effect.

The utility of the new generation of humanized mice to study HIV-1 infection: transmission, prevention, pathogenesis, and treatment

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Rowan Mark R

2011-08-01

Full Text Available Abstract Substantial improvements have been made in recent years in the ability to engraft human cells and tissues into immunodeficient mice. The use of human hematopoietic stem cells (HSCs leads to multi-lineage human hematopoiesis accompanied by production of a variety of human immune cell types. Population of murine primary and secondary lymphoid organs with human cells occurs, and long-term engraftment has been achieved. Engrafted cells are capable of producing human innate and adaptive immune responses, making these models the most physiologically relevant humanized animal models to date. New models have been successfully infected by a variety of strains of Human Immunodeficiency Virus Type 1 (HIV-1, accompanied by virus replication in lymphoid and non-lymphoid organs, including the gut-associated lymphoid tissue, the male and female reproductive tracts, and the brain. Multiple forms of virus-induced pathogenesis are present, and human T cell and antibody responses to HIV-1 are detected. These humanized mice are susceptible to a high rate of rectal and vaginal transmission of HIV-1 across an intact epithelium, indicating the potential to study vaccines and microbicides. Antiviral drugs, siRNAs, and hematopoietic stem cell gene therapy strategies have all been shown to be effective at reducing viral load and preventing or reversing helper T cell loss in humanized mice, indicating that they will serve as an important preclinical model to study new therapeutic modalities. HIV-1 has also been shown to evolve in response to selective pressures in humanized mice, thus showing that the model will be useful to study and/or predict viral evolution in response to drug or immune pressures. The purpose of this review is to summarize the findings reported to date on all new humanized mouse models (those transplanted with human HSCs in regards to HIV-1 sexual transmission, pathogenesis, anti-HIV-1 immune responses, viral evolution, pre- and post

The Oncolytic Virus MG1 Targets and Eliminates Cells Latently Infected With HIV-1: Implications for an HIV Cure.

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Ranganath, Nischal; Sandstrom, Teslin S; Burke Schinkel, Stephanie C; Côté, Sandra C; Angel, Jonathan B

2018-02-14

Cells latently infected with human immunodeficiency virus (HIV) evade immune- and drug-mediated clearance. These cells harbor intracellular signaling defects, including impairment of the antiviral type I interferon response. Such defects have also been observed in several cancers and have been exploited for the development of therapeutic oncolytic viruses, including the recombinant Maraba virus (MG1). We therefore hypothesized that MG1 would infect and eliminate cells latently infected with HIV-1, while sparing healthy uninfected cells. Preferential infection and elimination by MG1 was first demonstrated in cell lines latently infected with HIV-1. Following this, a reduction in HIV-1 DNA and inducible HIV-1 replication was observed following MG1 infection of latently infected, resting CD4+ T cells generated using an in vitro model of latency. Last, MG1 infection resulted in a reduction in HIV-1 DNA and inducible HIV-1 replication in memory CD4+ T cells isolated from effectively treated, HIV-1-infected individuals. Our results therefore highlight a novel approach to eliminate the latent HIV-1 reservoir. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

Regulatory CD4 T cells inhibit HIV-1 expression of other CD4 T cell subsets via interactions with cell surface regulatory proteins.

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Zhang, Mingce; Robinson, Tanya O; Duverger, Alexandra; Kutsch, Olaf; Heath, Sonya L; Cron, Randy Q

2018-03-01

During chronic HIV-1 infection, regulatory CD4 T cells (Tregs) frequently represent the largest subpopulation of CD4 T cell subsets, implying relative resistant to HIV-1. When HIV-1 infection of CD4 T cells was explored in vitro and ex vivo from patient samples, Tregs possessed lower levels of HIV-1 DNA and RNA in comparison with conventional effector and memory CD4 T cells. Moreover, Tregs suppressed HIV-1 expression in other CD4 T cells in an in vitro co-culture system. This suppression was mediated in part via multiple inhibitory surface proteins expressed on Tregs. Antibody blockade of CTLA-4, PD-1, and GARP on Tregs resulted in increased HIV-1 DNA integration and mRNA expression in neighboring CD4 T cells. Moreover, antibody blockade of Tregs inhibitory proteins resulted in increased HIV-1 LTR transcription in co-cultured CD4 T cells. Thus, Tregs inhibit HIV-1 infection of other CD4 T cell subsets via interactions with inhibitory cell surface proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

HIV-1 isolation from infected peripheral blood mononuclear cells.

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Dispinseri, Stefania; Saba, Elisa; Vicenzi, Elisa; Kootstra, Neeltje A; Schuitemaker, Hanneke; Scarlatti, Gabriella

2014-01-01

Human immunodeficiency virus 1 (HIV-1) isolation from peripheral blood mononuclear cells (PBMCs) allows retrieval of replication-competent viral variants. In order to impose the smallest possible selective pressure on the viral isolates, isolation must be carried out in primary cultures of cells and not in tumor derived cell lines. The procedure involves culture of PBMCs from an infected patient with phytohemagglutinin (PHA)-stimulated PBMC from seronegative donors, which provide susceptible target cells for HIV replication. HIV can be isolated from the bulk population of PBMCs or after cloning of the cells to obtain viral biological clones. Viral production is determined with p24 antigen (Ag) detection assays or with reverse transcriptase (RT) activity assay. Once isolated, HIV-1 can be propagated by infecting PHA-stimulated PBMCs from healthy donors. Aliquots from culture with a high production of virus are stored for later use.

HIV-1 and SIV Predominantly Use CCR5 Expressed on a Precursor Population to Establish Infection in T Follicular Helper Cells

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John Zaunders

2017-04-01

Full Text Available BackgroundT follicular helper (Tfh cells are increasingly recognized as a major reservoir of HIV infection that will likely need to be addressed in approaches to curing HIV. However, Tfh express minimal CCR5, the major coreceptor for HIV-1, and the mechanism by which they are infected is unclear. We have previously shown that macaque Tfh lack CCR5, but are infected in vivo with CCR5-using SIV at levels comparable to other memory CD4+ T cells. Similarly, human splenic Tfh cells are highly infected with HIV-1 DNA. Therefore, we set out to examine the mechanism of infection of Tfh cells.MethodologyTfh and other CD4+ T cell subsets from macaque lymph nodes and spleens, splenic Tfh from HIV+ subjects, and tonsillar Tfh from HIV-uninfected subjects were isolated by cell sorting prior to cell surface and molecular characterization. HIV proviral gp120 sequences were submitted to genotypic and phenotypic tropism assays. Entry of CCR5- and CXCR4-using viruses into Tfh from uninfected tonsillar tissue was measured using a fusion assay.ResultsPhylogenetic analysis, genotypic, and phenotypic analysis showed that splenic Tfh cells from chronic HIV+ subjects were predominantly infected with CCR5-using viruses. In macaques, purified CCR5+PD-1intermediate(int+ memory CD4+ T cells were shown to include pre-Tfh cells capable of differentiating in vitro to Tfh by upregulation of PD-1 and Bcl6, confirmed by qRT-PCR and single-cell multiplex PCR. Infected PD-1int cells survive, carry SIV provirus, and differentiate into PD-1hi Tfh after T cell receptor stimulation, suggesting a pathway for SIV infection of Tfh. In addition, a small subset of macaque and human PD-1hi Tfh can express low levels of CCR5, which makes them susceptible to infection. Fusion assays demonstrated CCR5-using HIV-1 entry into CCR5+ Tfh and pre-Tfh cells from human tonsils.ConclusionThe major route of infection of Tfh in macaques and humans appears to be via a CCR5-expressing pre-Tfh population

The involvement of plasmacytoid cells in HIV infection and pathogenesis.

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Aiello, Alessandra; Giannessi, Flavia; Percario, Zulema A; Affabris, Elisabetta

2018-04-01

Plasmacytoid dendritic cells (pDCs) are a unique dendritic cell subset that are specialized in type I interferon (IFN) production. pDCs are key players in the antiviral immune response and serve as bridge between innate and adaptive immunity. Although pDCs do not represent the main reservoir of the Human Immunodeficiency Virus (HIV), they are a crucial subset in HIV infection as they influence viral transmission, target cell infection and antigen presentation. pDCs act as inflammatory and immunosuppressive cells, thus contributing to HIV disease progression. This review provides a state of art analysis of the interactions between HIV and pDCs and their potential roles in HIV transmission, chronic immune activation and immunosuppression. A thorough understanding of the roles of pDCs in HIV infection will help to improve therapeutic strategies to fight HIV infection, and will further increase our knowledge on this important immune cell subset. Copyright © 2018 Elsevier Ltd. All rights reserved.

HIV-1 induces DCIR expression in CD4+ T cells.

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Alexandra A Lambert

2010-11-01

Full Text Available The C-type lectin receptor DCIR, which has been shown very recently to act as an attachment factor for HIV-1 in dendritic cells, is expressed predominantly on antigen-presenting cells. However, this concept was recently challenged by the discovery that DCIR can also be detected in CD4(+ T cells found in the synovial tissue from rheumatoid arthritis (RA patients. Given that RA and HIV-1 infections share common features such as a chronic inflammatory condition and polyclonal immune hyperactivation status, we hypothesized that HIV-1 could promote DCIR expression in CD4(+ T cells. We report here that HIV-1 drives DCIR expression in human primary CD4(+ T cells isolated from patients (from both aviremic/treated and viremic/treatment naive persons and cells acutely infected in vitro (seen in both virus-infected and uninfected cells. Soluble factors produced by virus-infected cells are responsible for the noticed DCIR up-regulation on uninfected cells. Infection studies with Vpr- or Nef-deleted viruses revealed that these two viral genes are not contributing to the mechanism of DCIR induction that is seen following acute infection of CD4(+ T cells with HIV-1. Moreover, we report that DCIR is linked to caspase-dependent (induced by a mitochondria-mediated generation of free radicals and -independent intrinsic apoptotic pathways (involving the death effector AIF. Finally, we demonstrate that the higher surface expression of DCIR in CD4(+ T cells is accompanied by an enhancement of virus attachment/entry, replication and transfer. This study shows for the first time that HIV-1 induces DCIR membrane expression in CD4(+ T cells, a process that might promote virus dissemination throughout the infected organism.

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

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Yasen, Aizezi; Herrera, Rossana; Rosbe, Kristina; Lien, Kathy; Tugizov, Sharof M.

2018-01-01

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. PMID:29277006

A Novel Toll-Like Receptor 9 Agonist, MGN1703, Enhances HIV-1 Transcription and NK Cell-Mediated Inhibition of HIV-1-Infected Autologous CD4+ T Cells.

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Offersen, Rasmus; Nissen, Sara Konstantin; Rasmussen, Thomas A; Østergaard, Lars; Denton, Paul W; Søgaard, Ole Schmeltz; Tolstrup, Martin

2016-05-01

Toll-like receptor (TLR) agonists are potent enhancers of innate antiviral immunity and may also reverse HIV-1 latency. Therefore, TLR agonists have a potential role in the context of a "shock-and-kill" approach to eradicate HIV-1. Our extensive preclinical evaluation suggests that a novel TLR9 agonist, MGN1703, may indeed perform both functions in an HIV-1 eradication trial. Peripheral blood mononuclear cells (PBMCs) from aviremic HIV-1-infected donors on antiretroviral therapy (ART) that were incubated with MGN1703 ex vivo exhibited increased secretion of interferon alpha (IFN-α) (P= 0.005) and CXCL10 (P= 0.0005) in culture supernatants. Within the incubated PBMC pool, there were higher proportions of CD69-positive CD56(dim)CD16(+)NK cells (P= 0.001) as well as higher proportions of CD107a-positive (P= 0.002) and IFN-γ-producing (P= 0.038) NK cells. Incubation with MGN1703 also increased the proportions of CD69-expressing CD4(+)and CD8(+)T cells. Furthermore, CD4(+)T cells within the pool of MGN1703-incubated PBMCs showed enhanced levels of unspliced HIV-1 RNA (P= 0.036). Importantly, MGN1703 increased the capacity of NK cells to inhibit virus spread within a culture of autologous CD4(+)T cells assessed by using an HIV-1 p24 enzyme-linked immunosorbent assay (ELISA) (P= 0.03). In conclusion, we show that MGN1703 induced strong antiviral innate immune responses, enhanced HIV-1 transcription, and boosted NK cell-mediated suppression of HIV-1 infection in autologous CD4(+)T cells. These findings support clinical testing of MGN1703 in HIV-1 eradication trials. We demonstrate that MGN1703 (a TLR9 agonist currently undergoing phase 3 clinical testing for the treatment of metastatic colorectal cancer) induces potent antiviral responses in immune effector cells from HIV-1-infected individuals on suppressive antiretroviral therapy. The significantly improved safety and tolerability profiles of MGN1703 versus TLR9 agonists of the CpG-oligodeoxynucleotide (CpG-ODN) family

Exogenous and endogenous hyaluronic acid reduces HIV infection of CD4+ T cells

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Li, Peilin; Fujimoto, Katsuya; Bourguingnon, Lilly; Yukl, Steven; Deeks, Steven; Wong, Joseph K

2014-01-01

Preventing mucosal transmission of HIV is critical to halting the HIV epidemic. Novel approaches to preventing mucosal transmission are needed. Hyaluronic acid (HA) is a major extracellular component of mucosa and the primary ligand for the cell surface receptor CD44. CD44 enhances HIV infection of CD4+ T cells, but the role of HA in this process is not clear. To study this, virions were generated with CD44 (HIVCD44) or without CD44 (HIVmock). Exogenous HA reduced HIV infection of unstimulated CD4+ T cells in a CD44-dependent manner. Conversely, hyaluronidase-mediated reduction of endogenous HA on the cell surface enhanced HIV binding to and infection of unstimulated CD4+ T cells. Exogenous HA treatment reduced activation of protein kinase C alpha via CD44 on CD4+ T cells during infection with HIVCD44. These results reveal new roles for HA during the interaction of HIV with CD4+ T cells that may be relevant to mucosal HIV transmission and could be exploitable as a future strategy to prevent HIV infection. PMID:24957217

HIV-1 transmission linkage in an HIV-1 prevention clinical trial

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Leitner, Thomas [Los Alamos National Laboratory; Campbell, Mary S [UNIV OF WASHINGTON; Mullins, James I [UNIV OF WASHINGTON; Hughes, James P [UNIV OF WASHINGTON; Wong, Kim G [UNIV OF WASHINGTON; Raugi, Dana N [UNIV OF WASHINGTON; Scrensen, Stefanie [UNIV OF WASHINGTON

2009-01-01

HIV-1 sequencing has been used extensively in epidemiologic and forensic studies to investigate patterns of HIV-1 transmission. However, the criteria for establishing genetic linkage between HIV-1 strains in HIV-1 prevention trials have not been formalized. The Partners in Prevention HSV/HIV Transmission Study (ClinicaITrials.gov NCT00194519) enrolled 3408 HIV-1 serodiscordant heterosexual African couples to determine the efficacy of genital herpes suppression with acyclovir in reducing HIV-1 transmission. The trial analysis required laboratory confirmation of HIV-1 linkage between enrolled partners in couples in which seroconversion occurred. Here we describe the process and results from HIV-1 sequencing studies used to perform transmission linkage determination in this clinical trial. Consensus Sanger sequencing of env (C2-V3-C3) and gag (p17-p24) genes was performed on plasma HIV-1 RNA from both partners within 3 months of seroconversion; env single molecule or pyrosequencing was also performed in some cases. For linkage, we required monophyletic clustering between HIV-1 sequences in the transmitting and seroconverting partners, and developed a Bayesian algorithm using genetic distances to evaluate the posterior probability of linkage of participants sequences. Adjudicators classified transmissions as linked, unlinked, or indeterminate. Among 151 seroconversion events, we found 108 (71.5%) linked, 40 (26.5%) unlinked, and 3 (2.0%) to have indeterminate transmissions. Nine (8.3%) were linked by consensus gag sequencing only and 8 (7.4%) required deep sequencing of env. In this first use of HIV-1 sequencing to establish endpoints in a large clinical trial, more than one-fourth of transmissions were unlinked to the enrolled partner, illustrating the relevance of these methods in the design of future HIV-1 prevention trials in serodiscordant couples. A hierarchy of sequencing techniques, analysis methods, and expert adjudication contributed to the linkage

Methamphetamine inhibits HIV-1 replication in CD4+ T cells by modulating anti-HIV-1 miRNA expression.

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Mantri, Chinmay K; Mantri, Jyoti V; Pandhare, Jui; Dash, Chandravanu

2014-01-01

Methamphetamine is the second most frequently used illicit drug in the United States. Methamphetamine abuse is associated with increased risk of HIV-1 acquisition, higher viral loads, and enhanced HIV-1 pathogenesis. Although a direct link between methamphetamine abuse and HIV-1 pathogenesis remains to be established in patients, methamphetamine has been shown to increase HIV-1 replication in macrophages, dendritic cells, and cells of HIV transgenic mice. Intriguingly, the effects of methamphetamine on HIV-1 replication in human CD4(+) T cells that serve as the primary targets of infection in vivo are not clearly understood. Therefore, we examined HIV-1 replication in primary CD4(+) T cells in the presence of methamphetamine in a dose-dependent manner. Our results demonstrate that methamphetamine had a minimal effect on HIV-1 replication at concentrations of 1 to 50 μmol/L. However, at concentrations >100 μmol/L, it inhibited HIV-1 replication in a dose-dependent manner. We also discovered that methamphetamine up-regulated the cellular anti-HIV-1 microRNAs (miR-125b, miR-150, and miR-28-5p) in CD4(+) T cells. Knockdown experiments illustrated that up-regulation of the anti-HIV miRNAs inhibited HIV-1 replication. These results are contrary to the paradigm that methamphetamine accentuates HIV-1 pathogenesis by increasing HIV-1 replication. Therefore, our findings underline the complex interaction between drug use and HIV-1 and necessitate comprehensive understanding of the effects of methamphetamine on HIV-1 pathogenesis. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

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

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Viraj Kulkarni

2017-11-01

Full Text Available 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.

Direct and dynamic detection of HIV-1 in living cells.

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Jonas Helma

Full Text Available In basic and applied HIV research, reliable detection of viral components is crucial to monitor progression of infection. While it is routine to detect structural viral proteins in vitro for diagnostic purposes, it previously remained impossible to directly and dynamically visualize HIV in living cells without genetic modification of the virus. Here, we describe a novel fluorescent biosensor to dynamically trace HIV-1 morphogenesis in living cells. We generated a camelid single domain antibody that specifically binds the HIV-1 capsid protein (CA at subnanomolar affinity and fused it to fluorescent proteins. The resulting fluorescent chromobody specifically recognizes the CA-harbouring HIV-1 Gag precursor protein in living cells and is applicable in various advanced light microscopy systems. Confocal live cell microscopy and super-resolution microscopy allowed detection and dynamic tracing of individual virion assemblies at the plasma membrane. The analysis of subcellular binding kinetics showed cytoplasmic antigen recognition and incorporation into virion assembly sites. Finally, we demonstrate the use of this new reporter in automated image analysis, providing a robust tool for cell-based HIV research.

Alterations in the nuclear proteome of HIV-1 infected T-cells

International Nuclear Information System (INIS)

DeBoer, Jason; Jagadish, Teena; Haverland, Nicole A.; Madson, Christian J.; Ciborowski, Pawel; Belshan, Michael

2014-01-01

Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified four clusters of protein–protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection. - Highlights: • We identify changes in the expression of nuclear proteins during HIV-1 infection. • 163 nuclear proteins were found differentially regulated during HIV-1 infection. • Bioinformatic analysis identified several nuclear pathways altered by HIV infection. • Candidate factors were validated in two independent cell lines

Alterations in the nuclear proteome of HIV-1 infected T-cells

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DeBoer, Jason [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Jagadish, Teena; Haverland, Nicole A. [Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198 (United States); Madson, Christian J. [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Ciborowski, Pawel [Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198 (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln 68583 (United States); Belshan, Michael, E-mail: michaelbelshan@creighton.edu [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln 68583 (United States)

2014-11-15

Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified four clusters of protein–protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection. - Highlights: • We identify changes in the expression of nuclear proteins during HIV-1 infection. • 163 nuclear proteins were found differentially regulated during HIV-1 infection. • Bioinformatic analysis identified several nuclear pathways altered by HIV infection. • Candidate factors were validated in two independent cell lines.

α4β7+ CD4+ Effector/Effector Memory T Cells Differentiate into Productively and Latently Infected Central Memory T Cells by Transforming Growth Factor β1 during HIV-1 Infection.

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Cheung, Ka-Wai; Wu, Tongjin; Ho, Sai Fan; Wong, Yik Chun; Liu, Li; Wang, Hui; Chen, Zhiwei

2018-04-15

HIV-1 transmission occurs mainly through mucosal tissues. During mucosal transmission, HIV-1 preferentially infects α 4 β 7 + gut-homing CCR7 - CD4 + effector/effector memory T cells (T EM ) and results in massive depletion of these cells and other subsets of T EM in gut-associated lymphoid tissues. However, besides being eliminated by HIV-1, the role of T EM during the early stage of infection remains inconclusive. Here, using in vitro -induced α 4 β 7 + gut-homing T EM (α 4 β 7 + T EM ), we found that α 4 β 7 + T EM differentiated into CCR7 + CD4 + central memory T cells (T CM ). This differentiation was HIV-1 independent but was inhibited by SB431542, a specific transforming growth factor β (TGF-β) receptor I kinase inhibitor. Consistently, T EM -to-T CM differentiation was observed in α 4 β 7 + T EM stimulated with TGF-β1 (TGF-β). The T CM properties of the TGF-β-induced T EM -derived T CM (α 4 β 7 + T CM ) were confirmed by their enhanced CCL19 chemotaxis and the downregulation of surface CCR7 upon T cell activation in vitro Importantly, the effect of TGF-β on T CM differentiation also held in T EM directly isolated from peripheral blood. To investigate the significance of the TGF-β-dependent T EM -to-T CM differentiation in HIV/AIDS pathogenesis, we observed that both productively and latently infected α 4 β 7 + T CM could differentiate from α 4 β 7 + T EM in the presence of TGF-β during HIV-1 infection. Collectively, this study not only provides a new insight for the plasticity of T EM but also suggests that the TGF-β-dependent T EM -to-T CM differentiation is a previously unrecognized mechanism for the formation of latently infected T CM after HIV-1 infection. IMPORTANCE HIV-1 is the causative agent of HIV/AIDS, which has led to millions of deaths in the past 30 years. Although the implementation of highly active antiretroviral therapy has remarkably reduced the HIV-1-related morbidity and mortality, HIV-1 is not eradicated in

A primary cell model of HIV-1 latency that uses activation through the T cell receptor and return to quiescence to establish latent infection

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Kim, Michelle; Hosmane, Nina N.; Bullen, C. Korin; Capoferri, Adam; Yang, Hung-Chih; Siliciano, Janet D.; Siliciano, Robert F.

