Cell-associated Human Immunodeficiency Virus Research Articles

Transcriptionally Active Defective HIV-1 Proviruses and Their Association With Immunological Nonresponse to Antiretroviral Therapy.

A subset of antiretroviral therapy-treated persons with human immunodeficiency virus (HIV), referred to as immunological nonresponders (INRs), fails to normalize CD4+ T-cell numbers. In a case-control study involving 26 INRs (CD4 < 250 cells/µL) and 25 immunological responders (IRs; CD4 ≥ 250 cells/µL), we evaluated the potential contribution of transcriptionally competent defective HIV-1 proviruses to poor CD4+ T-cell recovery. Compared to the responders, the INRs had higher levels of cell-associated HIV RNA (P = .034) and higher percentages of HLA-DR+ CD4+ T cells (P < .001). While not encoding replication-competent viruses, the RNA transcripts frequently encoded HIV-1 Gag-p17 and Nef proteins. These transcripts and/or resulting proteins may activate pathway(s) leading to the immunological nonresponse phenotype.

Immunologic and Virologic Parameters Associated With Human Immunodeficiency Virus (HIV) DNA Reservoir Size in People With HIV Receiving Antiretroviral Therapy.

A better understanding of the dynamics of human immunodeficiency virus (HIV) reservoirs in CD4+ T cells of people with HIV (PWH) receiving antiretroviral therapy (ART) is crucial for developing therapies to eradicate the virus. We conducted a study involving 28 aviremic PWH receiving ART with high and low levels of HIV DNA. We analyzed immunologic and virologic parameters and their association with the HIV reservoir size. The frequency of CD4+ T cells carrying HIV DNA was associated with higher pre-ART plasma viremia, lower pre-ART CD4+ T-cell counts, and lower pre-ART CD4/CD8 ratios. During ART, the High group maintained elevated levels of intact HIV proviral DNA, cell-associated HIV RNA, and inducible virion-associated HIV RNA. HIV sequence analysis showed no evidence for preferential accumulation of defective proviruses nor higher frequencies of clonal expansion in the High versus Low group. Phenotypic and functional T-cell analyses did not show enhanced immune-mediated virologic control in the Low versus High group. Of considerable interest, pre-ART innate immunity was significantly higher in the Low versus High group. Our data suggest that innate immunity at the time of ART initiation may play an important role in modulating the dynamics and persistence of viral reservoirs in PWH.

The BLT Humanized Mouse Model as a Tool for Studying Human Gamma Delta T Cell-HIV Interactions In Vivo.

Gamma-delta (γδ) T cells recognize antigens in a major histocompatibility complex (MHC) independent and have cytotoxic capability. Human immunodeficiency virus (HIV) infection reduces the proportion of the Vδ2 cell subset compared to the Vδ1 cell subset of γδ T cells in the blood in most infected individuals, except for elite controllers. The capacity of Vδ2 T cells to kill HIV-infected targets has been demonstrated in vitro, albeit in vivo confirmatory studies are lacking. Here, we provide the first characterization of γδ T cell-HIV interactions in bone marrow-liver-thymus (BLT) humanized mice and examined the immunotherapeutic potential of Vδ2 T cells in controlling HIV replication in vivo. We demonstrate a reduced proportion of Vδ2 T cells and an increased proportion of Vδ1 T cells in HIV-infected BLT humanized mice, like in HIV-positive individuals. HIV infection in BLT humanized mice also impaired the ex vivo expansion of Vδ2 T cells, like in HIV-positive individuals. Adoptive transfer of activated Vδ2 T cells did not control HIV replication during cell-associated HIV transmission in BLT humanized mice but instead exacerbated viremia, suggesting that Vδ2 T cells may serve as early targets for HIV replication. Our findings demonstrate that BLT humanized mice can model γδ T cell-HIV interactions in vivo.