2015-01-01

A mechanistic understanding of HIV-1 latency depends upon a model system that recapitulates the in vivo condition of latently infected, resting CD4+ T lymphocytes. Latency appears to be established after activated CD4+ T cells, the principal targets of HIV-1 infection, become productively infected and survive long enough to return to a resting memory state in which viral expression is inhibited by changes in the cellular environment. This protocol describes an ex vivo primary cell system that is generated under conditions that reflect the in vivo establishment of latency. Creation of these latency model cells takes 12 weeks and, once established, the cells can be maintained and used for several months. The resulting cell population contains both uninfected and latently infected cells. This primary cell model can be used to perform drug screens, study CTL responses to HIV-1, compare viral alleles, or to expand the ex vivo lifespan of cells from HIV-1 infected individuals for extended study. PMID:25375990

Properties of HTLV-I transformed CD8+ T-cells in response to HIV-1 infection.

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Gulzar, N; Shroff, A; Buberoglu, B; Klonowska, D; Kim, J E; Copeland, K F T

2010-10-25

HIV-1 infection studies of primary CD8(+) T-cells are hampered by difficulty in obtaining a significant number of targets for infection and low levels of productive infection. Further, there exists a paucity of CD8-expressing T-cell lines to address questions pertaining to the study of CD8(+) T-cells in the context of HIV-1 infection. In this study, a set of CD8(+) T-cell clones were originated through HTLV-I transformation in vitro, and the properties of these cells were examined. The clones were susceptible to T-cell tropic strains of the virus and exhibited HIV-1 production 20-fold greater than primary CD4(+) T-cells. Productive infection resulted in a decrease in expression of CD8 and CXCR4 molecules on the surface of the CD8(+) T-cell clones and antibodies to these molecules abrogated viral binding and replication. These transformed cells provide an important tool in the study of CD8(+) T-cells and may provide important insights into the mechanism(s) behind HIV-1 induced CD8(+) T-cell dysfunction. Copyright © 2010 Elsevier Inc. All rights reserved.

HIV gp120 binds to mannose receptor on vaginal epithelial cells and induces production of matrix metalloproteinases.

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Sashaina E Fanibunda

Full Text Available BACKGROUND: During sexual transmission of HIV in women, the virus breaches the multi-layered CD4 negative stratified squamous epithelial barrier of the vagina, to infect the sub-epithelial CD4 positive immune cells. However the mechanisms by which HIV gains entry into the sub-epithelial zone is hitherto unknown. We have previously reported human mannose receptor (hMR as a CD4 independent receptor playing a role in HIV transmission on human spermatozoa. The current study was undertaken to investigate the expression of hMR in vaginal epithelial cells, its HIV gp120 binding potential, affinity constants and the induction of matrix metalloproteinases (MMPs downstream of HIV gp120 binding to hMR. PRINCIPAL FINDINGS: Human vaginal epithelial cells and the immortalized vaginal epithelial cell line Vk2/E6E7 were used in this study. hMR mRNA and protein were expressed in vaginal epithelial cells and cell line, with a molecular weight of 155 kDa. HIV gp120 bound to vaginal proteins with high affinity, (Kd = 1.2±0.2 nM for vaginal cells, 1.4±0.2 nM for cell line and the hMR antagonist mannan dose dependently inhibited this binding. Both HIV gp120 binding and hMR exhibited identical patterns of localization in the epithelial cells by immunofluorescence. HIV gp120 bound to immunopurified hMR and affinity constants were 2.9±0.4 nM and 3.2±0.6 nM for vaginal cells and Vk2/E6E7 cell line respectively. HIV gp120 induced an increase in MMP-9 mRNA expression and activity by zymography, which could be inhibited by an anti-hMR antibody. CONCLUSION: hMR expressed by vaginal epithelial cells has high affinity for HIV gp120 and this binding induces production of MMPs. We propose that the induction of MMPs in response to HIV gp120 may lead to degradation of tight junction proteins and the extracellular matrix proteins in the vaginal epithelium and basement membrane, leading to weakening of the epithelial barrier; thereby facilitating transport of HIV across the

Brugia malayi Antigen (BmA Inhibits HIV-1 Trans-Infection but Neither BmA nor ES-62 Alter HIV-1 Infectivity of DC Induced CD4+ Th-Cells.

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Emily E I M Mouser

Full Text Available One of the hallmarks of HIV-1 disease is the association of heightened CD4+ T-cell activation with HIV-1 replication. Parasitic helminths including filarial nematodes have evolved numerous and complex mechanisms to skew, dampen and evade human immune responses suggesting that HIV-1 infection may be modulated in co-infected individuals. Here we studied the effects of two filarial nematode products, adult worm antigen from Brugia malayi (BmA and excretory-secretory product 62 (ES-62 from Acanthocheilonema viteae on HIV-1 infection in vitro. Neither BmA nor ES-62 influenced HIV-1 replication in CD4+ enriched T-cells, with either a CCR5- or CXCR4-using virus. BmA, but not ES-62, had the capacity to bind the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN thereby inhibiting HIV-1 trans-infection of CD4+ enriched T-cells. As for their effect on DCs, neither BmA nor ES-62 could enhance or inhibit DC maturation as determined by CD83, CD86 and HLA-DR expression, or the production of IL-6, IL-10, IL-12 and TNF-α. As expected, due to the unaltered DC phenotype, no differences were found in CD4+ T helper (Th cell phenotypes induced by DCs treated with either BmA or ES-62. Moreover, the HIV-1 susceptibility of the Th-cell populations induced by BmA or ES-62 exposed DCs was unaffected for both CCR5- and CXCR4-using HIV-1 viruses. In conclusion, although BmA has the potential capacity to interfere with HIV-1 transmission or initial viral dissemination through preventing the virus from interacting with DCs, no differences in the Th-cell polarizing capacity of DCs exposed to BmA or ES-62 were observed. Neither antigenic source demonstrated beneficial or detrimental effects on the HIV-1 susceptibility of CD4+ Th-cells induced by exposed DCs.

Role of Bruton's tyrosine kinase inhibitors in HIV-1-infected cells.

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Guendel, Irene; Iordanskiy, Sergey; Sampey, Gavin C; Van Duyne, Rachel; Calvert, Valerie; Petricoin, Emanuel; Saifuddin, Mohammed; Kehn-Hall, Kylene; Kashanchi, Fatah

2015-06-01

Many cellular cofactors have been documented to be critical for various stages of viral replication. Using high-throughput proteomic assays, we have previously identified Bruton's tyrosine kinase (BTK) as a host protein that was uniquely upregulated in the plasma membrane of human immunodeficiency virus (HIV-1)-infected T cells. Here, we have further characterized the BTK expression in HIV-1 infection and show that this cellular factor is specifically expressed in infected myeloid cells. Significant upregulation of the phosphorylated form of BTK was observed in infected cells. Using size exclusion chromatography, we found BTK to be virtually absent in the uninfected U937 cells; however, new BTK protein complexes were identified and distributed in both high molecular weight (∼600 kDa) and a small molecular weight complex (∼60-120 kDa) in the infected U1 cells. BTK levels were highest in cells either chronically expressing virus or induced/infected myeloid cells and that BTK translocated to the membrane following induction of the infected cells. BTK knockdown in HIV-1-infected cells using small interfering RNA (siRNA) resulted in selective death of infected, but not uninfected, cells. Using BTK-specific antibody and small-molecule inhibitors including LFM-A13 and a FDA-approved compound, ibrutinib (PCI-32765), we have found that HIV-1-infected cells are sensitive to apoptotic cell death and result in a decrease in virus production. Overall, our data suggests that HIV-1-infected cells are sensitive to treatments targeting BTK expressed in infected cells.

Langerhans Cells: the 'Yin and Yang' of HIV Restriction and Transmission.

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Mayr, Luzia; Su, Bin; Moog, Christiane

2017-03-01

Langerhans cells are specialized sentinels present in the epidermis expressing Langerin, a specific C-type lectin receptor involved in HIV capture and destruction. Recently, the specific mechanism leading to this HIV restriction was discovered. Nevertheless, Langerhans cells can be infected and the way HIV escapes this restriction needs to be unraveled. Copyright © 2017. Published by Elsevier Ltd.

The initial antibody response to HIV-1: induction of ineffective early B cell responses against GP41 by the transmitted/founder virus

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Chavez, Leslie L [Los Alamos National Laboratory; Perelson, Alan [Los Alamos National Laboratory

2008-01-01

A window of opportunity for immune responses to extinguish HIV -1 exists from the moment of transmission through establishment of the latent pool of HIV -I-infected cells. A critical time to study the initial immune responses to the transmitted/founder virus is the eclipse phase of HIV-1 infection (time from transmission to the first appearance of plasma virus) but, to date, this period has been logistically difficult to analyze. Studies in non-human primates challenged with chimeric simianhuman immunodeficiency virus have shown that neutralizing antibodies, when present at the time of infection, can prevent virus infection.

Transcriptional Reprogramming during Effector-to-Memory Transition Renders CD4+ T Cells Permissive for Latent HIV-1 Infection.

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Shan, Liang; Deng, Kai; Gao, Hongbo; Xing, Sifei; Capoferri, Adam A; Durand, Christine M; Rabi, S Alireza; Laird, Gregory M; Kim, Michelle; Hosmane, Nina N; Yang, Hung-Chih; Zhang, Hao; Margolick, Joseph B; Li, Linghua; Cai, Weiping; Ke, Ruian; Flavell, Richard A; Siliciano, Janet D; Siliciano, Robert F

2017-10-17

The latent reservoir for HIV-1 in resting memory CD4 + T cells is the major barrier to curing HIV-1 infection. Studies of HIV-1 latency have focused on regulation of viral gene expression in cells in which latent infection is established. However, it remains unclear how infection initially becomes latent. Here we described a unique set of properties of CD4 + T cells undergoing effector-to-memory transition including temporary upregulation of CCR5 expression and rapid downregulation of cellular gene transcription. These cells allowed completion of steps in the HIV-1 life cycle through integration but suppressed HIV-1 gene transcription, thus allowing the establishment of latency. CD4 + T cells in this stage were substantially more permissive for HIV-1 latent infection than other CD4 + T cells. Establishment of latent HIV-1 infection in CD4 + T could be inhibited by viral-specific CD8 + T cells, a result with implications for elimination of latent HIV-1 infection by T cell-based vaccines. Copyright © 2017. Published by Elsevier Inc.

T Follicular Helper Cells and B Cell Dysfunction in Aging and HIV-1 Infection.

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Pallikkuth, Suresh; de Armas, Lesley; Rinaldi, Stefano; Pahwa, Savita

2017-01-01

T follicular helper (Tfh) cells are a subset of CD4 T cells that provide critical signals to antigen-primed B cells in germinal centers to undergo proliferation, isotype switching, and somatic hypermutation to generate long-lived plasma cells and memory B cells during an immune response. The quantity and quality of Tfh cells therefore must be tightly controlled to prevent immune dysfunction in the form of autoimmunity and, on the other hand, immune deficiency. Both Tfh and B cell perturbations appear during HIV infection resulting in impaired antibody responses to vaccines such as seasonal trivalent influenza vaccine, also seen in biologic aging. Although many of the HIV-associated defects improve with antiretroviral therapy (ART), excess immune activation and antigen-specific B and T cell responses including Tfh function are still impaired in virologically controlled HIV-infected persons on ART. Interestingly, HIV infected individuals experience increased risk of age-associated pathologies. This review will discuss Tfh and B cell dysfunction in HIV infection and highlight the impact of chronic HIV infection and aging on Tfh-B cell interactions.

Cell-to-Cell Contact Results in a Selective Translocation of Maternal Human Immunodeficiency Virus Type 1 Quasispecies across a Trophoblastic Barrier by both Transcytosis and Infection

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Lagaye, S.; Derrien, M.; Menu, E.; Coïto, C.; Tresoldi, E.; Mauclère, P.; Scarlatti, G.; Chaouat, G.; Barré-Sinoussi, F.; Bomsel, M.

2001-01-01

Mother-to-child transmission can occur in utero, mainly intrapartum and postpartum in case of breastfeeding. In utero transmission is highly restricted and results in selection of viral variant from the mother to the child. We have developed an in vitro system that mimics the interaction between viruses, infected cells present in maternal blood, and the trophoblast, the first barrier protecting the fetus. Trophoblastic BeWo cells were grown as a tight polarized monolayer in a two-chamber system. Cell-free virions applied to the apical pole neither crossed the barrier nor productively infected BeWo cells. In contrast, apical contact with human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells (PBMCs) resulted in transcytosis of infectious virus across the trophoblastic monolayer and in productive infection correlating with the fusion of HIV-infected PBMCs with trophoblasts. We showed that viral variants are selected during these two steps and that in one case of in utero transmission, the predominant maternal viral variant characterized after transcytosis was phylogenetically indistinguishable from the predominant child's virus. Hence, the first steps of transmission of HIV-1 in utero appear to involve the interaction between HIV type 1-infected cells and the trophoblastic layer, resulting in the passage of infectious HIV by transcytosis and by fusion/infection, both leading to a selection of virus quasispecies. PMID:11312350

Antibodyomics: bioinformatics technologies for understanding B-cell immunity to HIV-1.

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Kwong, Peter D; Chuang, Gwo-Yu; DeKosky, Brandon J; Gindin, Tatyana; Georgiev, Ivelin S; Lemmin, Thomas; Schramm, Chaim A; Sheng, Zizhang; Soto, Cinque; Yang, An-Suei; Mascola, John R; Shapiro, Lawrence

2017-01-01

Numerous antibodies have been identified from HIV-1-infected donors that neutralize diverse strains of HIV-1. These antibodies may provide the basis for a B cell-mediated HIV-1 vaccine. However, it has been unclear how to elicit similar antibodies by vaccination. To address this issue, we have undertaken an informatics-based approach to understand the genetic and immunologic processes controlling the development of HIV-1-neutralizing antibodies. As DNA sequencing comprises the fastest growing database of biological information, we focused on incorporating next-generation sequencing of B-cell transcripts to determine the origin, maturation pathway, and prevalence of broadly neutralizing antibody lineages (Antibodyomics1, 2, 4, and 6). We also incorporated large-scale robotic analyses of serum neutralization to identify and quantify neutralizing antibodies in donor cohorts (Antibodyomics3). Statistical analyses furnish another layer of insight (Antibodyomics5), with physical characteristics of antibodies and their targets through molecular dynamics simulations (Antibodyomics7) and free energy perturbation analyses (Antibodyomics8) providing information-rich output. Functional interrogation of individual antibodies (Antibodyomics9) and synthetic antibody libraries (Antibodyomics10) also yields multi-dimensional data by which to understand and improve antibodies. Antibodyomics, described here, thus comprise resolution-enhancing tools, which collectively embody an information-driven discovery engine aimed toward the development of effective B cell-based vaccines. © 2017 The Authors. Immunological Reviews published by John Wiley & Sons Ltd.

Gene expression profiling of the host response to HIV-1 B, C, or A/E infection in monocyte-derived dendritic cells

International Nuclear Information System (INIS)

Solis, Mayra; Wilkinson, Peter; Romieu, Raphaelle; Hernandez, Eduardo; Wainberg, Mark A.; Hiscott, John

2006-01-01

Dendritic cells (DC) are among the first targets of human immunodeficiency virus type-1 (HIV-1) infection and in turn play a crucial role in viral transmission to T cells and in the regulation of the immune response. The major group of HIV-1 has diversified genetically based on variation in env sequences and comprise at least 11 subtypes. Because little is known about the host response elicited against different HIV-1 clade isolates in vivo, we sought to use gene expression profiling to identify genes regulated by HIV-1 subtypes B, C, and A/E upon de novo infection of primary immature monocyte-derived DC (iMDDCs). A total of 3700 immune-related genes were subjected to a significance analysis of microarrays (SAM); 656 genes were selected as significant and were further divided into 8 functional categories. Regardless of the time of infection, 20% of the genes affected by HIV-1 were involved in signal transduction, followed by 14% of the genes identified as transcription-related genes, and 7% were classified as playing a role in cell proliferation and cell cycle. Furthermore, 7% of the genes were immune response genes. By 72 h postinfection, genes upregulated by subtype B included the inhibitor of the matrix metalloproteinase TIMP2 and the heat shock protein 40 homolog (Hsp40) DNAJB1, whereas the IFN inducible gene STAT1, the MAPK1/ERK2 kinase regulator ST5, and the chemokine CXCL3 and SHC1 genes were induced by subtypes C and A/E. These analyses distinguish a temporally regulated host response to de novo HIV-1 infection in primary dendritic cells

Proteasome-independent degradation of HIV-1 in naturally non-permissive human placental trophoblast cells

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Barré-Sinoussi Françoise

2009-05-01

Full Text Available Abstract Background The human placenta-derived cell line BeWo has been demonstrated to be restrictive to cell-free HIV-1 infection. BeWo cells are however permissive to infection by VSV-G pseudotyped HIV-1, which enters cells by a receptor-independent mechanism, and to infection by HIV-1 via a cell-to-cell route. Results Here we analysed viral entry in wild type BeWo (CCR5+, CXCR4+ and BeWo-CD4+ (CD4+, CCR5+, CXCR4+ cells. We report that HIV-1 internalisation is not restricted in either cell line. Levels of internalised p24 antigen between VSV-G HIV-1 pseudotypes and R5 or X4 virions were comparable. We next analysed the fate of internalised virions; X4 and R5 HIV-1 virions were less stable over time in BeWo cells than VSV-G HIV-1 pseudotypes. We then investigated the role of the proteasome in restricting cell-free HIV-1 infection in BeWo cells using proteasome inhibitors. We observed an increase in the levels of VSV-G pseudotyped HIV-1 infection in proteasome-inhibitor treated cells, but the infection by R5-Env or X4-Env pseudotyped virions remains restricted. Conclusion Collectively these results suggest that cell-free HIV-1 infection encounters a surface block leading to a non-productive entry route, which either actively targets incoming virions for non-proteasomal degradation, and impedes their release into the cytoplasm, or causes the inactivation of mechanisms essential for viral replication.

Prevention of mother-to-child transmission of HIV-1 through breastfeeding by treating mothers with triple antiretroviral therapy in Dar es Salaam, Tanzania: the Mitra Plus study.

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Kilewo, Charles; Karlsson, Katarina; Ngarina, Matilda; Massawe, Augustine; Lyamuya, Eligius; Swai, Andrew; Lipyoga, Rosina; Mhalu, Fred; Biberfeld, Gunnel

2009-11-01

The main aim of this study was to reduce breast-milk transmission of HIV-1 by treating HIV-1-infected women with highly active antiretroviral therapy (HAART) during breastfeeding. Mitra Plus was an open-label, nonrandomized, prospective cohort study. HIV-1-infected pregnant women in Dar es Salaam were treated with zidovudine (ZDV) + lamivudine (3TC) + nevirapine (NVP). NVP was later replaced by nelfinavir for mothers with CD4 cell counts >200 cells per microliter or with adverse reaction to NVP. HAART was initiated at 34 weeks of gestation. For women with symptomatic HIV infection or CD4 cell counts below 200 cells per microliter, HAART was started earlier if possible. Treatment of the mothers was stopped at 6 months except for those mothers who needed HAART for their own health. The infants received ZDV + 3TC for 1 week after birth. Mothers were advised to exclusively breastfeed and to wean abruptly between 5 and 6 months. Transmission of HIV-1 was analyzed using the Kaplan-Meier survival technique. Cox regression was used for comparison with the breastfeeding population of the Petra trial arm A. There were 441 infants included in the analysis of HIV-1 transmission. The cumulative transmission of HIV-1 was 4.1 % [95% confidence interval (CI): 2.2 to 6.0] at 6 weeks, 5.0% (95% CI: 2.9 to 7.1) at 6 months, and 6.0% (95% CI: 3.7 to 8.3) at 18 months after delivery. The cumulative risk of HIV transmission between 6 weeks and 6 months was 1.0% and between 6 months and 18 months 1.1%. The cumulative HIV infection or death rate was 8.6% (95% CI: 6.0 to 11.2) at 6 months and 13.6% (95% CI: 10.3 to 16.9) at 18 months after delivery. Viral load at enrollment and duration of HAART before delivery were significantly associated with transmission but CD4 cell count at enrollment was not. The median time of breastfeeding was 24 weeks. The transmission in the Mitra Plus study was about half of the transmission in the breastfeeding population in the Petra trial arm A at 6 months

Factors associated with mother-to-child transmission of HIV-1 despite a maternal viral load EPF-ANRS CO1).

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Tubiana, Roland; Le Chenadec, Jerome; Rouzioux, Christine; Mandelbrot, Laurent; Hamrene, Karima; Dollfus, Catherine; Faye, Albert; Delaugerre, Constance; Blanche, Stephane; Warszawski, Josiane

2010-02-15

The rate of mother-to-child transmission (MTCT) of human immunodeficiency virus (HIV) type 1 is as low as 0.5% in non-breast-feeding mothers who delivered at term while receiving antiretroviral therapy with a plasma viral load <500 copies/mL. This situation accounted for 20% of the infected children born during the period 1997-2006 in the French Perinatal Cohort. We aimed to identify factors associated with such residual transmission risk. We performed a case-control study nested in the aforementioned subpopulation of the French Perinatal Cohort. Nineteen case patients (transmitters) and 60 control subjects (nontransmitters) were included. Case patients and control subjects did not differ by geographical origin, gestational age at HIV diagnosis, type of antiretroviral therapy received, or elective Cesarean delivery. Case patients were less often receiving treatment at the time that they conceived pregnancy than control subjects (16% vs 45%; P=.017). A lower proportion of case patients had a viral load <500 copies/mL, compared with control subjects, at 14 weeks (0% vs 38.1%; P=.02), 28 weeks (7.7% vs 62.1%; P=.005), and 32 weeks: (21.4% vs 71.1%; P=.004). The difference remained significant when we restricted analysis to the 10 of 16 intrapartum transmission cases. In a multivariate analysis at 30+/-4 weeks adjusted for viral load, CD4(+) T cell count, and time at antiretroviral therapy initiation, viral load was the only factor independently associated with MTCT of HIV (adjusted odds ratio, 23.2; 95% confidence interval, 3.5-553; P<.001). Early and sustained control of viral load is associated with a decreasing residual risk of MTCT of HIV-1. Guidelines should take into account not only CD4(+) T cell count and risk of preterm delivery, but also baseline HIV-1 load for deciding when to start antiretroviral therapy during pregnancy.

HIV-1 Resistant CDK2-Knockdown Macrophage-Like Cells Generated from 293T Cell-Derived Human Induced Pluripotent Stem Cells

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Kuan-Teh Jeang

2012-07-01

Full Text Available A major challenge in studies of human diseases involving macrophages is low yield and heterogeneity of the primary cells and limited ability of these cells for transfections and genetic manipulations. To address this issue, we developed a simple and efficient three steps method for somatic 293T cells reprogramming into monocytes and macrophage-like cells. First, 293T cells were reprogrammed into induced pluripotent stem cells (iPSCs through a transfection-mediated expression of two factors, Oct-4 and Sox2, resulting in a high yield of iPSC. Second, the obtained iPSC were differentiated into monocytes using IL-3 and M-CSF treatment. And third, monocytes were differentiated into macrophage-like cells in the presence of M-CSF. As an example, we developed HIV-1-resistant macrophage-like cells from 293T cells with knockdown of CDK2, a factor critical for HIV-1 transcription. Our study provides a proof-of-principle approach that can be used to study the role of host cell factors in HIV-1 infection of human macrophages.

TFH cells accumulate in mucosal tissues of humanized-DRAG mice and are highly permissive to HIV-1

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Allam, Atef; Majji, Sai; Peachman, Kristina; Jagodzinski, Linda; Kim, Jiae; Ratto-Kim, Silvia; Wijayalath, Wathsala; Merbah, Melanie; Kim, Jerome H.; Michael, Nelson L.; Alving, Carl R.; Casares, Sofia; Rao, Mangala

2015-01-01

CD4+ T follicular helper cells (TFH) in germinal centers are required for maturation of B-cells. While the role of TFH-cells has been studied in blood and lymph nodes of HIV-1 infected individuals, its role in the mucosal tissues has not been investigated. We show that the gut and female reproductive tract (FRT) of humanized DRAG mice have a high level of human lymphocytes and a high frequency of TFH (CXCR5+PD-1++) and precursor-TFH (CXCR5+PD-1+) cells. The majority of TFH-cells expressed CCR5 and CXCR3 and are the most permissive to HIV-1 infection. A single low-dose intravaginal HIV-1 challenge of humanized DRAG mice results in 100% infectivity with accumulation of TFH-cells mainly in the Peyer’s patches and FRT. The novel finding of TFH-cells in the FRT may contribute to the high susceptibility of DRAG mice to HIV-1 infection. This mouse model thus provides new opportunities to study TFH-cells and to evaluate HIV-1 vaccines. PMID:26034905

Mother-to-child transmission of HIV-2 infection from 1986 to 2007 in the ANRS French Perinatal Cohort EPF-CO1.

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Burgard, M; Jasseron, C; Matheron, S; Damond, F; Hamrene, K; Blanche, S; Faye, A; Rouzioux, C; Warszawski, J; Mandelbro, L

2010-10-01

Management of pregnant women with human immunodeficiency virus (HIV) type 2 infection remains unclear because of its low prevalence and important differences from HIV-1. Pregnant women monoinfected with HIV-2 or HIV-1 and their infants enrolled in the prospective, national, multicenter French Perinatal Cohort between 1986 and 2007. Overall, 2.6% (223/8660) of mothers were infected with HIV-2, and they accounted for 3.1% (367/ 11841) of the total births. Most were born in sub-Saharan Africa. A higher proportion of HIV-2-infected mothers than HIV-1-infected mothers had no symptoms, had received no antiretroviral therapy at conception (85.9% vs 66.7%), and had received no antiretroviral therapy during pregnancy (42.8% vs 19.9%), particularly highly active antiretroviral therapy (HAART) (79.7% vs 46.1%), and they had higher CD4 cell counts near delivery (median, 574 vs 452 cells/mm3; P < .01). If antiretroviral therapy was used, it was started at a later gestational age for HIV- 2-infected mothers (median, 28 vs 25 weeks; P < .01). HIV-2-infected mothers were more likely to deliver vaginally (67.9% vs 49.3%) and to breastfeed (3.6% vs 0.6%; P < .01), and their infants less frequently received postexposure prophylaxis. In the period 2000-2007, the proportion with viral load <100 copies/mL at delivery was 90.5% of HIV-2-infected mothers, compared with 76.2% of HIV-1-infected mothers (P=.1). There were 2 cases of transmission: 1 case in 1993 occurred following maternal primary infection, and the other case occurred postnatally in 2002 and involved a mother with severe immune deficiency. The mother-to-child transmission rate for HIV-2 was 0.6% (95% confidence interval, 0.07%-2.2%). Care for HIV-2-infected pregnant women rests on expert opinion. The mother-to-child transmission residual rate (0.07%-2.2%) argues for systematic treatment: protease inhibitor-based HAART for women requiring antiretrov

IL-15 STIMULATED NATURAL KILLER CELLS CLEAR HIV-1 INFECTED CELLS FOLLOWING LATENCY REVERSAL EX VIVO.

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Garrido, Carolina; Abad-Fernandez, Maria; Tuyishime, Marina; Pollara, Justin J; Ferrari, Guido; Soriano-Sarabia, Natalia; Margolis, David M

2018-03-28

Current efforts towards HIV eradication include approaches to augment immune recognition and elimination of persistently infected cells following latency reversal. Natural killer (NK) cells, the main effectors of the innate immune system, recognize and clear targets using different mechanisms than CD8 + T cells, offering an alternative or complementary approach for HIV clearance strategies. We assessed the impact of IL-15 treatment on NK cell function and the potential of stimulated NK cells to clear the HIV reservoir. We measured NK cell receptor expression, antibody-dependent cell-dependent cytotoxicity (ADCC), cytotoxicity, IFN-γ production and antiviral activity in autologous HIV replication systems. All NK cell functions were uniformly improved by IL-15, and more importantly, IL-15-treated NK cells were able to clear latently HIV infected cells after exposure to vorinostat, a clinically relevant latency reversing agent. We also demonstrate that NK cells from HIV infected individuals aviremic on antiretroviral therapy can be efficiently stimulated with IL-15. Our work opens a promising line of investigation towards future immunotherapies to clear persistent HIV infection using NK cells. IMPORTANCE In the search for an HIV cure, strategies to enhance immune function to allow recognition and clearance of HIV infected cells following latency reversal are being evaluated. Natural killer (NK) cells possess characteristics that can be exploited for immunotherapy against persistent HIV infection. We demonstrate that NK cells from HIV-positive donors can be strongly stimulated with IL-15, improving their antiviral and cytotoxic potential, and more importantly, clearing HIV infected cells after latency reversal with a clinically relevant drug. Our results encourage further investigation to design NK cell-based immunotherapies to achieve HIV eradication. Copyright © 2018 American Society for Microbiology.

The lectins griffithsin, cyanovirin-N and scytovirin inhibit HIV-1 binding to the DC-SIGN receptor and transfer to CD4+ cells

CSIR Research Space (South Africa)

Alexandre, Kabamba B

2012-02-01

Full Text Available It is generally believed that during the sexual transmission of HIV-1, the glycan-specific DC-SIGN receptor binds the virus and mediates its transfer to CD4(+) cells. The lectins griffithsin (GRFT), cyanovirin-N (CV-N) and scytovirin (SVN) inhibit...

The Natural Killer Cell Cytotoxic Function Is Modulated by HIV-1 Accessory Proteins

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Edward Barker

2011-07-01

Full Text Available Natural killer (NK cells’ major role in the control of viruses is to eliminate established infected cells. The capacity of NK cells to kill virus-infected cells is dependent on the interactions between ligands on the infected cell and receptors on the NK cell surface. Because of the importance of ligand-receptor interactions in modulating the NK cell cytotoxic response, HIV has developed strategies to regulate various NK cell ligands making the infected cell surprisingly refractory to NK cell lysis. This is perplexing because the HIV-1 accessory protein Vpr induces expression of ligands for the NK cell activating receptor, NKG2D. In addition, the accessory protein Nef removes the inhibitory ligands HLA-A and -B. The reason for the ineffective killing by NK cells despite the strong potential to eliminate infected cells is due to HIV-1 Vpu’s ability to down modulate the co-activation ligand, NTB-A, from the cell surface. Down modulation of NTB-A prevents efficient NK cell degranulation. This review will focus on the mechanisms through which the HIV-1 accessory proteins modulate their respective ligands, and its implication for NK cell killing of HIV-infected cells.

Distinct susceptibility of HIV vaccine vector-induced CD4 T cells to HIV infection

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Niu, Qingli; Hou, Wei; Churchyard, Gavin; Nitayaphan, Sorachai; Pitisuthithum, Punnee; Rerks-Ngarm, Supachai; Franchini, Genoveffa

2018-01-01

The concerns raised from adenovirus 5 (Ad5)-based HIV vaccine clinical trials, where excess HIV infections were observed in some vaccine recipients, have highlighted the importance of understanding host responses to vaccine vectors and the HIV susceptibility of vector-specific CD4 T cells in HIV vaccination. Our recent study reported that human Ad5-specific CD4 T cells induced by Ad5 vaccination (RV156A trial) are susceptible to HIV. Here we further investigated the HIV susceptibility of vector-specific CD4 T cells induced by ALVAC, a canarypox viral vector tested in the Thai trial RV144, as compared to Ad5 vector-specific CD4 T cells in the HVTN204 trial. We showed that while Ad5 vector-specific CD4 T cells were readily susceptible to HIV, ALVAC-specific CD4 T cells in RV144 PBMC were substantially less susceptible to both R5 and X4 HIV in vitro. The lower HIV susceptibility of ALVAC-specific CD4 T cells was associated with the reduced surface expression of HIV entry co-receptors CCR5 and CXCR4 on these cells. Phenotypic analyses identified that ALVAC-specific CD4 T cells displayed a strong Th1 phenotype, producing higher levels of IFN-γ and CCL4 (MIP-1β) but little IL-17. Of interest, ALVAC and Ad5 vectors induced distinct profiles of vector-specific CD8 vs. CD4 T-cell proliferative responses in PBMC, with ALVAC preferentially inducing CD8 T-cell proliferation, while Ad5 vector induced CD4 T-cell proliferation. Depletion of ALVAC-, but not Ad5-, induced CD8 T cells in PBMC led to a modest increase in HIV infection of vector-specific CD4 T cells, suggesting a role of ALVAC-specific CD8 T cells in protecting ALVAC-specific CD4 T cells from HIV. Taken together, our data provide strong evidence for distinct HIV susceptibility of CD4 T cells induced by different vaccine vectors and highlight the importance of better evaluating anti-vector responses in HIV vaccination. PMID:29474461

Role of Bruton’s Tyrosine Kinase inhibitors in HIV-1 infected cells

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Guendel, Irene; Iordanskiy, Sergey; Sampey, Gavin C; Van Duyne, Rachel; Calvert, Valerie; Petricoin, Emanuel; Saifuddin, Mohammed; Kehn-Hall, Kylene; Kashanchi, Fatah

2015-01-01

Many cellular cofactors have been documented to be critical for various stages of viral replication. Using high throughput proteomic assays, we have previously identified Bruton’s tyrosine kinase (BTK) as a host protein that was uniquely up-regulated in the plasma membrane of HIV-1 infected T-cells. Here, we have further characterized the BTK expression in HIV-1 infection and show that this cellular factor is specifically expressed in infected myeloid cells. Significant up-regulation of the phosphorylated form of BTK was observed in infected cells. Using size exclusion chromatography, we found BTK to be virtually absent in the uninfected U937 cells, however new BTK protein complexes were identified and distributed in both high molecular weight (~600 kDa) and a small molecular weight complex (~60–120 kDa) in the infected U1 cells. BTK levels were highest in cells either chronically expressing virus or induced/infected myeloid cells and that BTK translocated to the membrane following induction of the infected cells. BTK knockdown in HIV-1 infected cells using siRNA resulted in selective death of infected, but not uninfected, cells. Using BTK specific antibody and small molecule inhibitors including LFM-A13 and a FDA approved compound, Ibrutinib (PCI – 32765), we have found that HIV-1 infected cells are sensitive to apoptotic cell death and result in a decrease in virus production. Overall, our data suggests that HIV-1 infected cells are sensitive to treatments targeting BTK expressed in infected cells. PMID:25672887

Artificial 64-Residue HIV-1 Enhancer-Binding Peptide Is a Potent Inhibitor of Viral Replication in HIV-1-Infected Cells.

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Oufir, Mouhssin; Bisset, Leslie R; Hoffmann, Stefan R K; Xue, Gongda; Klauser, Stephan; Bergamaschi, Bianca; Gervaix, Alain; Böni, Jürg; Schüpbach, Jörg; Gutte, Bernd

2011-01-01

An artificial HIV-1 enhancer-binding peptide was extended by nine consecutive arginine residues at the C-terminus and by the nuclear localization signal of SV40 large T antigen at the N-terminus. The resulting synthetic 64-residue peptide was found to bind to the two enhancers of the HIV-1 long terminal repeat, cross the plasma membrane and the nuclear envelope of human cells, and suppress the HIV-1 enhancer-controlled expression of a green fluorescent protein reporter gene. Moreover, HIV-1 replication is inhibited by this peptide in HIV-1-infected CEM-GFP cells as revealed by HIV-1 p24 ELISA and real-time RT-PCR of HIV-1 RNA. Rapid uptake of this intracellular stable and inhibitory peptide into the cells implies that this peptide may have the potential to attenuate HIV-1 replication in vivo.

Artificial 64-Residue HIV-1 Enhancer-Binding Peptide Is a Potent Inhibitor of Viral Replication in HIV-1-Infected Cells

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Mouhssin Oufir

2011-01-01

Full Text Available An artificial HIV-1 enhancer-binding peptide was extended by nine consecutive arginine residues at the C-terminus and by the nuclear localization signal of SV40 large T antigen at the N-terminus. The resulting synthetic 64-residue peptide was found to bind to the two enhancers of the HIV-1 long terminal repeat, cross the plasma membrane and the nuclear envelope of human cells, and suppress the HIV-1 enhancer-controlled expression of a green fluorescent protein reporter gene. Moreover, HIV-1 replication is inhibited by this peptide in HIV-1-infected CEM-GFP cells as revealed by HIV-1 p24 ELISA and real-time RT-PCR of HIV-1 RNA. Rapid uptake of this intracellular stable and inhibitory peptide into the cells implies that this peptide may have the potential to attenuate HIV-1 replication in vivo.

Prevention of mother-to-child transmission of HIV guidelines: Nurses ...

African Journals Online (AJOL)

Prevention of mother-to-child transmission of HIV guidelines: Nurses' views at four primary ... lifelong antiretroviral therapy (ART) for all HIV-positive pregnant women regardless of CD4 cell count. ... Data were analysed using thematic analysis.

Cocaine modulates HIV-1 integration in primary CD4+ T cells: implications in HIV-1 pathogenesis in drug-abusing patients

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Addai, Amma B.; Pandhare, Jui; Paromov, Victor; Mantri, Chinmay K.; Pratap, Siddharth; Dash, Chandravanu

2015-01-01

Epidemiologic studies suggest that cocaine abuse worsens HIV-1 disease progression. Increased viral load has been suggested to play a key role for the accelerated HIV disease among cocaine-abusing patients. The goal of this study was to investigate whether cocaine enhances proviral DNA integration as a mechanism to increase viral load. We infected CD4+ T cells that are the primary targets of HIV-1 in vivo and treated the cells with physiologically relevant concentrations of cocaine (1 µM–100 µM). Proviral DNA integration in the host genome was measured by nested qPCR. Our results illustrated that cocaine from 1 µM through 50 µM increased HIV-1 integration in CD4+ T cells in a dose-dependent manner. As integration can be modulated by several early postentry steps of HIV-1 infection, we examined the direct effects of cocaine on viral integration by in vitro integration assays by use of HIV-1 PICs. Our data illustrated that cocaine directly increases viral DNA integration. Furthermore, our MS analysis showed that cocaine is able to enter CD4+ T cells and localize to the nucleus-. In summary, our data provide strong evidence that cocaine can increase HIV-1 integration in CD4+ T cells. Therefore, we hypothesize that increased HIV-1 integration is a novel mechanism by which cocaine enhances viral load and worsens disease progression in drug-abusing HIV-1 patients. PMID:25691383

HIV-1 Nef control of cell signalling molecules: multiple strategies to ...

Indian Academy of Sciences (India)

Unknown

The Nef protein of HIV-1 plays a fundamental role in the virus life cycle. ... to modulate the expression of key cellular receptors important for cell ... of the Src family kinases, leading to an effect on host cell function is likely to ..... Bad, Nef serves to balance the apoptosis inducing effects ..... ties of vpu, env, and nef; J. Virol.

Cell death by pyroptosis drives CD4 T-cell depletion in HIV-1 infection

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Doitsh, Gilad; Galloway, Nicole L. K.; Geng, Xin; Yang, Zhiyuan; Monroe, Kathryn M.; Zepeda, Orlando; Hunt, Peter W.; Hatano, Hiroyu; Sowinski, Stefanie; Muñoz-Arias, Isa; Greene, Warner C.

2014-01-01

The pathway causing CD4 T-cell death in HIV-infected hosts remains poorly understood although apoptosis has been proposed as a key mechanism. We now show that caspase-3-mediated apoptosis accounts for the death of only a small fraction of CD4 T cells corresponding to those that are both activated and productively infected. The remaining over 95% of quiescent lymphoid CD4 T cells die by caspase-1-mediated pyroptosis triggered by abortive viral infection. Pyroptosis corresponds to an intensely inflammatory form of programmed cell death in which cytoplasmic contents and pro-inflammatory cytokines, including IL-1β, are released. This death pathway thus links the two signature events in HIV infection--CD4 T-cell depletion and chronic inflammation--and creates a pathogenic vicious cycle in which dying CD4 T cells release inflammatory signals that attract more cells to die. This cycle can be broken by caspase 1 inhibitors shown to be safe in humans, raising the possibility of a new class of `anti-AIDS' therapeutics targeting the host rather than the virus.

HIV-1 Tat depresses DNA-PKCS expression and DNA repair, and sensitizes cells to ionizing radiation

International Nuclear Information System (INIS)

Sun Yi; Huang Yuechen; Xu Qinzhi; Wang Huiping; Bai Bei; Sui Jianli; Zhou Pingkun

2006-01-01

Purpose There is accumulating evidence that cancer patients with human immmunodeficiency virus-1/acquired immunodeficency syndrome (HIV-1/AIDS) have more severe tissue reactions and often develop cutaneous toxic effects when subjected to radiotherapy. Here we explored the effects of the HIV-1 Tat protein on cellular responses to ionizing radiation. Methods and Materials Two Tat-expressing cell lines, TT2 and TE671-Tat, were derived from human rhabdomyosarcoma cells by transfecting with the HIV-1 tat gene. Radiosensitivity was determined using colony-forming ability. Gene expression was assessed by cDNA microarray and immunohybridization. The Comet assay and γ-H2AX foci were use to detect DNA double-strand breaks (DSBs) and repair. Radiation-induced cell cycle changes were detected by flow cytometry. Results The radiosensitivity of TT2 and TE671-Tat cells was significantly increased as compared with parental TE671 cells or the control TE671-pCI cells. Tat also increased proliferation activity. The comet assay and γH2AX foci detection revealed a decreased capacity to repair radiation-induced DNA DSBs in Tat-expressing cells. Microarray assay demonstrated that the DNA repair gene DNA-PKcs, and cell cycle-related genes Cdc20, Cdc25C, KIF2C and CTS1 were downregulated in Tat-expressing cells. Depression of DNA-PKcs in Tat-expressing cells was further confirmed by RT-PCR and immuno-hybridization analysis. Tat-expressing cells exhibited a prolonged S phase arrest after 4 Gy γ-irradiation, and a noticeable delay in the initiation and elimination of radiation-induced G 2 /M arrest as compared with parental cells. In addition, the G 2 /M arrest was incomplete in TT2 cells. Moreover, HIV-1 Tat resulted in a constitutive overexpression of cyclin B1 protein. Conclusion HIV-1 Tat protein sensitizes cells to ionizing radiation via depressing DNA repair and dysregulating cell cycle checkpoints. These observations provide new insight into the increased tissue reactions of AIDS

Homeostatic proliferation fails to efficiently reactivate HIV-1 latently infected central memory CD4+ T cells.

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Alberto Bosque

2011-10-01

Full Text Available Homeostatic proliferation ensures the longevity of central memory T-cells by inducing cell proliferation in the absence of cellular differentiation or activation. This process is governed mainly by IL-7. Central memory T-cells can also be stimulated via engagement of the T-cell receptor, leading to cell proliferation but also activation and differentiation. Using an in vitro model of HIV-1 latency, we have examined in detail the effects of homeostatic proliferation on latently infected central memory T cells. We have also used antigenic stimulation via anti-CD3/anti-CD28 antibodies and established a comparison with a homeostatic proliferation stimulus, to evaluate potential differences in how either treatment affects the dynamics of latent virus populations. First, we show that homeostatic proliferation, as induced by a combination of IL-2 plus IL-7, leads to partial reactivation of latent HIV-1 but is unable to reduce the size of the reservoir in vitro. Second, latently infected cells are able to homeostatically proliferate in the absence of viral reactivation or cell differentiation. These results indicate that IL-2 plus IL-7 may induce a detrimental effect by favoring the maintenance of the latent HIV-1 reservoir. On the other hand, antigenic stimulation efficiently reactivated latent HIV-1 in cultured central memory cells and led to depletion of the latently infected cells via virus-induced cell death.

Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells

International Nuclear Information System (INIS)

Iordanskiy, Sergey; Van Duyne, Rachel; Sampey, Gavin C; Woodson, Caitlin M; Fry, Kelsi; Saifuddin, Mohammed; Guo, Jia; Wu, Yuntao; Romerio, Fabio; Kashanchi, Fatah

2015-01-01

The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4"+ T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4"+ T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4"+ T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the “Shock and Kill” strategy for latently HIV-1 infected cells. - Highlights: • X-ray irradiation (IR) increases

Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells

Energy Technology Data Exchange (ETDEWEB)

Iordanskiy, Sergey [School of Systems Biology, Laboratory of Molecular Virology, George Mason University, Manassas, VA 20110 (United States); Van Duyne, Rachel [School of Systems Biology, Laboratory of Molecular Virology, George Mason University, Manassas, VA 20110 (United States); Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Sampey, Gavin C; Woodson, Caitlin M; Fry, Kelsi; Saifuddin, Mohammed; Guo, Jia; Wu, Yuntao [School of Systems Biology, Laboratory of Molecular Virology, George Mason University, Manassas, VA 20110 (United States); Romerio, Fabio [Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201 (United States); Kashanchi, Fatah, E-mail: fkashanc@gmu.edu [School of Systems Biology, Laboratory of Molecular Virology, George Mason University, Manassas, VA 20110 (United States)

2015-11-15

The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4{sup +} T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4{sup +} T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4{sup +} T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the “Shock and Kill” strategy for latently HIV-1 infected cells. - Highlights: • X-ray irradiation

HMGB1 Is Involved in IFN-α Production and TRAIL Expression by HIV-1-Exposed Plasmacytoid Dendritic Cells: Impact of the Crosstalk with NK Cells.