Cell-Associated Human Immunodeficiency Virus (HIV) Ribonucleic Acid Has a Circadian Cycle in Males With HIV on Antiretroviral Therapy.

Circadian transcription factors that regulate cell-autonomous circadian clocks can also increase human immunodeficiency virus (HIV) transcription in vitro. We aimed to determine whether circadian variation in HIV transcription exists in people with HIV (PWH) on antiretroviral therapy (ART). We performed a prospective observational study of male PWH on ART, sampling blood every 4 hours for 24 hours. Using quantitative polymerase chain reaction, we quantified expression of circadian-associated genes, HIV deoxyribonucleic acid (DNA), and cell-associated unspliced (CA-US) ribonucleic acid (RNA) in peripheral blood CD4+ T cells. Plasma sex hormones were quantified alongside plasma and salivary cortisol. The primary outcome was to identify temporal variations in CA-US HIV RNA using a linear mixed-effect regression framework and maximum likelihood estimation. Salivary and plasma cortisol, and circadian genes including Clock, Bmal1, and Per3, varied with a circadian rhythm. Cell-associated unspliced HIV RNA and the ratio of CA-US HIV RNA/DNA in CD4+ T cells also demonstrated circadian variations, with no variation in HIV DNA. Circulating estradiol was highly predictive of CA-US HIV RNA variation in vivo. Cell-associated unspliced HIV RNA in PWH on ART varies temporally with a circadian rhythm. These findings have implications for the design of clinical trials and biomarkers to assess HIV cure interventions.

Predictors of Cell-Associated Human Immunodeficiency Virus (HIV)-1 DNA Over 1 Year in Very Early Treated Infants

Younger age of antiretroviral therapy (ART) initiation is associated with smaller viral reservoirs in perinatally acquired HIV-1 infection, but there is wide variability among early-treated infants. Predictors of this variability are not fully described. Sixty-three neonates diagnosed with HIV-1 <48 hours after birth in Johannesburg, South Africa, were started on ART as soon as possible. Fifty-nine (94%) infants received nevirapine prophylaxis from birth until ART start. Viably preserved peripheral blood mononuclear cells (PBMCs) collected at regular intervals to 48 weeks, and from mothers at enrollment, were tested using integrase-targeted, semi-nested, real-time quantitative hydrolysis probe (TaqMan) PCR assays to quantify total HIV-1 subtype C viral DNA (vDNA). Predictors were investigated using generalized estimating equation regression. Thirty-one (49.2%) infants initiated ART <48 hours, 24 (38.1%) <14 days, and 8 (12.7%) >14 days of birth. Three-quarters were infected despite maternal antenatal ART (however, only 9.5% of women had undetectable viral load closest to delivery) and 86% were breastfed. Higher infant CD4+ T-cell percentage and viral load <100 000 copies/mL pre-ART were associated with lower vDNA in the first 48 weeks after ART start. No antenatal maternal ART and breastfeeding were also associated with lower vDNA. Older age at ART initiation had a discernible negative impact when initiated >14 days. Among very early treated infants, higher CD4+ T-cell percentage and viral load <100 000 copies/mL pre-ART, infection occurring in the absence of maternal antenatal ART, and breastfeeding were associated with lower levels of HIV-1 DNA in the first 48 weeks of treatment. Clinical Trials Registration. clinicaltrials.gov (NCT02431975).

Prolonged administration of maraviroc reactivates latent HIV in vivo but it does not prevent antiretroviral-free viral rebound