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Héla Saïdi

2016-02-01

Full Text Available Plasmacytoid dendritic cells (pDCs are innate sensors of viral infections and important mediators of antiviral innate immunity through their ability to produce large amounts of IFN-α. Moreover, Toll-like receptor 7 (TLR7 and 9 (TLR9 ligands, such as HIV and CpG respectively, turn pDCs into TRAIL-expressing killer pDCs able to lyse HIV-infected CD4+ T cells. NK cells can regulate antiviral immunity by modulating pDC functions, and pDC production of IFN-α as well as cell-cell contact is required to promote NK cell functions. Impaired pDC-NK cell crosstalk was reported in the setting of HIV-1 infection, but the impact of HIV-1 on TRAIL expression and innate antiviral immunity during this crosstalk is unknown. Here, we report that low concentrations of CCR5-tropic HIV-1Ba-L promote the release of pro-inflammatory cytokines such as IFN-α, TNF-α, IFN-γ and IL-12, and CCR5-interacting chemokines (MIP-1α and MIP-1β in NK-pDCs co-cultures. At high HIV-1BaL concentrations, the addition of NK cells did not promote the release of these mediators, suggesting that once efficiently triggered by the virus, pDCs could not integrate new activating signals delivered by NK cells. However, high HIV-1BaL concentrations were required to trigger IFN-α-mediated TRAIL expression at the surface of both pDCs and NK cells during their crosstalk. Interestingly, we identified the alarmin HMGB1, released at pDC-NK cell synapse, as an essential trigger for the secretion of IFN-α and IFN-related soluble mediators during the interplay of HIV-1 exposed pDCs with NK cells. Moreover, HMGB1 was found crucial for mTRAIL translocation to the plasma membrane of both pDCs and NK cells during their crosstalk following pDC exposure to HIV-1. Data from serum analyses of circulating HMGB1, HMGB1-specific antibodies, sTRAIL and IP-10 in a cohort of 67 HIV-1+ patients argue for the in vivo relevance of these observations. Altogether, these findings identify HMGB1 as a trigger for IFN

HMGB1 Is Involved in IFN-α Production and TRAIL Expression by HIV-1-Exposed Plasmacytoid Dendritic Cells: Impact of the Crosstalk with NK Cells.

Science.gov (United States)

Saïdi, Héla; Bras, Marlène; Formaglio, Pauline; Melki, Marie-Thérèse; Charbit, Bruno; Herbeuval, Jean-Philippe; Gougeon, Marie-Lise

2016-02-01

Plasmacytoid dendritic cells (pDCs) are innate sensors of viral infections and important mediators of antiviral innate immunity through their ability to produce large amounts of IFN-α. Moreover, Toll-like receptor 7 (TLR7) and 9 (TLR9) ligands, such as HIV and CpG respectively, turn pDCs into TRAIL-expressing killer pDCs able to lyse HIV-infected CD4+ T cells. NK cells can regulate antiviral immunity by modulating pDC functions, and pDC production of IFN-α as well as cell-cell contact is required to promote NK cell functions. Impaired pDC-NK cell crosstalk was reported in the setting of HIV-1 infection, but the impact of HIV-1 on TRAIL expression and innate antiviral immunity during this crosstalk is unknown. Here, we report that low concentrations of CCR5-tropic HIV-1Ba-L promote the release of pro-inflammatory cytokines such as IFN-α, TNF-α, IFN-γ and IL-12, and CCR5-interacting chemokines (MIP-1α and MIP-1β) in NK-pDCs co-cultures. At high HIV-1BaL concentrations, the addition of NK cells did not promote the release of these mediators, suggesting that once efficiently triggered by the virus, pDCs could not integrate new activating signals delivered by NK cells. However, high HIV-1BaL concentrations were required to trigger IFN-α-mediated TRAIL expression at the surface of both pDCs and NK cells during their crosstalk. Interestingly, we identified the alarmin HMGB1, released at pDC-NK cell synapse, as an essential trigger for the secretion of IFN-α and IFN-related soluble mediators during the interplay of HIV-1 exposed pDCs with NK cells. Moreover, HMGB1 was found crucial for mTRAIL translocation to the plasma membrane of both pDCs and NK cells during their crosstalk following pDC exposure to HIV-1. Data from serum analyses of circulating HMGB1, HMGB1-specific antibodies, sTRAIL and IP-10 in a cohort of 67 HIV-1+ patients argue for the in vivo relevance of these observations. Altogether, these findings identify HMGB1 as a trigger for IFN

Cooperativity of HIV-Specific Cytolytic CD4 T Cells and CD8 T Cells in Control of HIV Viremia

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Johnson, Susan; Eller, Michael; Teigler, Jeffrey E.; Maloveste, Sebastien M.; Schultz, Bruce T.; Soghoian, Damien Z.; Lu, Richard; Oster, Alexander F.; Chenine, Agnès-Laurence; Alter, Galit; Dittmer, Ulf; Marovich, Mary; Robb, Merlin L.; Michael, Nelson L.; Bolton, Diane

2015-01-01

ABSTRACT CD4+ T cells play a pivotal role in the control of chronic viral infections. Recently, nontraditional CD4+ T cell functions beyond helper effects have been described, and a role for cytolytic CD4+ T cells in the control of HIV infection has been suggested. We define here the transcriptional, phenotypic, and functional profiles of HIV-specific cytolytic CD4+ T cells. Fluidigm BioMark and multiparameter flow cytometric analysis of HIV-specific cytolytic CD4+ T cells revealed a distinct transcriptional signature compared to Th1 CD4+ cells but shared similar features with HIV-specific cytolytic CD8+ T cells. Furthermore, HIV-specific cytolytic CD4+ T cells showed comparable killing activity relative to HIV-specific CD8+ T cells and worked cooperatively in the elimination of virally infected cells. Interestingly, we found that cytolytic CD4+ T cells emerge early during acute HIV infection and tightly follow acute viral load trajectory. This emergence was associated to the early viral set point, suggesting an involvement in early control, in spite of CD4 T cell susceptibility to HIV infection. Our data suggest cytolytic CD4+ T cells as an independent subset distinct from Th1 cells that show combined activity with CD8+ T cells in the long-term control of HIV infection. IMPORTANCE The ability of the immune system to control chronic HIV infection is of critical interest to both vaccine design and therapeutic approaches. Much research has focused on the effect of the ability of CD8+ T cells to control the virus, while CD4+ T cells have been overlooked as effectors in HIV control due to the fact that they are preferentially infected. We show here that a subset of HIV-specific CD4+ T cells cooperate in the cytolytic control of HIV replication. Moreover, these cells represent a distinct subset of CD4+ T cells showing significant transcriptional and phenotypic differences compared to HIV-specific Th1 cells but with similarities to CD8+ T cells. These findings are

B cell, CD8 + T cell and gamma delta T cell infiltration alters alveolar immune cell homeostasis in HIV-infected Malawian adults [version 2; referees: 1 approved, 2 approved with reservations

Directory of Open Access Journals (Sweden)

Andrew Mwale

2017-12-01

Full Text Available Background: HIV infection is associated with increased risk to lower respiratory tract infections (LRTI. However, the impact of HIV infection on immune cell populations in the lung is not well defined. We sought to comprehensively characterise the impact of HIV infection on immune cell populations in the lung. Methods: Twenty HIV-uninfected controls and 17 HIV-1 infected ART-naïve adults were recruited from Queen Elizabeth Central Hospital, Malawi. Immunophenotyping of lymphocyte and myeloid cell populations was done on bronchoalveolar lavage fluid and peripheral blood cells. Results: We found that the numbers of CD8 + T cells, B cells and gamma delta T cells were higher in BAL fluid of HIV-infected adults compared to HIV-uninfected controls (all p<0.05. In contrast, there was no difference in the numbers of alveolar CD4 + T cells in HIV-infected adults compared to HIV-uninfected controls (p=0.7065. Intermediate monocytes were the predominant monocyte subset in BAL fluid (HIV-, 63%; HIV+ 81%, while the numbers of classical monocytes was lower in HIV-infected individuals compared to HIV-uninfected adults (1 × 10 5 vs. 2.8 × 10 5 cells/100ml of BAL fluid, p=0.0001. The proportions of alveolar macrophages and myeloid dendritic cells was lower in HIV-infected adults compared to HIV-uninfected controls (all p<0.05. Conclusions: Chronic HIV infection is associated with broad alteration of immune cell populations in the lung, but does not lead to massive depletion of alveolar CD4 + T cells. Disruption of alveolar immune cell homeostasis likely explains in part the susceptibility for LRTIs in HIV-infected adults.

R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.

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Cavarelli, Mariangela; Foglieni, Chiara; Rescigno, Maria; Scarlatti, Gabriella

2013-05-01

The gastrointestinal tract is a principal route of entry and site of persistence of human immunodeficiency virus type 1 (HIV-1). The intestinal mucosa, being rich of cells that are the main target of the virus, represents a primary site of viral replication and CD4(+) T-cell depletion. Here, we show both in vitro and ex vivo that HIV-1 of R5 but not X4 phenotype is capable of selectively triggering dendritic cells (DCs) to migrate within 30 min between intestinal epithelial cells to sample virions and transfer infection to target cells. The engagement of the chemokine receptor 5 on DCs and the viral envelope, regardless of the genetic subtype, drive DC migration. Viruses penetrating through transient opening of the tight junctions likely create a paracellular gradient to attract DCs. The formation of junctions with epithelial cells may initiate a haptotactic process of DCs and at the same time favour cell-to-cell viral transmission. Our findings indicate that HIV-1 translocation across the intestinal mucosa occurs through the selective engagement of DCs by R5 viruses, and may guide the design of new prevention strategies. Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

Surface Transmission or Polarized Egress? Lessons Learned from HTLV Cell-to-Cell Transmission

Science.gov (United States)

Jin, Jing; Sherer, Nathan; Mothes, Walther

2010-01-01

Commentary on Pais-Correia, A.M.; Sachse, M.; Guadagnini, S.; Robbiati, V.; Lasserre, R.; Gessain, A.; Gout, O.; Alcover, A.; Thoulouze, M.I. Biofilm-like extracellular viral assemblies mediate HTLV-1 cell-to-cell transmission at virological synapses. Nat. Med. 2010, 16, 83–89. PMID:21994650

Syphilis and HIV-1 co-infection: influence on CD4 T cell count, HIV-1 viral load and treatment response

DEFF Research Database (Denmark)

Kofoed, Kristian; Gerstoft, Jan; Mathiesen, Lars Reinhardt

2006-01-01

OBJECTIVES: To assess the effect of human immunodeficiency virus (HIV)-1 and syphilis coinfection on HIV-ribonucleic acid (RNA) viral load, CD4 cell count, and the response in rapid plasmin reagin (RPR) to treatment of the syphilis infection. STUDY DESIGN: Cases of syphilis diagnosed during 1 year...... in HIV-infected patients in Copenhagen were included. HIV-RNA, CD4 cell counts, and RPR-serology were measured before, during, and after syphilis. RESULTS: Forty-one patients were included. CD4 cell count decreased significantly during infection in patients with primary and secondary stages of syphilis...... (mean 106 cells/mm, P = 0.03). Treatment of syphilis was associated with an increase in the CD4 cell count and a decrease in HIV-RNA in the overall group (mean 66 cells/mm and -0.261 RNA log10 copies/ml, P = 0.02 and 0.04). The serological response rates for 15 patients treated with penicillin and 25...

Photo-translocation of anti-HIV-1 drugs into TZM-bl cells

CSIR Research Space (South Africa)

Khanyile, T

2013-04-01

Full Text Available Targeted drug delivery into HIV-1 infected cells offers a reduction in toxicity and side effect. Using a femtosecond (fs) laser of different beam shapes anti-HIV-1 drugs are efficiently delivered into TZM-bl cells....

Characteristics and quantities of HIV host cells in human genital tract secretions.

Science.gov (United States)

Politch, Joseph A; Marathe, Jai; Anderson, Deborah J

2014-12-15

Human immunodeficiency virus (HIV)-infected leukocytes have been detected in genital secretions from HIV-infected men and women and may play an important role in the sexual transmission of HIV. However, they have been largely overlooked in studies on mechanisms of HIV transmission and in the design and testing of HIV vaccine and microbicide candidates. This article describes the characteristics and quantities of leukocytes in male and female genital secretions under various conditions and also reviews evidence for the involvement of HIV-infected cells in both horizontal and vertical cell-associated HIV transmission. Additional research is needed in this area to better target HIV prevention strategies. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

TREX1 Knockdown Induces an Interferon Response to HIV that Delays Viral Infection in Humanized Mice

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Lee Adam Wheeler

2016-05-01

Full Text Available Despite their antiviral effect, the in vivo effect of interferons on HIV transmission is difficult to predict, because interferons also activate and recruit HIV-susceptible cells to sites of infection. HIV does not normally induce type I interferons in infected cells, but does if TREX1 is knocked down. Here, we investigated the effect of topical TREX1 knockdown and local interferon production on HIV transmission in human cervicovaginal explants and humanized mice. In explants in which TREX1 was knocked down, HIV induced interferons, which blocked infection. In humanized mice, even though TREX1 knockdown increased infiltrating immune cells, it delayed viral replication for 3–4 weeks. Similarly intravaginal application of type I interferons the day before HIV infection induced interferon responsive genes, reduced inflammation, and decreased viral replication. However, intravenous interferon enhanced inflammation and infection. Thus, in models of human sexual transmission, a localized interferon response inhibits HIV transmission but systemic interferons do not.

Clonal expansion of genome-intact HIV-1 in functionally polarized Th1 CD4+ T cells.

Science.gov (United States)

Lee, Guinevere Q; Orlova-Fink, Nina; Einkauf, Kevin; Chowdhury, Fatema Z; Sun, Xiaoming; Harrington, Sean; Kuo, Hsiao-Hsuan; Hua, Stephane; Chen, Hsiao-Rong; Ouyang, Zhengyu; Reddy, Kavidha; Dong, Krista; Ndung'u, Thumbi; Walker, Bruce D; Rosenberg, Eric S; Yu, Xu G; Lichterfeld, Mathias

2017-06-30

HIV-1 causes a chronic, incurable disease due to its persistence in CD4+ T cells that contain replication-competent provirus, but exhibit little or no active viral gene expression and effectively resist combination antiretroviral therapy (cART). These latently infected T cells represent an extremely small proportion of all circulating CD4+ T cells but possess a remarkable long-term stability and typically persist throughout life, for reasons that are not fully understood. Here we performed massive single-genome, near-full-length next-generation sequencing of HIV-1 DNA derived from unfractionated peripheral blood mononuclear cells, ex vivo-isolated CD4+ T cells, and subsets of functionally polarized memory CD4+ T cells. This approach identified multiple sets of independent, near-full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4+ T cells harboring intact HIV-1. Intact, near-full-genome HIV-1 DNA sequences that were derived from such clonally expanded CD4+ T cells constituted 62% of all analyzed genome-intact sequences in memory CD4 T cells, were preferentially observed in Th1-polarized cells, were longitudinally detected over a duration of up to 5 years, and were fully replication- and infection-competent. Together, these data suggest that clonal proliferation of Th1-polarized CD4+ T cells encoding for intact HIV-1 represents a driving force for stabilizing the pool of latently infected CD4+ T cells.

Kaposi's-sarcoma-associated-herpesvirus-activated dendritic cells promote HIV-1 trans-infection and suppress CD4+ T cell proliferation

International Nuclear Information System (INIS)

Liu, Wan; Qin, Yan; Bai, Lei; Lan, Ke; Wang, Jian-Hua

2013-01-01

Infection of Kaposi's sarcoma-associated herpesvirus (KSHV) is commonly occurred in AIDS patients. KSHV and HIV-1 act cooperatively in regulating infection with each other and in human carcinogenesis. Dendritic cells (DCs), as the pivotal cells in host immunity, may be modulated by both viruses, for immunoevasion and dissemination, therefore, the interaction between DCs and each virus has been a prior focus for pathogenesis elucidation. Here, we assessed the potential effect of KSHV on DC–HIV-1 interaction. We found that KSHV stimulation could promote maturation of monocyte-derived DCs (MDDCs) and impaired the ability of MDDCs to drive proliferation of resting CD4 + T cells, demonstrating the immunosuppression induced by KSHV. More importantly, KSHV-stimulated MDDCs could capture more HIV-1 and efficiently transferred these infectious viruses to Hut/CCR5 T cell line. Our results reveal the novel modulation of DC-mediated HIV-1 dissemination by KSHV, and highlight the importance of studying DC–HIV-1 interaction to elucidate HIV/AIDS pathogenesis. - Highlights: ► KSHV impaired the ability of MDDCs to drive proliferation of resting CD4 + T cells. ► KSHV stimulation matured MDDCs and enhanced HIV-1 endocytosis. ► KSHV stimulated MDDCs increased ICAM-1 expression and tighten contact with T cells. ► KSHV-stimulated MDDCs promoted HIV-1 trans-infection of CD4 + T cells

Anti-HIV-1 B cell responses are dependent on B cell precursor frequency and antigen-binding affinity.

Science.gov (United States)

Dosenovic, Pia; Kara, Ervin E; Pettersson, Anna-Klara; McGuire, Andrew T; Gray, Matthew; Hartweger, Harald; Thientosapol, Eddy S; Stamatatos, Leonidas; Nussenzweig, Michel C

2018-04-16

The discovery that humans can produce potent broadly neutralizing antibodies (bNAbs) to several different epitopes on the HIV-1 spike has reinvigorated efforts to develop an antibody-based HIV-1 vaccine. Antibody cloning from single cells revealed that nearly all bNAbs show unusual features that could help explain why it has not been possible to elicit them by traditional vaccination and instead would require a sequence of different immunogens. This idea is supported by experiments with genetically modified immunoglobulin (Ig) knock-in mice. Sequential immunization with a series of specifically designed immunogens was required to shepherd the development of bNAbs. However, knock-in mice contain superphysiologic numbers of bNAb precursor-expressing B cells, and therefore how these results can be translated to a more physiologic setting remains to be determined. Here we make use of adoptive transfer experiments using knock-in B cells that carry a synthetic intermediate in the pathway to anti-HIV-1 bNAb development to examine how the relationship between B cell receptor affinity and precursor frequency affects germinal center (GC) B cell recruitment and clonal expansion. Immunization with soluble HIV-1 antigens can recruit bNAb precursor B cells to the GC when there are as few as 10 such cells per mouse. However, at low precursor frequencies, the extent of clonal expansion is directly proportional to the affinity of the antigen for the B cell receptor, and recruitment to GCs is variable and dependent on recirculation.

Functional characteristics of HIV-1 subtype C compatible with increased heterosexual transmissibility

DEFF Research Database (Denmark)

Walter, Brandon L; Armitage, Andrew E; Graham, Stephen C

2009-01-01

BACKGROUND: Despite the existence of over 50 subtypes and circulating recombinant forms of HIV-1, subtype C dominates the heterosexual pandemic causing approximately 56% of all infections. OBJECTIVE: To evaluate whether viral genetic factors may contribute to the observed subtype-C predominance. ....... CONCLUSION: As CD4-CCR5-T cells are key targets for genital HIV infection and cervical selection can favor compact V1-V2 loops and 316T, which increase viral infectivity, we propose that these conserved subtype-C motifs may contribute to transmission and spread of this subtype....

HIV-1 isolation from infected peripheral blood mononuclear cells

NARCIS (Netherlands)

Dispinseri, Stefania; Saba, Elisa; Vicenzi, Elisa; Kootstra, Neeltje A.; Schuitemaker, Hanneke; Scarlatti, Gabriella

2014-01-01

Human immunodeficiency virus 1 (HIV-1) isolation from peripheral blood mononuclear cells (PBMCs) allows retrieval of replication-competent viral variants. In order to impose the smallest possible selective pressure on the viral isolates, isolation must be carried out in primary cultures of cells and

Exposure to apoptotic activated CD4+ T cells induces maturation and APOBEC3G-mediated inhibition of HIV-1 infection in dendritic cells.

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Venkatramanan Mohanram

Full Text Available Dendritic cells (DCs are activated by signaling via pathogen-specific receptors or exposure to inflammatory mediators. Here we show that co-culturing DCs with apoptotic HIV-infected activated CD4(+ T cells (ApoInf or apoptotic uninfected activated CD4(+ T cells (ApoAct induced expression of co-stimulatory molecules and cytokine release. In addition, we measured a reduced HIV infection rate in DCs after co-culture with ApoAct. A prerequisite for reduced HIV infection in DCs was activation of CD4(+ T cells before apoptosis induction. DCs exposed to ApoAct or ApoInf secreted MIP-1α, MIP-1β, MCP-1, and TNF-α; this effect was retained in the presence of exogenous HIV. The ApoAct-mediated induction of co-stimulatory CD86 molecules and reduction of HIV infection in DCs were partially abrogated after blocking TNF-α using monoclonal antibodies. APOBEC3G expression in DCs was increased in co-cultures of DCs and ApoAct but not by apoptotic resting CD4(+ T cells (ApoRest. Silencing of APOBEC3G in DC abrogated the HIV inhibitory effect mediated by ApoAct. Sequence analyses of an env region revealed significant induction of G-to-A hypermutations in the context of GG or GA dinucleotides in DNA isolated from DCs exposed to HIV and ApoAct. Thus, ApoAct-mediated DC maturation resulted in induction of APOBEC3G that was important for inhibition of HIV-infection in DCs. These findings underscore the complexity of differential DC responses evoked upon interaction with resting as compared with activated dying cells during HIV infection.

Expanded breadth of the T-cell response to mosaic HIV-1 envelope DNA vaccination

Energy Technology Data Exchange (ETDEWEB)

Korber, Bette [Los Alamos National Laboratory; Fischer, William [Los Alamos National Laboratory; Wallstrom, Timothy [Los Alamos National Laboratory

2009-01-01

An effective AIDS vaccine must control highly diverse circulating strains of HIV-1. Among HIV -I gene products, the envelope (Env) protein contains variable as well as conserved regions. In this report, an informatic approach to the design of T-cell vaccines directed to HIV -I Env M group global sequences was tested. Synthetic Env antigens were designed to express mosaics that maximize the inclusion of common potential Tcell epitope (PTE) 9-mers and minimize the inclusion of rare epitopes likely to elicit strain-specific responses. DNA vaccines were evaluated using intracellular cytokine staining (ICS) in inbred mice with a standardized panel of highly conserved 15-mer PTE peptides. I, 2 and 3 mosaic sets were developed that increased theoretical epitope coverage. The breadth and magnitude ofT-cell immunity stimulated by these vaccines were compared to natural strain Env's; additional comparisons were performed on mutant Env's, including gpl60 or gpl45 with or without V regions and gp41 deletions. Among them, the 2 or 3 mosaic Env sets elicited the optimal CD4 and CD8 responses. These responses were most evident in CD8 T cells; the 3 mosaic set elicited responses to an average of 8 peptide pools compared to 2 pools for a set of3 natural Env's. Synthetic mosaic HIV -I antigens can therefore induce T-cell responses with expanded breadth and may facilitate the development of effective T -cell-based HIV -1 vaccines.

Psoralen/UV inactivation of HIV-1-infected cells for use in cytologic and immunologic procedures

International Nuclear Information System (INIS)

Watson, A.J.; Klaniecki, J.; Hanson, C.V.

1990-01-01

A rapid procedure for the inactivation of HIV-1-infected cells using psoralen and ultraviolet (UV) light is described. Exposure of HIV-1-infected cells to 5 micrograms/ml psoralen followed by UV irradiation (320-380 nm) for 5 minutes yields cells that are noninfectious as assessed by extended infectivity assays. The psoralen/UV inactivation procedure described is effective with cells chronically or acutely infected with HIV-1 and is unaffected by cell densities up to 12 x 10(6)/ml. At 5 micrograms/ml psoralen does little damage to cellular permeability as shown by the ability of treated cells to exclude trypan blue and propidium iodide. Psoralen/UV treatment of HIV-1-infected cells does not cause a significant decrease in the reactivity of HIV-1 core and envelope antigens or cellular antigens to monoclonal antibodies. Experiments are presented demonstrating the use of these cells for flow cytometry studies and for cell surface labeling using the lactoperoxidase 125 I iodination procedure

HIV-1-infected monocyte-derived dendritic cells do not undergo maturation but can elicit IL-10 production and T cell regulation

Science.gov (United States)

Granelli-Piperno, Angela; Golebiowska, Angelika; Trumpfheller, Christine; Siegal, Frederick P.; Steinman, Ralph M.

2004-05-01

Dendritic cells (DCs) undergo maturation during virus infection and thereby become potent stimulators of cell-mediated immunity. HIV-1 replicates in immature DCs, but we now find that infection is not accompanied by many components of maturation in either infected cells or uninfected bystanders. The infected cultures do not develop potent stimulating activity for the mixed leukocyte reaction (MLR), and the DCs producing HIV-1 gag p24 do not express CD83 and DC-lysosome-associated membrane protein maturation markers. If different maturation stimuli are applied to DCs infected with HIV-1, the infected cells selectively fail to mature. When DCs from HIV-1-infected patients are infected and cultured with autologous T cells, IL-10 was produced in 6 of 10 patients. These DC-T cell cocultures could suppress another immune response, the MLR. The regulation was partially IL-10-dependent and correlated in extent with the level of IL-10 produced. Suppressor cells only developed from infected patients, rather than healthy controls, and the DCs had to be exposed to live virus rather than HIV-1 gag peptides or protein. These results indicate that HIV-1-infected DCs have two previously unrecognized means to evade immune responses: maturation can be blocked reducing the efficacy of antigen presentation from infected cells, and T cell-dependent suppression can be induced.

Mucosal stromal fibroblasts markedly enhance HIV infection of CD4+ T cells.

Directory of Open Access Journals (Sweden)

Jason A Neidleman

2017-02-01

Full Text Available Understanding early events of HIV transmission within mucosal tissues is vital for developing effective prevention strategies. Here, we report that primary stromal fibroblasts isolated from endometrium, cervix, foreskin, male urethra, and intestines significantly increase HIV infection of CD4+ T cells-by up to 37-fold for R5-tropic HIV and 100-fold for X4-tropic HIV-without themselves becoming infected. Fibroblasts were more efficient than dendritic cells at trans-infection and mediate this response in the absence of the DC-SIGN and Siglec-1 receptors. In comparison, mucosal epithelial cells secrete antivirals and inhibit HIV infection. These data suggest that breaches in the epithelium allow external or luminal HIV to escape an antiviral environment to access the infection-favorable environment of the stromal fibroblasts, and suggest that resident fibroblasts have a central, but previously unrecognized, role in HIV acquisition at mucosal sites. Inhibiting fibroblast-mediated enhancement of HIV infection should be considered as a novel prevention strategy.

Exosomes from Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Cells License Quiescent CD4+ T Lymphocytes To Replicate HIV-1 through a Nef- and ADAM17-Dependent Mechanism

OpenAIRE

Arenaccio, Claudia; Chiozzini, Chiara; Columba-Cabezas, Sandra; Manfredi, Francesco; Affabris, Elisabetta; Baur, Andreas; Federico, Maurizio

2014-01-01

Resting CD4+ T lymphocytes resist human immunodeficiency virus (HIV) infection. Here, we provide evidence that exosomes from HIV-1-infected cells render resting human primary CD4+ T lymphocytes permissive to HIV-1 replication. These results were obtained with transwell cocultures of HIV-1-infected cells with quiescent CD4+ T lymphocytes in the presence of inhibitors of exosome release and were confirmed using exosomes purified from supernatants of HIV-1-infected primary CD4+ T lymphocytes. We...

Impact of host cell variation on the neutralization of HIV-1 in vitro.

Science.gov (United States)

Polonis, Victoria R; Schuitemaker, Hanneke; Bunnik, Evelien M; Brown, Bruce K; Scarlatti, Gabriella

2009-09-01

In this review we present current advances in our understanding of HIV-1 neutralization assays that employ primary cell types, as compared with those that utilize cell lines and the newer, more standardized pseudovirus assays. A commentary on the challenges of standardizing in-vitro neutralization assays using primary cells is included. The data from reporter cell line neutralization assays may agree with results observed in primary cells; however, exceptions have recently been reported. Multiple variables exist in primary cell assays using peripheral blood mononuclear cells from HIV-seronegative donors; in-vitro neutralization titers can vary significantly based on the donor cells used for assay targets and for virus propagation. Thus, more research is required to achieve validated primary cell neutralization assays. HIV-vaccine-induced antibody performance in the current neutralization assays may function as a 'gatekeeper' for HIV-1 subunit vaccine advancement. Development of standardized platforms for reproducible measurement of in-vitro neutralization is therefore a high priority. Given the considerable variation in results obtained from some widely applied HIV neutralization platforms, parallel evaluation of new antibodies using different host cells for assay targets, as well as virus propagation, is recommended until immune correlates of protection are identified.

Nuclear retention of multiply spliced HIV-1 RNA in resting CD4+ T cells.

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Kara G Lassen

2006-07-01

Full Text Available HIV-1 latency in resting CD4+ T cells represents a major barrier to virus eradication in patients on highly active antiretroviral therapy (HAART. We describe here a novel post-transcriptional block in HIV-1 gene expression in resting CD4+ T cells from patients on HAART. This block involves the aberrant localization of multiply spliced (MS HIV-1 RNAs encoding the critical positive regulators Tat and Rev. Although these RNAs had no previously described export defect, we show that they exhibit strict nuclear localization in resting CD4+ T cells from patients on HAART. Overexpression of the transcriptional activator Tat from non-HIV vectors allowed virus production in these cells. Thus, the nuclear retention of MS HIV-1 RNA interrupts a positive feedback loop and contributes to the non-productive nature of infection of resting CD4+ T cells. To define the mechanism of nuclear retention, proteomic analysis was used to identify proteins that bind MS HIV-1 RNA. Polypyrimidine tract binding protein (PTB was identified as an HIV-1 RNA-binding protein differentially expressed in resting and activated CD4+ T cells. Overexpression of PTB in resting CD4+ T cells from patients on HAART allowed cytoplasmic accumulation of HIV-1 RNAs. PTB overexpression also induced virus production by resting CD4+ T cells. Virus culture experiments showed that overexpression of PTB in resting CD4+ T cells from patients on HAART allowed release of replication-competent virus, while preserving a resting cellular phenotype. Whether through effects on RNA export or another mechanism, the ability of PTB to reverse latency without inducing cellular activation is a result with therapeutic implications.

The impact of inflammation and immune activation on B cell differentiation during HIV-1 infection

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Nicolas eRuffin

2012-01-01

Full Text Available HIV-1 infection is characterized by continuous antigenic stimulation, chronic immune activation and impaired survival of T and B cells. A decline of resting memory B cells has previously been reported to occur in both children and adults infected with HIV-1; these cells are responsible for mounting and maintaining an adequate serological response to antigens previously encountered in life through natural infection or vaccination. Further understanding of the mechanisms leading to impaired B cell differentiation and germinal center reaction might be essential to design new HIV vaccines and therapies that could improve humoral immune responses in HIV-1 infected individuals. In the present article we summarize the literature and present our view on critical mechanisms of B cell development which are impaired during HIV-1 infection. We also discuss the impact of microbial translocation, a driving force for persistent inflammation during HIV-1 infection, on survival of terminally differentiated B cells and how the altered expression of cytokines/chemokines pivotal for communication between T and B cells in lymphoid tissues may impair formation of memory B cells.

Excision of HIV-1 proviral DNA by recombinant cell permeable tre-recombinase.

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Lakshmikanth Mariyanna

Full Text Available Over the previous years, comprehensive studies on antiretroviral drugs resulted in the successful introduction of highly active antiretroviral therapy (HAART into clinical practice for treatment of HIV/AIDS. However, there is still need for new therapeutic approaches, since HAART cannot eradicate HIV-1 from the infected organism and, unfortunately, can be associated with long-term toxicity and the development of drug resistance. In contrast, novel gene therapy strategies may have the potential to reverse the infection by eradicating HIV-1. For example, expression of long terminal repeat (LTR-specific recombinase (Tre-recombinase has been shown to result in chromosomal excision of proviral DNA and, in consequence, in the eradication of HIV-1 from infected cell cultures. However, the delivery of Tre-recombinase currently depends on the genetic manipulation of target cells, a process that is complicating such therapeutic approaches and, thus, might be undesirable in a clinical setting. In this report we demonstrate that E.coli expressed Tre-recombinases, tagged either with the protein transduction domain (PTD from the HIV-1 Tat trans-activator or the translocation motif (TLM of the Hepatitis B virus PreS2 protein, were able to translocate efficiently into cells and showed significant recombination activity on HIV-1 LTR sequences. Tre activity was observed using episomal and stable integrated reporter constructs in transfected HeLa cells. Furthermore, the TLM-tagged enzyme was able to excise the full-length proviral DNA from chromosomal integration sites of HIV-1-infected HeLa and CEM-SS cells. The presented data confirm Tre-recombinase activity on integrated HIV-1 and provide the basis for the non-genetic transient application of engineered recombinases, which may be a valuable component of future HIV eradication strategies.

The global transmission network of HIV-1.

Science.gov (United States)

Wertheim, Joel O; Leigh Brown, Andrew J; Hepler, N Lance; Mehta, Sanjay R; Richman, Douglas D; Smith, Davey M; Kosakovsky Pond, Sergei L

2014-01-15

Human immunodeficiency virus type 1 (HIV-1) is pandemic, but its contemporary global transmission network has not been characterized. A better understanding of the properties and dynamics of this network is essential for surveillance, prevention, and eventual eradication of HIV. Here, we apply a simple and computationally efficient network-based approach to all publicly available HIV polymerase sequences in the global database, revealing a contemporary picture of the spread of HIV-1 within and between countries. This approach automatically recovered well-characterized transmission clusters and extended other clusters thought to be contained within a single country across international borders. In addition, previously undescribed transmission clusters were discovered. Together, these clusters represent all known modes of HIV transmission. The extent of international linkage revealed by our comprehensive approach demonstrates the need to consider the global diversity of HIV, even when describing local epidemics. Finally, the speed of this method allows for near-real-time surveillance of the pandemic's progression.

Hepatitis C virus cell-cell transmission and resistance to direct-acting antiviral agents

DEFF Research Database (Denmark)

Xiao, Fei; Fofana, Isabel; Heydmann, Laura

2014-01-01

Hepatitis C virus (HCV) is transmitted between hepatocytes via classical cell entry but also uses direct cell-cell transfer to infect neighboring hepatocytes. Viral cell-cell transmission has been shown to play an important role in viral persistence allowing evasion from neutralizing antibodies....... In contrast, the role of HCV cell-cell transmission for antiviral resistance is unknown. Aiming to address this question we investigated the phenotype of HCV strains exhibiting resistance to direct-acting antivirals (DAAs) in state-of-the-art model systems for cell-cell transmission and spread. Using HCV...... genotype 2 as a model virus, we show that cell-cell transmission is the main route of viral spread of DAA-resistant HCV. Cell-cell transmission of DAA-resistant viruses results in viral persistence and thus hampers viral eradication. We also show that blocking cell-cell transmission using host...

Kaposi's-sarcoma-associated-herpesvirus-activated dendritic cells promote HIV-1 trans-infection and suppress CD4{sup +} T cell proliferation

Energy Technology Data Exchange (ETDEWEB)

Liu, Wan; Qin, Yan; Bai, Lei [Key Laboratory of Molecular Virology and Immunology, Institute Pasteur of Shanghai, the Chinese Academy of Sciences, Shanghai (China); Graduate School of the Chinese Academy of Sciences, Beijing (China); Lan, Ke [Key Laboratory of Molecular Virology and Immunology, Institute Pasteur of Shanghai, the Chinese Academy of Sciences, Shanghai (China); Wang, Jian-Hua, E-mail: Jh_wang@sibs.ac.cn [Key Laboratory of Molecular Virology and Immunology, Institute Pasteur of Shanghai, the Chinese Academy of Sciences, Shanghai (China)

2013-06-05

Infection of Kaposi's sarcoma-associated herpesvirus (KSHV) is commonly occurred in AIDS patients. KSHV and HIV-1 act cooperatively in regulating infection with each other and in human carcinogenesis. Dendritic cells (DCs), as the pivotal cells in host immunity, may be modulated by both viruses, for immunoevasion and dissemination, therefore, the interaction between DCs and each virus has been a prior focus for pathogenesis elucidation. Here, we assessed the potential effect of KSHV on DC–HIV-1 interaction. We found that KSHV stimulation could promote maturation of monocyte-derived DCs (MDDCs) and impaired the ability of MDDCs to drive proliferation of resting CD4{sup +} T cells, demonstrating the immunosuppression induced by KSHV. More importantly, KSHV-stimulated MDDCs could capture more HIV-1 and efficiently transferred these infectious viruses to Hut/CCR5 T cell line. Our results reveal the novel modulation of DC-mediated HIV-1 dissemination by KSHV, and highlight the importance of studying DC–HIV-1 interaction to elucidate HIV/AIDS pathogenesis. - Highlights: ► KSHV impaired the ability of MDDCs to drive proliferation of resting CD4{sup +} T cells. ► KSHV stimulation matured MDDCs and enhanced HIV-1 endocytosis. ► KSHV stimulated MDDCs increased ICAM-1 expression and tighten contact with T cells. ► KSHV-stimulated MDDCs promoted HIV-1 trans-infection of CD4{sup +} T cells.

Incorporation of podoplanin into HIV released from HEK-293T cells, but not PBMC, is required for efficient binding to the attachment factor CLEC-2

Science.gov (United States)

2010-01-01

Background Platelets are associated with HIV in the blood of infected individuals and might modulate viral dissemination, particularly if the virus is directly transmitted into the bloodstream. The C-type lectin DC-SIGN and the novel HIV attachment factor CLEC-2 are expressed by platelets and facilitate HIV transmission from platelets to T-cells. Here, we studied the molecular mechanisms behind CLEC-2-mediated HIV-1 transmission. Results Binding studies with soluble proteins indicated that CLEC-2, in contrast to DC-SIGN, does not recognize the viral envelope protein, but a cellular factor expressed on kidney-derived 293T cells. Subsequent analyses revealed that the cellular mucin-like membranous glycoprotein podoplanin, a CLEC-2 ligand, was expressed on 293T cells and incorporated into virions released from these cells. Knock-down of podoplanin in 293T cells by shRNA showed that virion incorporation of podoplanin was required for efficient CLEC-2-dependent HIV-1 interactions with cell lines and platelets. Flow cytometry revealed no evidence for podoplanin expression on viable T-cells and peripheral blood mononuclear cells (PBMC). Podoplanin was also not detected on HIV-1 infected T-cells. However, apoptotic bystander cells in HIV-1 infected cultures reacted with anti-podoplanin antibodies, and similar results were obtained upon induction of apoptosis in a cell line and in PBMCs suggesting an unexpected link between apoptosis and podoplanin expression. Despite the absence of detectable podoplanin expression, HIV-1 produced in PBMC was transmitted to T-cells in a CLEC-2-dependent manner, indicating that T-cells might express an as yet unidentified CLEC-2 ligand. Conclusions Virion incorporation of podoplanin mediates CLEC-2 interactions of HIV-1 derived from 293T cells, while incorporation of a different cellular factor seems to be responsible for CLEC-2-dependent capture of PBMC-derived viruses. Furthermore, evidence was obtained that podoplanin expression is

Incorporation of podoplanin into HIV released from HEK-293T cells, but not PBMC, is required for efficient binding to the attachment factor CLEC-2

Directory of Open Access Journals (Sweden)

Münch Jan

2010-05-01

Full Text Available Abstract Background Platelets are associated with HIV in the blood of infected individuals and might modulate viral dissemination, particularly if the virus is directly transmitted into the bloodstream. The C-type lectin DC-SIGN and the novel HIV attachment factor CLEC-2 are expressed by platelets and facilitate HIV transmission from platelets to T-cells. Here, we studied the molecular mechanisms behind CLEC-2-mediated HIV-1 transmission. Results Binding studies with soluble proteins indicated that CLEC-2, in contrast to DC-SIGN, does not recognize the viral envelope protein, but a cellular factor expressed on kidney-derived 293T cells. Subsequent analyses revealed that the cellular mucin-like membranous glycoprotein podoplanin, a CLEC-2 ligand, was expressed on 293T cells and incorporated into virions released from these cells. Knock-down of podoplanin in 293T cells by shRNA showed that virion incorporation of podoplanin was required for efficient CLEC-2-dependent HIV-1 interactions with cell lines and platelets. Flow cytometry revealed no evidence for podoplanin expression on viable T-cells and peripheral blood mononuclear cells (PBMC. Podoplanin was also not detected on HIV-1 infected T-cells. However, apoptotic bystander cells in HIV-1 infected cultures reacted with anti-podoplanin antibodies, and similar results were obtained upon induction of apoptosis in a cell line and in PBMCs suggesting an unexpected link between apoptosis and podoplanin expression. Despite the absence of detectable podoplanin expression, HIV-1 produced in PBMC was transmitted to T-cells in a CLEC-2-dependent manner, indicating that T-cells might express an as yet unidentified CLEC-2 ligand. Conclusions Virion incorporation of podoplanin mediates CLEC-2 interactions of HIV-1 derived from 293T cells, while incorporation of a different cellular factor seems to be responsible for CLEC-2-dependent capture of PBMC-derived viruses. Furthermore, evidence was obtained that

Mother-to-child transmission of HIV-1: advances and controversies of the twentieth centuries.

Science.gov (United States)

Scarlatti, G

2004-01-01

Mother-to-child transmission (MTCT) is the overwhelming source of HIV-1 infection in young children. According to the World Health Organization (WHO), during the year 2003, despite effective antiretroviral (ARV) therapy, there were approximately 700,000 new infections in children worldwide, the majority of whom were from resource-limited countries. Alternative protocols to the long-course and complex regimens of ARV drugs, which in high-income countries have almost eradicated HIV MTCT, have been shown to reduce early transmission rates by 38-50%. However, the accumulation of drug resistance and the long-term toxicities of ARVs mean that alternative approaches need to be developed. Furthermore, transmission via breastfeeding, which accounts for one third of all transmission events, can reduce the benefits of short-course therapies given to women for the prevention of MTCT. The complex mechanisms and determinants of HIV-1 MTCT and its prevention in the different routes of transmission are still not completely understood. Despite the large contribution that many international agencies have made during the past 10-15 years in support of observational and intervention trials, as well as basic scientific research, HIV-1 MTCT intervention trials and basic research often are not integrated, leading to the generation of a fragmented picture. Maternal RNA levels, CD4+ T-cell counts, mode of delivery and gestational age were shown to be independent factors associated with transmission. However, these markers are only partial surrogates and cannot be used as absolute predictors of MTCT of HIV-1. Studies on the role of viral characteristics, immune response and host genomic polymorphisms did not always achieve conclusive results. Although CCR5-using viruses are preferentially carried by HIV-1 infected women as well as transmitted to their infants, the 32-basepair deletion of the CCR5 gene was not shown to influence perinatal MTCT. X4 viruses are apparently hampered in MTCT

The regulated secretory pathway in CD4(+ T cells contributes to human immunodeficiency virus type-1 cell-to-cell spread at the virological synapse.

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Clare Jolly

2011-09-01

Full Text Available Direct cell-cell spread of Human Immunodeficiency Virus type-1 (HIV-1 at the virological synapse (VS is an efficient mode of dissemination between CD4(+ T cells but the mechanisms by which HIV-1 proteins are directed towards intercellular contacts is unclear. We have used confocal microscopy and electron tomography coupled with functional virology and cell biology of primary CD4(+ T cells from normal individuals and patients with Chediak-Higashi Syndrome and report that the HIV-1 VS displays a regulated secretion phenotype that shares features with polarized secretion at the T cell immunological synapse (IS. Cell-cell contact at the VS re-orientates the microtubule organizing center (MTOC and organelles within the HIV-1-infected T cell towards the engaged target T cell, concomitant with polarization of viral proteins. Directed secretion of proteins at the T cell IS requires specialized organelles termed secretory lysosomes (SL and we show that the HIV-1 envelope glycoprotein (Env localizes with CTLA-4 and FasL in SL-related compartments and at the VS. Finally, CD4(+ T cells that are disabled for regulated secretion are less able to support productive cell-to-cell HIV-1 spread. We propose that HIV-1 hijacks the regulated secretory pathway of CD4(+ T cells to enhance its dissemination.

Vpx overcomes a SAMHD1-independent block to HIV reverse transcription that is specific to resting CD4 T cells.

Science.gov (United States)

Baldauf, Hanna-Mari; Stegmann, Lena; Schwarz, Sarah-Marie; Ambiel, Ina; Trotard, Maud; Martin, Margarethe; Burggraf, Manja; Lenzi, Gina M; Lejk, Helena; Pan, Xiaoyu; Fregoso, Oliver I; Lim, Efrem S; Abraham, Libin; Nguyen, Laura A; Rutsch, Frank; König, Renate; Kim, Baek; Emerman, Michael; Fackler, Oliver T; Keppler, Oliver T

2017-03-07

Early after entry into monocytes, macrophages, dendritic cells, and resting CD4 T cells, HIV encounters a block, limiting reverse transcription (RT) of the incoming viral RNA genome. In this context, dNTP triphosphohydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) has been identified as a restriction factor, lowering the concentration of dNTP substrates to limit RT. The accessory lentiviral protein X (Vpx) proteins from the major simian immunodeficiency virus of rhesus macaque, sooty mangabey, and HIV-2 (SIVsmm/SIVmac/HIV-2) lineage packaged into virions target SAMHD1 for proteasomal degradation, increase intracellular dNTP pools, and facilitate HIV cDNA synthesis. We find that virion-packaged Vpx proteins from a second SIV lineage, SIV of red-capped mangabeys or mandrills (SIVrcm/mnd-2), increased HIV infection in resting CD4 T cells, but not in macrophages, and, unexpectedly, acted in the absence of SAMHD1 degradation, dNTP pool elevation, or changes in SAMHD1 phosphorylation. Vpx rcm/mnd-2 virion incorporation resulted in a dramatic increase of HIV-1 RT intermediates and viral cDNA in infected resting CD4 T cells. These analyses also revealed a barrier limiting HIV-1 infection of resting CD4 T cells at the level of nuclear import. Single amino acid changes in the SAMHD1-degrading Vpx mac239 allowed it to enhance early postentry steps in a Vpx rcm/mnd-2-like fashion. Moreover, Vpx enhanced HIV-1 infection of SAMHD1-deficient resting CD4 T cells of a patient with Aicardi-Goutières syndrome. These results indicate that Vpx, in addition to SAMHD1, overcomes a previously unappreciated restriction for lentiviruses at the level of RT that acts independently of dNTP concentrations and is specific to resting CD4 T cells.

Engineering HIV-1-resistant T-cells from short-hairpin RNA-expressing hematopoietic stem/progenitor cells in humanized BLT mice.

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Gene-Errol E Ringpis

Full Text Available Down-regulation of the HIV-1 coreceptor CCR5 holds significant potential for long-term protection against HIV-1 in patients. Using the humanized bone marrow/liver/thymus (hu-BLT mouse model which allows investigation of human hematopoietic stem/progenitor cell (HSPC transplant and immune system reconstitution as well as HIV-1 infection, we previously demonstrated stable inhibition of CCR5 expression in systemic lymphoid tissues via transplantation of HSPCs genetically modified by lentiviral vector transduction to express short hairpin RNA (shRNA. However, CCR5 down-regulation will not be effective against existing CXCR4-tropic HIV-1 and emergence of resistant viral strains. As such, combination approaches targeting additional steps in the virus lifecycle are required. We screened a panel of previously published shRNAs targeting highly conserved regions and identified a potent shRNA targeting the R-region of the HIV-1 long terminal repeat (LTR. Here, we report that human CD4(+ T-cells derived from transplanted HSPC engineered to co-express shRNAs targeting CCR5 and HIV-1 LTR are resistant to CCR5- and CXCR4- tropic HIV-1-mediated depletion in vivo. Transduction with the combination vector suppressed CXCR4- and CCR5- tropic viral replication in cell lines and peripheral blood mononuclear cells in vitro. No obvious cytotoxicity or interferon response was observed. Transplantation of combination vector-transduced HSPC into hu-BLT mice resulted in efficient engraftment and subsequent stable gene marking and CCR5 down-regulation in human CD4(+ T-cells within peripheral blood and systemic lymphoid tissues, including gut-associated lymphoid tissue, a major site of robust viral replication, for over twelve weeks. CXCR4- and CCR5- tropic HIV-1 infection was effectively inhibited in hu-BLT mouse spleen-derived human CD4(+ T-cells ex vivo. Furthermore, levels of gene-marked CD4(+ T-cells in peripheral blood increased despite systemic infection with either

Increasing procaspase 8 expression using repurposed drugs to induce HIV infected cell death in ex vivo patient cells.

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Rahul Sampath

Full Text Available HIV persists because a reservoir of latently infected CD4 T cells do not express viral proteins and are indistinguishable from uninfected cells. One approach to HIV cure suggests that reactivating HIV will activate cytotoxic pathways; yet when tested in vivo, reactivating cells do not die sufficiently to reduce cell-associated HIV DNA levels. We recently showed that following reactivation from latency, HIV infected cells generate the HIV specific cytotoxic protein Casp8p41 which is produced by HIV protease cleaving procaspase 8. However, cell death is prevented, possibly due to low procaspase 8 expression. Here, we tested whether increasing procaspase 8 levels in CD4 T cells will produce more Casp8p41 following HIV reactivation, causing more reactivated cells to die. Screening 1277 FDA approved drugs identified 168 that increased procaspase 8 expression by at least 1.7-fold. Of these 30 were tested for anti-HIV effects in an acute HIVIIIb infection model, and 9 drugs at physiologic relevant levels significantly reduced cell-associated HIV DNA. Primary CD4 T cells from ART suppressed HIV patients were treated with one of these 9 drugs and reactivated with αCD3/αCD28. Four drugs significantly increased Casp8p41 levels following HIV reactivation, and decreased total cell associated HIV DNA levels (flurbiprofen: p = 0.014; doxycycline: p = 0.044; indomethacin: p = 0.025; bezafibrate: P = 0.018 without effecting the viability of uninfected cells. Thus procaspase 8 levels can be increased pharmacologically and, in the context of HIV reactivation, increase Casp8p41 causing death of reactivating cells and decreased HIV DNA levels. Future studies will be required to define the clinical utility of this or similar approaches.

TLR-4 engagement of dendritic cells confers a BST-2/tetherin-mediated restriction of HIV-1 infection to CD4+ T cells across the virological synapse

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Blanchet Fabien P

2013-01-01

Full Text Available Abstract Background Dendritic cells and their subsets, located at mucosal surfaces, are among the first immune cells to encounter disseminating pathogens. The cellular restriction factor BST-2/tetherin (also known as CD317 or HM1.24 potently restricts HIV-1 release by retaining viral particles at the cell surface in many cell types, including primary cells such as macrophages. However, BST-2/tetherin does not efficiently restrict HIV-1 infection in immature dendritic cells. Results We now report that BST-2/tetherin expression in myeloid (myDC and monocyte-derived dendritic cells (DC can be significantly up-regulated by IFN-α treatment and TLR-4 engagement with LPS. In contrast to HeLa or 293T cells, infectious HIV-1 release in immature DC and IFN-α–matured DC was only modestly affected in the absence of Vpu compared to wild-type viruses. Strikingly, immunofluorescence analysis revealed that BST-2/tetherin was excluded from HIV containing tetraspanin-enriched microdomains (TEMs in both immature DC and IFN-α–matured DC. In contrast, in LPS-mediated mature DC, BST-2/tetherin exerted a significant restriction in transfer of HIV-1 infection to CD4+ T cells. Additionally, LPS, but not IFN-α stimulation of immature DC, leads to a dramatic redistribution of cellular restriction factors to the TEM as well as at the virological synapse between DC and CD4+ T cells. Conclusions In conclusion, we demonstrate that TLR-4 engagement in immature DC significantly up-regulates the intrinsic antiviral activity of BST-2/tetherin, during cis-infection of CD4+ T cells across the DC/T cell virological synapse. Manipulating the function and potency of cellular restriction factors such as BST-2/tetherin to HIV-1 infection, has implications in the design of antiviral therapeutic strategies.

14-3-3 theta binding to cell cycle regulatory factors is enhanced by HIV-1 Vpr

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Sakai Keiko

2008-04-01

Full Text Available Abstract Background Despite continuing advances in our understanding of AIDS pathogenesis, the mechanism of CD4+ T cell depletion in HIV-1-infected individuals remains unclear. The HIV-1 Vpr accessory protein causes cell death, likely through a mechanism related to its ability to arrest cells in the G2,M phase. Recent evidence implicated the scaffold protein, 14-3-3, in Vpr cell cycle blockade. Results We found that in human T cells, 14-3-3 plays an active role in mediating Vpr-induced cell cycle arrest and reveal a dramatic increase in the amount of Cdk1, Cdc25C, and CyclinB1 bound to 14-3-3 θ during Vprv-induced G2,M arrest. By contrast, a cell-cycle-arrest-dead Vpr mutant failed to augment 14-3-3 θ association with Cdk1 and CyclinB1. Moreover, G2,M arrest caused by HIV-1 infection strongly correlated with a disruption in 14-3-3 θ binding to centrosomal proteins, Plk1 and centrin. Finally, Vpr caused elevated levels of CyclinB1, Plk1, and Cdk1 in a complex with the nuclear transport and spindle assembly protein, importin β. Conclusion Thus, our data reveal a new facet of Vpr-induced cell cycle arrest involving previously unrecognized abnormal rearrangements of multiprotein assemblies containing key cell cycle regulatory proteins. Reviewers This article was reviewed by David Kaplan, Nathaniel R. Landau and Yan Zhou.

Noninvasive micromanipulation of live HIV-1 infected cells via laser light

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Mthunzi, Patience

2015-12-01

Live mammalian cells from various tissues of origin can be aseptically and noninvasively micromanipulated via lasers of different regimes. Laser-driven techniques are therefore paving a path toward the advancement of human immuno-deficiency virus (HIV-1) investigations. Studies aimed at the interaction of laser light, nanomaterials, and biological materials can also lead to an understanding of a wealth of disease conditions and result in photonics-based therapies and diagnostic tools. Thus, in our research, both continuous wave and pulsed lasers operated at varying wavelengths are employed, as they possess special properties that allow classical biomedical applications. This paper discusses photo-translocation of antiretroviral drugs into HIV-1 permissive cells and preliminary results of low-level laser therapy (LLLT) in HIV-1 infected cells.

Laser irradiation reduces HIV-1 infection in TZM-bl cells

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Lugongolo, Masixole Y

2016-10-01

Full Text Available HIV-1 epidemic remains a major health challenge. This study explores the effects of low level laser therapy on HIV-1 infected cells. Infection is reduced by irradiation and the mechanism needs to be investigated further....

Quantitative live-cell imaging of human immunodeficiency virus (HIV-1) assembly.

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Baumgärtel, Viola; Müller, Barbara; Lamb, Don C

2012-05-01

Advances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site.

Optimizing HIV-1-specific CD8+ T-cell induction by recombinant BCG in prime-boost regimens with heterologous viral vectors.

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Hopkins, Richard; Bridgeman, Anne; Bourne, Charles; Mbewe-Mvula, Alice; Sadoff, Jerald C; Both, Gerald W; Joseph, Joan; Fulkerson, John; Hanke, Tomáš

2011-12-01

The desire to induce HIV-1-specific responses soon after birth to prevent breast milk transmission of HIV-1 led us to propose a vaccine regimen which primes HIV-1-specific T cells using a recombinant Mycobacterium bovis bacillus Calmette-Guérin (rBCG) vaccine. Because attenuated live bacterial vaccines are typically not sufficiently immunogenic as stand-alone vaccines, rBCG-primed T cells will likely require boost immunization(s). Here, we compared modified Danish (AERAS-401) and Pasteur lysine auxotroph (222) strains of BCG expressing the immunogen HIVA for their potency to prime HIV-1-specific responses in adult BALB/c mice and examined four heterologous boosting HIVA vaccines for their immunogenic synergy. We found that both BCG.HIVA(401) and BCG.HIVA(222) primed HIV-1-specific CD8(+) T-cell-mediated responses. The strongest boosts were delivered by human adenovirus-vectored HAdV5.HIVA and sheep atadenovirus-vectored OAdV7.HIVA vaccines, followed by poxvirus MVA.HIVA; the weakest was plasmid pTH.HIVA DNA. The prime-boost regimens induced T cells capable of efficient in vivo killing of sensitized target cells. We also observed that the BCG.HIVA(401) and BCG.HIVA(222) vaccines have broadly similar immunologic properties, but display a number of differences mainly detected through distinct profiles of soluble intercellular signaling molecules produced by immune splenocytes in response to both HIV-1- and BCG-specific stimuli. These results encourage further development of the rBCG prime-boost regimen. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of cell surface targets for HIV-1 therapeutics using genetic screens

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Dunn, Stephen J.; Khan, Imran H.; Chan, Ursula A.; Scearce, Robin L.; Melara, Claudia L.; Paul, Amber M.; Sharma, Vikram; Bih, Fong-Yih; Holzmayer, Tanya A.; Luciw, Paul A.; Abo, Arie

2004-01-01

Human immunodeficiency virus (HIV) drugs designed to interfere with obligatory utilization of certain host cell factors by virus are less likely to encounter development of resistant strains than drugs directed against viral components. Several cellular genes required for productive infection by HIV were identified by the use of genetic suppressor element (GSE) technology as potential targets for anti-HIV drug development. Fragmented cDNA libraries from various pools of human peripheral blood mononuclear cells (PBMC) were expressed in vitro in human immunodeficiency virus type 1 (HIV-1)-susceptible cell lines and subjected to genetic screens to identify GSEs that interfered with viral replication. After three rounds of selection, more than 15 000 GSEs were sequenced, and the cognate genes were identified. The GSEs that inhibited the virus were derived from a diverse set of genes including cell surface receptors, cytokines, signaling proteins, transcription factors, as well as genes with unknown function. Approximately 2.5% of the identified genes were previously shown to play a role in the HIV-1 life cycle; this finding supports the biological relevance of the assay. GSEs were derived from the following 12 cell surface proteins: CXCR4, CCR4, CCR7, CD11C, CD44, CD47, CD68, CD69, CD74, CSF3R, GABBR1, and TNFR2. Requirement of some of these genes for viral infection was also investigated by using RNA interference (RNAi) technology; accordingly, 10 genes were implicated in early events of the viral life cycle, before viral DNA synthesis. Thus, these cell surface proteins represent novel targets for the development of therapeutics against HIV-1 infection and AIDS

Bacterial vaginosis associated with increased risk of female-to-male HIV-1 transmission: a prospective cohort analysis among African couples.

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Craig R Cohen

-1 transmission to male partners. Several limitations may affect the generalizability of our results including: all participants underwent couples HIV counseling and testing and enrolled in an HIV-1 prevention trial, and index participants had a baseline CD4 count ≥ 250 cells/mm³ and were HSV-2 seropositive. Given the high prevalence of BV and the association of BV with increased risk of both female HIV-1 acquisition and transmission found in our study, if this association proves to be causal, BV could be responsible for a substantial proportion of new HIV-1 infections in Africa. Normalization of vaginal flora in HIV-1-infected women could mitigate female-to-male HIV-1 transmission.ClinicalTrials.com NCT00194519.

TSPAN7, effector of actin nucleation required for dendritic cell-mediated transfer of HIV-1 to T cells.

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Ménager, Mickaël M

2017-06-15

Dendritic cells (DCs) have essential roles in early detection of pathogens and activation of both innate and adaptive immune responses. Whereas human DCs are resistant to productive HIV-1 replication, they have a unique ability to take up virus and transmit it efficiently to T lymphocytes. By doing that, HIV-1 may evade, at least in part, the first line of defense of the immune system, exploiting DCs instead to facilitate rapid infection of a large pool of immune cells. While performing an shRNA screen in human primary monocyte-derived DCs, to gain insights into this cell biological process, we discovered the role played by tetraspanin-7 (TSPAN7). This member of the tetraspanin family appears to be a positive regulator of actin nucleation and stabilization, through the ARP2/3 complex. By doing so, TSPAN7 limits HIV-1 endocytosis and maintains viral particles on actin-rich dendrites for an efficient transfer toward T lymphocytes. While studying the function of TSPAN7 in the control of actin nucleation, we also discovered the existence in DCs of two opposing forces at the plasma membrane: actin nucleation, a protrusive force which seems to counterbalance actomyosin contraction. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Preferential susceptibility of Th9 and Th2 CD4+ T cells to X4-tropic HIV-1 infection.

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Orlova-Fink, Nina; Chowdhury, Fatema Z; Sun, Xiaoming; Harrington, Sean; Rosenberg, Eric S; Yu, Xu G; Lichterfeld, Mathias

2017-10-23

The functional polarization of CD4 T cells determines their antimicrobial effector profile, but may also impact the susceptibility to infection with HIV-1. Here, we analyzed the susceptibility of CD4 T cells with different functional polarization to infection with X4 and R5-tropic HIV-1. CD4 T cells with a Th1, Th2, Th17, and Th9 polarization were subjected to in-vitro infection assays with X4, R5, or vesicular stomatitis virus-G protein-pseudotyped HIV-1. In addition, we sorted differentially polarized CD4 T-cell subsets from individuals treated with antiretroviral therapy and analyzed the tropism of viral env sequences. Th9-polarized CD4 T cells and, to a lesser extent, Th2-polarized CD4 T cells expressed higher surface levels of CXCR4, and are more permissive to X4-tropic infection in vitro. In contrast, Th1 and Th17 CD4 T cells exhibited stronger surface expression of CCR5, and were more susceptible to infection with R5-tropic viruses. Correspondingly, the distribution of X4-tropic viral sequences in antiretroviral therapy-treated HIV-1-infected patients was biased toward Th9/Th2 cells, whereas R5-tropic sequences were more frequently observed in Th17 cells. CD4 T-cell polarization is associated with a distinct susceptibility to X4 and R5-tropic HIV-1 infection.

Upregulation of Glucose Uptake and Hexokinase Activity of Primary Human CD4+ T Cells in Response to Infection with HIV-1

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Maia Kavanagh Williamson

2018-03-01

Full Text Available Infection of primary CD4+ T cells with HIV-1 coincides with an increase in glycolysis. We investigated the expression of glucose transporters (GLUT and glycolytic enzymes in human CD4+ T cells in response to infection with HIV-1. We demonstrate the co-expression of GLUT1, GLUT3, GLUT4, and GLUT6 in human CD4+ T cells after activation, and their concerted overexpression in HIV-1 infected cells. The investigation of glycolytic enzymes demonstrated activation-dependent expression of hexokinases HK1 and HK2 in human CD4+ T cells, and a highly significant increase in cellular hexokinase enzyme activity in response to infection with HIV-1. HIV-1 infected CD4+ T cells showed a marked increase in expression of HK1, as well as the functionally related voltage-dependent anion channel (VDAC protein, but not HK2. The elevation of GLUT, HK1, and VDAC expression in HIV-1 infected cells mirrored replication kinetics and was dependent on virus replication, as evidenced by the use of reverse transcription inhibitors. Finally, we demonstrated that the upregulation of HK1 in HIV-1 infected CD4+ T cells is independent of the viral accessory proteins Vpu, Vif, Nef, and Vpr. Though these data are consistent with HIV-1 dependency on CD4+ T cell glucose metabolism, a cellular response mechanism to infection cannot be ruled out.

Glycosylphosphatidylinositol-Anchored Anti-HIV scFv Efficiently Protects CD4 T Cells from HIV-1 Infection and Deletion in hu-PBL Mice

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Ye, Chaobaihui; Wang, Weiming; Cheng, Liang; Li, Guangming; Wen, Michael; Wang, Qi; Zhang, Qing; Li, Dan

2016-01-01

ABSTRACT Despite success in viral inhibition and CD4 T cell recovery by highly active antiretroviral treatment (HAART), HIV-1 is still not curable due to the persistence of the HIV-1 reservoir during treatment. One patient with acute myeloid leukemia who received allogeneic hematopoietic stem cell transplantation from a homozygous CCR5 Δ32 donor has had no detectable viremia for 9 years after HAART cessation. This case has inspired a field of HIV-1 cure research focusing on engineering HIV-1 resistance in permissive cells. Here, we employed a glycosylphosphatidylinositol (GPI)-scFv X5 approach to confer resistance of human primary CD4 T cells to HIV-1. We showed that primary CD4 T cells expressing GPI-scFv X5 were resistant to CCR5 (R5)-, CXCR4 (X4)-, and dual-tropic HIV-1 and had a survival advantage compared to control cells ex vivo. In a hu-PBL mouse study, GPI-scFv X5-transduced CD4 T cells were selected in peripheral blood and lymphoid tissues upon HIV-1 infection. Finally, GPI-scFv X5-transduced CD4 T cells, after being cotransfused with HIV-infected cells, showed significantly reduced viral loads and viral RNA copy numbers relative to CD4 cells in hu-PBL mice compared to mice with GPI-scFv AB65-transduced CD4 T cells. We conclude that GPI-scFv X5-modified CD4 T cells could potentially be used as a genetic intervention against both R5- and X4-tropic HIV-1 infections. IMPORTANCE Blocking of HIV-1 entry is one of most promising approaches for therapy. Genetic disruption of the HIV-1 coreceptor CCR5 by nucleases in T cells is under 2 clinical trials and leads to reduced viremia in patients. However, the emergence of viruses using the CXCR4 coreceptor is a concern for therapies applying single-coreceptor disruption. Here, we report that HIV-1-permissive CD4 T cells engineered with GPI-scFv X5 are resistant to R5-, X4-, or dual-tropic virus infection ex vivo. In a preclinical study using hu-PBL mice, we show that CD4 T cells were protected and that GPI-scFv X5

Dendritic cell maturation, but not type I interferon exposure, restricts infection by HTLV-1, and viral transmission to T-cells.

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Gergès Rizkallah

2017-04-01

Full Text Available Human T lymphotropic Virus type 1 (HTLV-1 is the etiological agent of Adult T cell Leukemia/Lymphoma (ATLL and HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP. Both CD4+ T-cells and dendritic cells (DCs infected with HTLV-1 are found in peripheral blood from HTLV-1 carriers. We previously demonstrated that monocyte-derived IL-4 DCs are more susceptible to HTLV-1 infection than autologous primary T-cells, suggesting that DC infection precedes T-cell infection. However, during blood transmission, breast-feeding or sexual transmission, HTLV-1 may encounter different DC subsets present in the blood, the intestinal or genital mucosa respectively. These different contacts may impact HTLV-1 ability to infect DCs and its subsequent transfer to T-cells. Using in vitro monocyte-derived IL-4 DCs, TGF-β DCs and IFN-α DCs that mimic DCs contacting HTLV-1 in vivo, we show here that despite their increased ability to capture HTLV-1 virions, IFN-α DCs restrict HTLV-1 productive infection. Surprisingly, we then demonstrate that it is not due to the antiviral activity of type-I interferon produced by IFN-α DCs, but that it is likely to be linked to a distinct trafficking route of HTLV-1 in IL-4 DCs vs. IFN-α DCs. Finally, we demonstrate that, in contrast to IL-4 DCs, IFN-α DCs are impaired in their capacity to transfer HTLV-1 to CD4 T-cells, both after viral capture and trans-infection and after their productive infection. In conclusion, the nature of the DCs encountered by HTLV-1 upon primo-infection and the viral trafficking route through the vesicular pathway of these cells determine the efficiency of viral transmission to T-cells, which may condition the fate of infection.

Clinical Control of HIV-1 by Cytotoxic T Cells Specific for Multiple Conserved Epitopes.

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Murakoshi, Hayato; Akahoshi, Tomohiro; Koyanagi, Madoka; Chikata, Takayuki; Naruto, Takuya; Maruyama, Rie; Tamura, Yoshiko; Ishizuka, Naoki; Gatanaga, Hiroyuki; Oka, Shinichi; Takiguchi, Masafumi

2015-05-01

Identification and characterization of CD8(+) T cells effectively controlling HIV-1 variants are necessary for the development of AIDS vaccines and for studies of AIDS pathogenesis, although such CD8(+) T cells have been only partially identified. In this study, we sought to identify CD8(+) T cells controlling HIV-1 variants in 401 Japanese individuals chronically infected with HIV-1 subtype B, in which protective alleles HLA-B*57 and HLA-B*27 are very rare, by using comprehensive and exhaustive methods. We identified 13 epitope-specific CD8(+) T cells controlling HIV-1 in Japanese individuals, though 9 of these epitopes were not previously reported. The breadths of the T cell responses to the 13 epitopes were inversely associated with plasma viral load (P = 2.2 × 10(-11)) and positively associated with CD4 count (P = 1.2 × 10(-11)), indicating strong synergistic effects of these T cells on HIV-1 control in vivo. Nine of these epitopes were conserved among HIV-1 subtype B-infected individuals, whereas three out of four nonconserved epitopes were cross-recognized by the specific T cells. These findings indicate that these 12 epitopes are strong candidates for antigens for an AIDS vaccine. The present study highlighted a strategy to identify CD8(+) T cells controlling HIV-1 and demonstrated effective control of HIV-1 by those specific for 12 conserved or cross-reactive epitopes. HLA-B*27-restricted and HLA-B*57-restricted cytotoxic T lymphocytes (CTLs) play a key role in controlling HIV-1 in Caucasians and Africans, whereas it is unclear which CTLs control HIV-1 in Asian countries, where HLA-B*57 and HLA-B*27 are very rare. A recent study showed that HLA-B*67:01 and HLA-B*52:01-C*12:02 haplotypes were protective alleles in Japanese individuals, but it is unknown whether CTLs restricted by these alleles control HIV-1. In this study, we identified 13 CTLs controlling HIV-1 in Japan by using comprehensive and exhaustive methods. They included 5 HLA-B*52:01-restricted

Polymorphisms in DC-SIGN and L-SIGN genes are associated with HIV-1 vertical transmission in a Northeastern Brazilian population.

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da Silva, Ronaldo Celerino; Segat, Ludovica; Zanin, Valentina; Arraes, Luiz Claudio; Crovella, Sergio

2012-11-01

DC-SIGN and L-SIGN are receptors expressed on specialized macrophages in decidua, (Hofbauer and placental capillary endothelial cells), known to interact with several pathogens, including HIV-1. To disclose the possible involvement of these molecules in the susceptibility to HIV vertical transmission, we analyzed DC-SIGN and L-SIGN gene single nucleotide polymorphisms (SNPs) in 192 HIV-1 positive children and 58 HIV-1 negative children all born to HIV-1 positive mothers, as well as 96 healthy uninfected children not exposed to HIV-1, all from Northeast Brazil. The frequency of three SNPs in the DC-SIGN promoter (-139G>A, -201G>T and -336A>G) were significantly different when comparing HIV positive children with HIV-1 exposed uninfected children, indicating an association with susceptibility to HIV-1 vertical transmission. This genetic association suggests that DC-SIGN molecule may play a role in susceptibility to HIV-1 infection through vertical transmission. Copyright © 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Induction of human immunodeficiency virus (HIV-1 envelope specific cell-mediated immunity by a non-homologous synthetic peptide.

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Ammar Achour

2007-11-01

Full Text Available Cell mediated immunity, including efficient CTL response, is required to prevent HIV-1 from cell-to-cell transmission. In previous investigations, we have shown that B1 peptide derived by Fourier transformation of HIV-1 primary structures and sharing no sequence homology with the parent proteins was able to generate antiserum which recognizes envelope and Tat proteins. Here we have investigated cellular immune response towards a novel non-homologous peptide, referred to as cA1 peptide.The 20 amino acid sequence of cA1 peptide was predicted using the notion of peptide hydropathic properties; the peptide is encoded by the complementary anti-sense DNA strand to the sense strand of previously described non-homologous A1 peptide. In this report we demonstrate that the cA1 peptide can be a target for major histocompatibility complex (MHC class I-restricted cytotoxic T lymphocytes in HIV-1-infected or envelope-immunized individuals. The cA1 peptide is recognized in association with different MHC class I allotypes and could prime in vitro CTLs, derived from gp160-immunized individuals capable to recognize virus variants.For the first time a theoretically designed immunogen involved in broad-based cell-immune memory activation is described. Our findings may thus contribute to the advance in vaccine research by describing a novel strategy to develop a synthetic AIDS vaccine.

Demonstration of a novel HIV-1 restriction phenotype from a human T cell line.

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Yanxing Han

2008-07-01

Full Text Available Although retroviruses may invade host cells, a productive infection can be established only after the virus counteracts inhibition from different types of host restriction factors. Fv1, APOBEC3G/F, TRIM5alpha, ZAP, and CD317 inhibit the replication of different retroviruses by interfering with viral uncoating, reverse transcription, nuclear import, RNA stability, and release. In humans, although APOBEC3G/3F and CD317 block HIV-1 replication, their antiviral activities are neutralized by viral proteins Vif and Vpu. So far, no human gene has been found to effectively block wild type HIV-1 replication under natural condition. Thus, identification of such a gene product would be of great medical importance for the development of HIV therapies.In this study, we discovered a new type of host restriction against the wild type HIV-1 from a CD4/CXCR4 double-positive human T cell line. We identified a CEM-derived cell line (CEM.NKR that is highly resistant to productive HIV-1 infection. Viral production was reduced by at least 1000-fold when compared to the other permissive human T cell lines such as H9, A3.01, and CEM-T4. Importantly, this resistance was evident at extremely high multiplicity of infection. Further analyses demonstrated that HIV-1 could finish the first round of replication in CEM.NKR cells, but the released virions were poorly infectious. These virions could enter the target cells, but failed to initiate reverse transcription. Notably, this restriction phenotype was also present in CEM.NKR and 293T heterokaryons.These results clearly indicate that CEM.NKR cells express a HIV inhibitory gene(s. Further characterization of this novel gene product(s will reveal a new antiretroviral mechanism that directly inactivates wild type HIV-1.

Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation.

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Alexander Zhyvoloup

2017-07-01

Full Text Available HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation.

Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation.

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Zhyvoloup, Alexander; Melamed, Anat; Anderson, Ian; Planas, Delphine; Lee, Chen-Hsuin; Kriston-Vizi, Janos; Ketteler, Robin; Merritt, Andy; Routy, Jean-Pierre; Ancuta, Petronela; Bangham, Charles R M; Fassati, Ariberto

2017-07-01

HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation.

Electron tomography of HIV-1 infection in gut-associated lymphoid tissue.

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Ladinsky, Mark S; Kieffer, Collin; Olson, Gregory; Deruaz, Maud; Vrbanac, Vladimir; Tager, Andrew M; Kwon, Douglas S; Bjorkman, Pamela J

2014-01-01

Critical aspects of HIV-1 infection occur in mucosal tissues, particularly in the gut, which contains large numbers of HIV-1 target cells that are depleted early in infection. We used electron tomography (ET) to image HIV-1 in gut-associated lymphoid tissue (GALT) of HIV-1-infected humanized mice, the first three-dimensional ultrastructural examination of HIV-1 infection in vivo. Human immune cells were successfully engrafted in the mice, and following infection with HIV-1, human T cells were reduced in GALT. Virions were found by ET at all stages of egress, including budding immature virions and free mature and immature viruses. Immuno-electron microscopy verified the virions were HIV-1 and showed CD4 sequestration in the endoplasmic reticulum of infected cells. Observation of HIV-1 in infected GALT tissue revealed that most HIV-1-infected cells, identified by immunolabeling and/or the presence of budding virions, were localized to intestinal crypts with pools of free virions concentrated in spaces between cells. Fewer infected cells were found in mucosal regions and the lamina propria. The preservation quality of reconstructed tissue volumes allowed details of budding virions, including structures interpreted as host-encoded scission machinery, to be resolved. Although HIV-1 virions released from infected cultured cells have been described as exclusively mature, we found pools of both immature and mature free virions within infected tissue. The pools could be classified as containing either mostly mature or mostly immature particles, and analyses of their proximities to the cell of origin supported a model of semi-synchronous waves of virion release. In addition to HIV-1 transmission by pools of free virus, we found evidence of transmission via virological synapses. Three-dimensional EM imaging of an active infection within tissue revealed important differences between cultured cell and tissue infection models and furthered the ultrastructural understanding of

Electron tomography of HIV-1 infection in gut-associated lymphoid tissue.

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Mark S Ladinsky

2014-01-01

Full Text Available Critical aspects of HIV-1 infection occur in mucosal tissues, particularly in the gut, which contains large numbers of HIV-1 target cells that are depleted early in infection. We used electron tomography (ET to image HIV-1 in gut-associated lymphoid tissue (GALT of HIV-1-infected humanized mice, the first three-dimensional ultrastructural examination of HIV-1 infection in vivo. Human immune cells were successfully engrafted in the mice, and following infection with HIV-1, human T cells were reduced in GALT. Virions were found by ET at all stages of egress, including budding immature virions and free mature and immature viruses. Immuno-electron microscopy verified the virions were HIV-1 and showed CD4 sequestration in the endoplasmic reticulum of infected cells. Observation of HIV-1 in infected GALT tissue revealed that most HIV-1-infected cells, identified by immunolabeling and/or the presence of budding virions, were localized to intestinal crypts with pools of free virions concentrated in spaces between cells. Fewer infected cells were found in mucosal regions and the lamina propria. The preservation quality of reconstructed tissue volumes allowed details of budding virions, including structures interpreted as host-encoded scission machinery, to be resolved. Although HIV-1 virions released from infected cultured cells have been described as exclusively mature, we found pools of both immature and mature free virions within infected tissue. The pools could be classified as containing either mostly mature or mostly immature particles, and analyses of their proximities to the cell of origin supported a model of semi-synchronous waves of virion release. In addition to HIV-1 transmission by pools of free virus, we found evidence of transmission via virological synapses. Three-dimensional EM imaging of an active infection within tissue revealed important differences between cultured cell and tissue infection models and furthered the ultrastructural

Increased T cell trafficking as adjunct therapy for HIV-1

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Wolinsky, Steven M.; McLean, Angela R.

2018-01-01

Although antiretroviral drug therapy suppresses human immunodeficiency virus-type 1 (HIV-1) to undetectable levels in the blood of treated individuals, reservoirs of replication competent HIV-1 endure. Upon cessation of antiretroviral therapy, the reservoir usually allows outgrowth of virus and approaches to targeting the reservoir have had limited success. Ongoing cycles of viral replication in regions with low drug penetration contribute to this persistence. Here, we use a mathematical model to illustrate a new approach to eliminating the part of the reservoir attributable to persistent replication in drug sanctuaries. Reducing the residency time of CD4 T cells in drug sanctuaries renders ongoing replication unsustainable in those sanctuaries. We hypothesize that, in combination with antiretroviral drugs, a strategy to orchestrate CD4 T cell trafficking could contribute to a functional cure for HIV-1 infection. PMID:29499057

HIV-associated disruption of tight and adherens junctions of oral epithelial cells facilitates HSV-1 infection and spread.

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Irna Sufiawati

Full Text Available Herpes simplex virus (HSV types 1 and 2 are the most common opportunistic infections in HIV/AIDS. In these immunocompromised individuals, HSV-1 reactivates and replicates in oral epithelium, leading to oral disorders such as ulcers, gingivitis, and necrotic lesions. Although the increased risk of HSV infection may be mediated in part by HIV-induced immune dysfunction, direct or indirect interactions of HIV and HSV at the molecular level may also play a role. In this report we show that prolonged interaction of the HIV proteins tat and gp120 and cell-free HIV virions with polarized oral epithelial cells leads to disruption of tight and adherens junctions of epithelial cells through the mitogen-activated protein kinase signaling pathway. HIV-induced disruption of oral epithelial junctions facilitates HSV-1 paracellular spread between the epithelial cells. Furthermore, HIV-associated disruption of adherens junctions exposes sequestered nectin-1, an adhesion protein and critical receptor for HSV envelope glycoprotein D (gD. Exposure of nectin-1 facilitates binding of HSV-1 gD, which substantially increases HSV-1 infection of epithelial cells with disrupted junctions over that of cells with intact junctions. Exposed nectin-1 from disrupted adherens junctions also increases the cell-to-cell spread of HSV-1 from infected to uninfected oral epithelial cells. Antibodies to nectin-1 and HSV-1 gD substantially reduce HSV-1 infection and cell-to-cell spread, indicating that HIV-promoted HSV infection and spread are mediated by the interaction of HSV gD with HIV-exposed nectin-1. Our data suggest that HIV-associated disruption of oral epithelial junctions may potentiate HSV-1 infection and its paracellular and cell-to-cell spread within the oral mucosal epithelium. This could be one of the possible mechanisms of rapid development of HSV-associated oral lesions in HIV-infected individuals.

Intracellular high mobility group B1 protein (HMGB1) represses HIV-1 LTR-directed transcription in a promoter- and cell-specific manner

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Naghavi, Mojgan H.; Nowak, Piotr; Andersson, Jan; Soennerborg, Anders; Yang Huan; Tracey, Kevin J.; Vahlne, Anders

2003-01-01

We investigated whether the high mobility group B 1 (HMGB1), an abundant nuclear protein in all mammalian cells, affects HIV-1 transcription. Intracellular expression of human HMGB1 repressed HIV-1 gene expression in epithelial cells. This inhibitory effect of HMGB1 was caused by repression of long terminal repeat (LTR)-mediated transcription. Other viral promoters/enhancers, including simian virus 40 or cytomegalovirus, were not inhibited by HMGB1. In addition, HMGB1 inhibition of HIV-1 subtype C expression was dependent on the number of NFκB sites in the LTR region. The inhibitory effect of HMGB1 on viral gene expression observed in HeLa cells was confirmed by an upregulation of viral replication in the presence of antisense HMGB1 in monocytic cells. In contrast to what was found in HeLa cells and monocytic cells, endogenous HMGB1 expression did not affect HIV-1 replication in unstimulated Jurkat cells. Thus, intracellular HMGB1 affects HIV-1 LTR-directed transcription in a promoter- and cell-specific manner

Polyploidy and Mitotic Cell Death Are Two Distinct HIV-1 Vpr-Driven Outcomes in Renal Tubule Epithelial Cells.

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Payne, Emily H; Ramalingam, Dhivya; Fox, Donald T; Klotman, Mary E

2018-01-15

Prior studies have found that HIV, through the Vpr protein, promotes genome reduplication (polyploidy) in infection-surviving epithelial cells within renal tissue. However, the temporal progression and molecular regulation through which Vpr promotes polyploidy have remained unclear. Here we define a sequential progression to Vpr-mediated polyploidy in human renal tubule epithelial cells (RTECs). We found that as in many cell types, Vpr first initiates G 2 cell cycle arrest in RTECs. We then identified a previously unreported cascade of Vpr-dependent events that lead to renal cell survival and polyploidy. Specifically, we found that a fraction of G 2 -arrested RTECs reenter the cell cycle. Following this cell cycle reentry, two distinct outcomes occur. Cells that enter complete mitosis undergo mitotic cell death due to extra centrosomes and aberrant division. Conversely, cells that abort mitosis undergo endoreplication to become polyploid. We further show that multiple small-molecule inhibitors of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, including those that target ATR, ATM, and mTOR, indirectly prevent Vpr-mediated polyploidy by preventing G 2 arrest. In contrast, an inhibitor that targets DNA-dependent protein kinase (DNA-PK) specifically blocks the Vpr-mediated transition from G 2 arrest to polyploidy. These findings outline a temporal, molecularly regulated path to polyploidy in HIV-positive renal cells. IMPORTANCE Current cure-focused efforts in HIV research aim to elucidate the mechanisms of long-term persistence of HIV in compartments. The kidney is recognized as one such compartment, since viral DNA and mRNA persist in the renal tissues of HIV-positive patients. Further, renal disease is a long-term comorbidity in the setting of HIV. Thus, understanding the regulation and impact of HIV infection on renal cell biology will provide important insights into this unique HIV compartment. Our work identifies mechanisms that distinguish

Plasmacytoid dendritic cells accumulate and secrete interferon alpha in lymph nodes of HIV-1 patients.

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Clara Lehmann

2010-06-01

Full Text Available Circulating plasmacytoid dendritic cells (pDC decline during HIV-1 infection, but at the same time they express markedly higher levels of interferon alpha (IFNalpha, which is associated with HIV-1 disease progression. Here we show an accumulation of pDC in lymph nodes (LN of treatment-naïve HIV-1 patients. This phenomenon was associated with elevated expression of the LN homing marker, CCR7, on pDC in peripheral blood of HIV-1 patients, which conferred increased migratory capacity in response to CCR7 ligands in ex vivo functional assays. LN-homed pDC of HIV-1 patients presented higher CD40 and lower BDCA2 levels, but unchanged CD83 and CD86 expression. In addition, these cells expressed markedly higher amounts of IFNalpha compared to uninfected individuals, and were undergoing faster rates of cell death. These results demonstrate for the first time that in asymptomatic, untreated HIV-1 patients circulating pDC up-regulate CCR7 expression, accumulate in lymph nodes, and express high amounts of IFNalpha before undergoing cell death. Since IFNalpha inhibits cell proliferation and modulates immune responses, chronically high levels of this cytokine in LN of HIV-1 patients may impair differentiation and immune function of bystander CD4(+ T cells, thus playing into the mechanisms of AIDS immunopathogenesis.

Mucosal safety of PHI-443 and stampidine as a combination microbicide to prevent genital transmission of HIV-1.

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D'Cruz, Osmond J; Uckun, Fatih M

2007-10-01

the secretion of proinflammatory cytokines and chemokines by co-cultures of human genital epithelia and Langerhans cells. These attributes are particularly useful for the clinical development of PHI-443 and stampidine as a combination microbicide and as a prophylactic anti-HIV agent to curb genital transmission of HIV-1 by semen.

CD4 cell count response to first-line combination ART in HIV-2+ patients compared with HIV-1+ patients

DEFF Research Database (Denmark)

Wittkop, Linda; Arsandaux, Julie; Trevino, Ana

2017-01-01

Background: CD4 cell recovery following first-line combination ART (cART) is poorer in HIV-2+ than in HIV-1+ patients. Only large comparisons may allow adjustments for demographic and pretreatment plasma viral load (pVL). Methods: ART-naive HIV+ adults from two European multicohort collaborations...

HIV-specific antibodies but not t-cell responses are associated with protection in seronegative partners of HIV-1-infected individuals in Cambodia.

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Nguyen, Marie; Pean, Polidy; Lopalco, Lucia; Nouhin, Janin; Phoung, Viseth; Ly, Nary; Vermisse, Pierre; Henin, Yvette; Barré-Sinoussi, Françoise; Burastero, Samuele E; Reynes, Jean-Marc; Carcelain, Guislaine; Pancino, Gianfranco

2006-08-01

To study biological factors related to protection against HIV-1 infection in Cambodia, we recruited 48 partners of HIV-1-infected patients who remained uninfected (exposed uninfected individuals, EUs) despite unprotected sexual intercourse for more than 1 year and 49 unexposed controls (UCs). HIV-1-specific antibodies (IgA anti-gp41 and IgG anti-CD4-gp120 complex), T-cell responses, and cellular factors that may be involved in protection (peripheral blood mononuclear cell [PBMC] resistance to HIV-1 infection and beta-chemokine production) were evaluated. Anti-HIV-1 antibodies were higher in EUs than those in UCs (P = 0.01 and P = 0.04 for anti-gp41 and anti-CD4-gp120, respectively). We observed a decreased susceptibility to a primary Cambodian isolate, HIV-1KH019, in EU PBMCs as compared with UC PBMCs (P = 0.03). A weak T-cell response to one pool of HIV-1 Gag peptides was found by ELISpot in 1 of 19 EUs. Whereas T-cell specific immunity was not associated to protection, our results suggest that HIV-specific humoral immunity and reduced cell susceptibility to infection may contribute to protection against HIV-1 infection in Cambodian EUs.

Selective Loss of Early Differentiated, Highly Functional PD1high CD4 T Cells with HIV Progression.

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Robert M Paris

Full Text Available The role of PD-1 expression on CD4 T cells during HIV infection is not well understood. Here, we describe the differential expression of PD-1 in CD127high CD4 T cells within the early/intermediate differentiated (EI (CD27highCD45RAlow T cell population among uninfected and HIV-infected subjects, with higher expression associated with decreased viral replication (HIV-1 viral load. A significant loss of circulating PD-1highCTLA-4low CD4 T cells was found specifically in the CD127highCD27highCD45RAlow compartment, while initiation of antiretroviral treatment, particularly in subjects with advanced disease, reversed these dynamics. Increased HIV-1 Gag DNA was also found in PD-1high compared to PD-1low ED CD4 T cells. In line with an increased susceptibility to HIV infection, PD-1 expression in this CD4 T cell subset was associated with increased activation and expression of the HIV co-receptor, CCR5. Rather than exhaustion, this population produced more IFN-g, MIP1-a, IL-4, IL-10, and IL-17a compared to PD-1low EI CD4 T cells. In line with our previous findings, PD-1high EI CD4 T cells were also characterized by a high expression of CCR7, CXCR5 and CCR6, a phenotype associated with increased in vitro B cell help. Our data show that expression of PD-1 on early-differentiated CD4 T cells may represent a population that is highly functional, more susceptible to HIV infection and selectively lost in chronic HIV infection.

HIV-1 intersection with CD4 T cell vesicle exocytosis: intercellular communication goes viral

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Helena eSoares

2014-09-01

Full Text Available In cells of the immune system the secretion of extracellular vesicles is modulated through cellular activation. In particular, T cell activation is achieved through cell-cell contacts with antigen presenting cells and the consequent formation of a specialized signaling junction called the immunological synapse. Recent works on CD4 T cells have elucidated that cognate antigen recognition by the T cell receptor (TCR engages two distinct exocytic events. The first, involves the exocytic targeting of signaling molecules at the synaptic membrane and drives the functional architecture of the immunological synapse. The second, enlists the extracellular secretion of the TCR itself, once the functional architecture of the immunological synapse is accomplished. HIV-1, a human lymphotropic virus, has evolved sophisticated mechanisms to co-opt CD4 T cell physiology. Notably, it has become apparent that HIV-1 intersects the regulated secretory system of CD4 T cells in order to bud from the plasma membrane of the infected cell and to promote bystander cell death. Here, I review the relevance of CD4 vesicle exocytosis to immune regulation and to HIV-1 pathogenesis and discuss their potential therapeutic applications.

Escape of HIV-1-infected dendritic cells from TRAIL-mediated NK cell cytotoxicity during NK-DC cross-talk--a pivotal role of HMGB1.

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Marie-Thérèse Melki

2010-04-01

Full Text Available Early stages of Human Immunodeficiency Virus-1 (HIV-1 infection are associated with local recruitment and activation of important effectors of innate immunity, i.e. natural killer (NK cells and dendritic cells (DCs. Immature DCs (iDCs capture HIV-1 through specific receptors and can disseminate the infection to lymphoid tissues following their migration, which is associated to a maturation process. This process is dependent on NK cells, whose role is to keep in check the quality and the quantity of DCs undergoing maturation. If DC maturation is inappropriate, NK cells will kill them ("editing process" at sites of tissue inflammation, thus optimizing the adaptive immunity. In the context of a viral infection, NK-dependent killing of infected-DCs is a crucial event required for early elimination of infected target cells. Here, we report that NK-mediated editing of iDCs is impaired if DCs are infected with HIV-1. We first addressed the question of the mechanisms involved in iDC editing, and we show that cognate NK-iDC interaction triggers apoptosis via the TNF-related apoptosis-inducing ligand (TRAIL-Death Receptor 4 (DR4 pathway and not via the perforin pathway. Nevertheless, once infected with HIV-1, DC(HIV become resistant to NK-induced TRAIL-mediated apoptosis. This resistance occurs despite normal amounts of TRAIL released by NK cells and comparable DR4 expression on DC(HIV. The escape of DC(HIV from NK killing is due to the upregulation of two anti-apoptotic molecules, the cellular-Flice like inhibitory protein (c-FLIP and the cellular inhibitor of apoptosis 2 (c-IAP2, induced by NK-DC(HIV cognate interaction. High-mobility group box 1 (HMGB1, an alarmin and a key mediator of NK-DC cross-talk, was found to play a pivotal role in NK-dependent upregulation of c-FLIP and c-IAP2 in DC(HIV. Finally, we demonstrate that restoration of DC(HIV susceptibility to NK-induced TRAIL killing can be obtained either by silencing c-FLIP and c-IAP2 by specific

Superior control of HIV-1 replication by CD8+ T cells targeting conserved epitopes: implications for HIV vaccine design.

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Pratima Kunwar

Full Text Available A successful HIV vaccine will likely induce both humoral and cell-mediated immunity, however, the enormous diversity of HIV has hampered the development of a vaccine that effectively elicits both arms of the adaptive immune response. To tackle the problem of viral diversity, T cell-based vaccine approaches have focused on two main strategies (i increasing the breadth of vaccine-induced responses or (ii increasing vaccine-induced responses targeting only conserved regions of the virus. The relative extent to which set-point viremia is impacted by epitope-conservation of CD8(+ T cell responses elicited during early HIV-infection is unknown but has important implications for vaccine design. To address this question, we comprehensively mapped HIV-1 CD8(+ T cell epitope-specificities in 23 ART-naïve individuals during early infection and computed their conservation score (CS by three different methods (prevalence, entropy and conseq on clade-B and group-M sequence alignments. The majority of CD8(+ T cell responses were directed against variable epitopes (p<0.01. Interestingly, increasing breadth of CD8(+ T cell responses specifically recognizing conserved epitopes was associated with lower set-point viremia (r = - 0.65, p = 0.009. Moreover, subjects possessing CD8(+ T cells recognizing at least one conserved epitope had 1.4 log10 lower set-point viremia compared to those recognizing only variable epitopes (p = 0.021. The association between viral control and the breadth of conserved CD8(+ T cell responses may be influenced by the method of CS definition and sequences used to determine conservation levels. Strikingly, targeting variable versus conserved epitopes was independent of HLA type (p = 0.215. The associations with viral control were independent of functional avidity of CD8(+ T cell responses elicited during early infection. Taken together, these data suggest that the next-generation of T-cell based HIV-1 vaccines should focus

CD4- and dynamin-dependent endocytosis of HIV-1 into plasmacytoid dendritic cells

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Pritschet, Kathrin; Donhauser, Norbert; Schuster, Philipp; Ries, Moritz; Haupt, Sabrina; Kittan, Nicolai A.; Korn, Klaus [Institute of Clinical and Molecular Virology, National Reference Centre for Retroviruses, Friedrich-Alexander-Universitaet Erlangen-Nuernberg, 91054 Erlangen (Germany); Poehlmann, Stefan [Institute of Virology, Hannover Medical School, 30625 Hannover (Germany); Holland, Gudrun; Bannert, Norbert [Robert Koch-Institute, Center for Biological Security 4, 13353 Berlin (Germany); Bogner, Elke [Institute of Virology, Charite University Hospital, 10117 Berlin (Germany); Schmidt, Barbara, E-mail: baschmid@viro.med.uni-erlangen.de [Institute of Clinical and Molecular Virology, National Reference Centre for Retroviruses, Friedrich-Alexander-Universitaet Erlangen-Nuernberg, 91054 Erlangen (Germany)

2012-02-20

Chronic immune activation, triggered by plasmacytoid dendritic cell (PDC) interferon (IFN)-alpha production, plays an important role in HIV-1 pathogenesis. As the entry of HIV-1 seems to be important for the activation of PDC, we directly characterized the viral entry into these cells using immuno-electron microscopy, cellular fractionation, confocal imaging, and functional experiments. After attachment to PDC, viruses were taken up in an energy-dependent manner. The virions were located in compartments positive for caveolin; early endosomal antigen 1; Rab GTPases 5, 7 and 9; lysosomal-associated membrane protein 1. PDC harbored more virus in endocytic vesicles than CD4+ T cells (p < 0.05). Blocking CD4 inhibited the uptake of virions into cytosolic and endosomal compartments. Dynasore, an inhibitor of dynamin-dependent endocytosis, not the fusion inhibitor T-20, reduced the HIV-1 induced IFN-alpha production. Altogether, our morphological and functional data support the role of endocytosis for the entry and IFN-alpha induction of HIV-1 in PDC.

Fluorescent reporter signals, EGFP and DsRed, encoded in HIV-1 facilitate the detection of productively infected cells and cell-associated viral replication levels

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Kazutaka eTerahara

2012-01-01

Full Text Available Flow cytometric analysis is a reliable and convenient method for investigating molecules at the single cell level. Previously, recombinant human immunodeficiency virus type 1 (HIV-1 strains were constructed that express a fluorescent reporter, either enhanced green fluorescent protein or DsRed, which allow the monitoring of HIV-1-infected cells by flow cytometry. The present study further investigated the potential of these recombinant viruses in terms of whether the HIV-1 fluorescent reporters would be helpful in evaluating viral replication based on fluorescence intensity. When primary CD4+ T cells were infected with recombinant viruses, the fluorescent reporter intensity measured by flow cytometry was associated with the level of CD4 downmodulation and Gag p24 expression in infected cells. Interestingly, some HIV-1-infected cells, in which CD4 was only moderately downmodulated, were reporter-positive but Gag p24-negative. Furthermore, when the activation status of primary CD4+ T cells was modulated by T cell receptor-mediated stimulation, we confirmed the preferential viral production upon strong stimulation and showed that the intensity of the fluorescent reporter within a proportion of HIV-1-infected cells was correlated with the viral replication level. These findings indicate that a fluorescent reporter encoded within HIV-1 is useful for the sensitive detection of productively-infected cells at different stages of infection and for evaluating cell-associated viral replication at the single cell level.

Multivalent dendrimeric compounds containing carbohydrates expressed on immune cells inhibit infection by primary isolates of HIV-1

International Nuclear Information System (INIS)

Rosa Borges, Andrew; Wieczorek, Lindsay; Johnson, Benitra; Benesi, Alan J.; Brown, Bruce K.; Kensinger, Richard D.; Krebs, Fred C.; Wigdahl, Brian; Blumenthal, Robert; Puri, Anu; McCutchan, Francine E.; Birx, Deborah L.; Polonis, Victoria R.; Schengrund, Cara-Lynne

2010-01-01

Specific glycosphingolipids (GSL), found on the surface of target immune cells, are recognized as alternate cell surface receptors by the human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein. In this study, the globotriose and 3'-sialyllactose carbohydrate head groups found on two GSL were covalently attached to a dendrimer core to produce two types of unique multivalent carbohydrates (MVC). These MVC inhibited HIV-1 infection of T cell lines and primary peripheral blood mononuclear cells (PBMC) by T cell line-adapted viruses or primary isolates, with IC 50 s ranging from 0.1 to 7.4 μg/ml. Inhibition of Env-mediated membrane fusion by MVC was also observed using a dye-transfer assay. These carbohydrate compounds warrant further investigation as a potential new class of HIV-1 entry inhibitors. The data presented also shed light on the role of carbohydrate moieties in HIV-1 virus-host cell interactions. -- Research Highlights: →Multivalent carbohydrates (MVCs) inhibited infection of PBMCs by HIV-1. →MVCs inhibited infection by T cell line-adapted viruses. →MVCs inhibited infection by primary isolates of HIV-1. →MVCs inhibited Env-mediated membrane fusion.

SAMHD1 restricts HIV-1 replication and regulates interferon production in mouse myeloid cells.

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Ruonan Zhang

Full Text Available SAMHD1 restricts the replication of HIV-1 and other retroviruses in human myeloid and resting CD4(+ T cells and that is counteracted in SIV and HIV-2 by the Vpx accessory protein. The protein is a phosphohydrolase that lowers the concentration of deoxynucleoside triphosphates (dNTP, blocking reverse transcription of the viral RNA genome. Polymorphisms in the gene encoding SAMHD1 are associated with Aicardi-Goutières Syndrome, a neurological disorder characterized by increased type-I interferon production. SAMHD1 is conserved in mammals but its role in restricting virus replication and controlling interferon production in non-primate species is not well understood. We show that SAMHD1 is catalytically active and expressed at high levels in mouse spleen, lymph nodes, thymus and lung. siRNA knock-down of SAMHD1 in bone marrow-derived macrophages increased their susceptibility to HIV-1 infection. shRNA knock-down of SAMHD1 in the murine monocytic cell-line RAW264.7 increased its susceptibility to HIV-1 and murine leukemia virus and increased the levels of the dNTP pool. In addition, SAMHD1 knock-down in RAW264.7 cells induced the production of type-I interferon and several interferon-stimulated genes, modeling the situation in Aicardi-Goutières Syndrome. Our findings suggest that the role of SAMHD1 in restricting viruses is conserved in the mouse. The RAW264.7 cell-line serves as a useful tool to study the antiviral and innate immune response functions of SAMHD1.

Rapid selection of escape mutants by the first CD8 T cell responses in acute HIV-1 infection

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Korber, Bette Tina Marie [Los Alamos National Laboratory

2008-01-01

The recent failure of a vaccine that primes T cell responses to control primary HIV-1 infection has raised doubts about the role of CD8+ T cells in early HIV-1 infection. We studied four patients who were identified shortly after HIV-1 infection and before seroconversion. In each patient there was very rapid selection of multiple HIV-1 escape mutants in the transmitted virus by CD8 T cells, including examples of complete fixation of non-synonymous substitutions within 2 weeks. Sequencing by single genome amplification suggested that the high rate of virus replication in acute infection gave a selective advantage to virus molecules that contained simultaneous and gained sequential T cell escape mutations. These observations show that whilst early HIV-1 specific CD8 T cells can act against virus, rapid escape means that these T cell responses are unlikely to benefit the patient and may in part explain why current HIV-1 T cell vaccines may not be protective.

SupT1 Cell Infusion as a Possible Cell-Based Therapy for HIV: Results from a Pilot Study in Hu-PBMC BRGS Mice

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Jonathan Fior

2016-04-01

Full Text Available In a previous in vitro study, the SupT1 cell line was explored as a decoy target for HIV-1, proposing SupT1 cell infusion as a possible cell-based therapy for HIV. In the present work, the previous in vitro model was translated into an in vivo setting. Specifically, Hu-PBMC BRGS mice were infected with a high input of HIV-1 LAI (100,000 TCID50, and 40 million 30 Gy-irradiated SupT1 cells were infused weekly for 4 weeks as a therapy. Blood samples were taken to monitor CD4+ T cell count and viral load, and mice were monitored daily for signs of illness. At the earliest time point analyzed (Week 1, there was a significantly lower plasma viral load (~10-fold in all animals treated with SupT1 cell infusion, associated with a higher CD4+ T cell count. At later time points, infection proceeded with robust viral replication and evident CD4+ T cell depletion, except in one mouse that showed complete suppression of viral replication and preservation of CD4+ T cell count. No morbidity or mortality was associated with SupT1 cell infusion. The interesting tendencies observed in the generated data suggest that this approach should be further investigated as a possible cell-based HIV therapy.

Apoptosis and telomeres shortening related to HIV-1 induced oxidative stress in an astrocytoma cell line

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Mollace Vincenzo

2009-05-01

Full Text Available Abstract Background Oxidative stress plays a key role in the neuropathogenesis of Human Immunodeficiency Virus-1 (HIV-1 infection causing apoptosis of astroglia cells and neurons. Recent data have shown that oxidative stress is also responsible for the acceleration of human fibroblast telomere shortening in vitro. In the present study we analyzed the potential relations occurring between free radicals formation and telomere length during HIV-1 mediated astroglial death. Results To this end, U373 human astrocytoma cells have been directly exposed to X4-using HIV-1IIIB strain, for 1, 3 or 5 days and treated (where requested with N-acetylcysteine (NAC, a cysteine donor involved in the synthesis of glutathione (GSH, a cellular antioxidant and apoptosis has been evaluated by FACS analysis. Quantitative-FISH (Q-FISH has been employed for studying the telomere length while intracellular reduced/oxidized glutathione (GSH/GSSG ratio has been determined by High-Performance Liquid Chromatography (HPLC. Incubation of U373 with HIV-1IIIB led to significant induction of cellular apoptosis that was reduced in the presence of 1 mM NAC. Moreover, NAC improved the GSH/GSSG, a sensitive indicator of oxidative stress, that significantly decreased after HIV-1IIIB exposure in U373. Analysis of telomere length in HIV-1 exposed U373 showed a statistically significant telomere shortening, that was completely reverted in NAC-treated U373. Conclusion Our results support the role of HIV-1-mediated oxidative stress in astrocytic death and the importance of antioxidant compounds in preventing these cellular damages. Moreover, these data indicate that the telomere structure, target for oxidative damage, could be the key sensor of cell apoptosis induced by oxidative stress after HIV infection.

In utero and intra-partum HIV-1 transmission and acute HIV-1 infection during pregnancy: using the BED capture enzyme-immunoassay as a surrogate marker for acute infection.

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Marinda, Edmore T; Moulton, Lawrence H; Humphrey, Jean H; Hargrove, John W; Ntozini, Robert; Mutasa, Kuda; Levin, Jonathan

2011-08-01

The BED assay was developed to estimate the proportion of recent HIV infections in a population. We used the BED assay as a proxy for acute infection to quantify the associated risk of mother-to-child-transmission (MTCT) during pregnancy and delivery. Design A total of 3773 HIV-1 sero-positive women were tested within 96 h of delivery using the BED assay, and CD4 cell count measurements were taken. Mothers were classified according to their likelihood of having recently seroconverted. The risk of MTCT in utero and intra-partum was assessed comparing different groups defined by BED and CD4 cell count, adjusting for background factors using multinomial logistic models. Compared with women with BED ≥ 0.8/CD4 ≥ 350 (typical of HIV-1 chronic patients) there was insufficient evidence to conclude that women presenting with BED pregnancy.

A Peptide Derived from the HIV-1 gp120 Coreceptor-Binding Region Promotes Formation of PAP248-286 Amyloid Fibrils to Enhance HIV-1 Infection.

Directory of Open Access Journals (Sweden)

Jinquan Chen

Full Text Available Semen is a major vehicle for HIV transmission. Prostatic acid phosphatase (PAP fragments, such as PAP248-286, in human semen can form amyloid fibrils to enhance HIV infection. Other endogenous or exogenous factors present during sexual intercourse have also been reported to promote the formation of seminal amyloid fibrils.Here, we demonstrated that a synthetic 15-residue peptide derived from the HIV-1 gp120 coreceptor-binding region, designated enhancing peptide 2 (EP2, can rapidly self-assemble into nanofibers. These EP2-derivated nanofibers promptly accelerated the formation of semen amyloid fibrils by PAP248-286, as shown by Thioflavin T (ThT and Congo red assays. The amyloid fibrils presented similar morphology, assessed via transmission electron microscopy (TEM, in the presence or absence of EP2. Circular dichroism (CD spectroscopy revealed that EP2 accelerates PAP248-286 amyloid fibril formation by promoting the structural transition of PAP248-286 from a random coil into a cross-β-sheet. Newly formed semen amyloid fibrils effectively enhanced HIV-1 infection in TZM-bl cells and U87 cells by promoting the binding of HIV-1 virions to target cells.Nanofibers composed of EP2 promote the formation of PAP248-286 amyloid fibrils and enhance HIV-1 infection.

An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients.

Directory of Open Access Journals (Sweden)

Celsa A Spina

Full Text Available The possibility of HIV-1 eradication has been limited by the existence of latently infected cellular reservoirs. Studies to examine control of HIV latency and potential reactivation have been hindered by the small numbers of latently infected cells found in vivo. Major conceptual leaps have been facilitated by the use of latently infected T cell lines and primary cells. However, notable differences exist among cell model systems. Furthermore, screening efforts in specific cell models have identified drug candidates for "anti-latency" therapy, which often fail to reactivate HIV uniformly across different models. Therefore, the activity of a given drug candidate, demonstrated in a particular cellular model, cannot reliably predict its activity in other cell model systems or in infected patient cells, tested ex vivo. This situation represents a critical knowledge gap that adversely affects our ability to identify promising treatment compounds and hinders the advancement of drug testing into relevant animal models and clinical trials. To begin to understand the biological characteristics that are inherent to each HIV-1 latency model, we compared the response properties of five primary T cell models, four J-Lat cell models and those obtained with a viral outgrowth assay using patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models. Our results indicate that no single in vitro cell model alone is able to capture accurately the ex vivo response characteristics of latently infected T cells from patients. Most cell models demonstrated that sensitivity to HIV reactivation was skewed toward or against specific drug classes. Protein kinase C agonists and PHA reactivated latent HIV uniformly across models, although drugs in most other classes did not.

An In-Depth Comparison of Latent HIV-1 Reactivation in Multiple Cell Model Systems and Resting CD4+ T Cells from Aviremic Patients

Science.gov (United States)

Spina, Celsa A.; Anderson, Jenny; Archin, Nancie M.; Bosque, Alberto; Chan, Jonathan; Famiglietti, Marylinda; Greene, Warner C.; Kashuba, Angela; Lewin, Sharon R.; Margolis, David M.; Mau, Matthew; Ruelas, Debbie; Saleh, Suha; Shirakawa, Kotaro; Siliciano, Robert F.; Singhania, Akul; Soto, Paula C.; Terry, Valeri H.; Verdin, Eric; Woelk, Christopher; Wooden, Stacey; Xing, Sifei; Planelles, Vicente

2013-01-01

The possibility of HIV-1 eradication has been limited by the existence of latently infected cellular reservoirs. Studies to examine control of HIV latency and potential reactivation have been hindered by the small numbers of latently infected cells found in vivo. Major conceptual leaps have been facilitated by the use of latently infected T cell lines and primary cells. However, notable differences exist among cell model systems. Furthermore, screening efforts in specific cell models have identified drug candidates for “anti-latency” therapy, which often fail to reactivate HIV uniformly across different models. Therefore, the activity of a given drug candidate, demonstrated in a particular cellular model, cannot reliably predict its activity in other cell model systems or in infected patient cells, tested ex vivo. This situation represents a critical knowledge gap that adversely affects our ability to identify promising treatment compounds and hinders the advancement of drug testing into relevant animal models and clinical trials. To begin to understand the biological characteristics that are inherent to each HIV-1 latency model, we compared the response properties of five primary T cell models, four J-Lat cell models and those obtained with a viral outgrowth assay using patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models. Our results indicate that no single in vitro cell model alone is able to capture accurately the ex vivo response characteristics of latently infected T cells from patients. Most cell models demonstrated that sensitivity to HIV reactivation was skewed toward or against specific drug classes. Protein kinase C agonists and PHA reactivated latent HIV uniformly across models, although drugs in most other classes did not. PMID:24385908

Identification of conserved subdominant HIV Type 1 CD8(+) T Cell epitopes restricted within common HLA Supertypes for therapeutic HIV Type 1 vaccines

DEFF Research Database (Denmark)

Karlsson, Ingrid; Kløverpris, Henrik; Jensen, Kristoffer Jarlov

2012-01-01

The high HIV-1 prevalence, up to 4.6% in Guinea-Bissau, West Africa, makes it a relevant location for testing of therapeutic vaccines. With the aim of performing a clinical study in Guinea-Bissau, after first testing the vaccine for safety in Denmark, Europe, we here describe the design...... of a universal epitope peptide-based T cell vaccine with relevance for any geographic locations. The two major obstacles when designing such a vaccine are the high diversities of the HIV-1 genome and of the human major histocompatibility complex (MHC) class I. We selected 15 CD8-restricted epitopes predicted......-specific, HLA-restricted T cell specificities using peptide-MHC class I tetramer labeling of CD8(+) T cells from HIV-1-infected individuals. The selected vaccine epitopes are infrequently targeted in HIV-1-infected individuals from both locations. Moreover, we HLA-typed HIV-1-infected individuals...