Human immunodeficiency virus (HIV) remains incurable due to latent viral reservoirs established in non-activated CD4 T cells that cannot be eliminated via antiretroviral therapy. Current efforts to cure HIV are focused on identifying drugs that will induce viral gene expression in latently infected cells, commonly known as latency reversing agents (LRAs). Some drugs have been shown to reactivate latent HIV but do not cause a reduction in reservoir size. Therefore, finding new LRAs or new combinations or increasing the round of stimulations is needed to cure HIV. However, the effects of these drugs on viral rebound after prolonged treatment have not been evaluated. In a previous clinical trial, antiretroviral therapy intensification with maraviroc for 48 weeks caused an increase in residual viremia and episomal two LTR-DNA circles suggesting that maraviroc could reactivate latent HIV. We amended the initial clinical trial to explore additional virologic parameters in stored samples and to evaluate the time to viral rebound during analytical treatment interruption in three patients. Maraviroc induced an increase in cell-associated HIV RNA during the administration of the drug. However, there was a rapid rebound of viremia after antiretroviral therapy discontinuation. HIV-specific T cell response was slightly enhanced. These results show that maraviroc can reactivate latent HIV in vivo but further studies are required to efficiently reduce the reservoir size.

Human Immunodeficiency Virus (HIV)-Infected CCR6+ Rectal CD4+ T Cells and HIV Persistence On Antiretroviral Therapy.

BackgroundIdentifying where human immunodeficiency virus (HIV) persists in people living with HIV and receiving antiretroviral therapy is critical to develop cure strategies. We assessed the relationship of HIV persistence to expression of chemokine receptors and their chemokines in blood (n = 48) and in rectal (n = 20) and lymph node (LN; n = 8) tissue collected from people living with HIV who were receiving suppressive antiretroviral therapy.MethodsCell-associated integrated HIV DNA, unspliced HIV RNA, and chemokine messenger RNA were quantified by quantitative polymerase chain reaction. Chemokine receptor expression on CD4+ T cells was determined using flow cytometry.ResultsIntegrated HIV DNA levels in CD4+ T cells, CCR6+CXCR3+ memory CD4+ T-cell frequency, and CCL20 expression (ligand for CCR6) were highest in rectal tissue, where HIV-infected CCR6+ T cells accounted for nearly all infected cells (median, 89.7%). Conversely in LN tissue, CCR6+ T cells were infrequent, and there was a statistically significant association of cell-associated HIV DNA and RNA with CCL19, CCL21, and CXCL13 chemokines.ConclusionsHIV-infected CCR6+ CD4+ T cells accounted for the majority of infected cells in rectal tissue. The different relationships between HIV persistence and T-cell subsets and chemokines in rectal and LN tissue suggest that different tissue-specific strategies may be required to eliminate HIV persistence and that assessment of biomarkers for HIV persistence may not be generalizable between blood and other tissues.

Activation of Latent HIV-1 T Cell Reservoirs with a Combination of Innate Immune and Epigenetic Regulators.

The presence of T cell reservoirs in which human immunodeficiency virus (HIV) establishes latency by integrating into the host genome represents a major obstacle to an HIV cure and has prompted the development of strategies aimed at the eradication of HIV from latently infected cells. The "shock-and-kill" strategy is one of the most pursued approaches to the elimination of viral reservoirs. Although several latency-reversing agents (LRAs) have shown promising reactivation activity, they have failed to eliminate the cellular reservoir. In this study, we evaluated a novel immune system-mediated approach to clearing the HIV reservoir, based on a combination of innate immune stimulation and epigenetic reprogramming. The combination of the STING agonist cGAMP (cyclic GMP-AMP) and the FDA-approved histone deacetylase inhibitor resminostat resulted in a significant increase in HIV proviral reactivation and specific apoptosis in HIV-infected cells in vitro Reductions in the proportion of HIV-harboring cells and the total amount of HIV DNA were also observed in CD4+ central memory T (TCM) cells, a primary cell model of latency, where resminostat alone or together with cGAMP induced high levels of selective cell death. Finally, high levels of cell-associated HIV RNA were detected ex vivo in peripheral blood mononuclear cells (PBMCs) and CD4+ T cells from individuals on suppressive antiretroviral therapy (ART). Although synergism was not detected in PBMCs with the combination, viral RNA expression was significantly increased in CD4+ T cells. Collectively, these results represent a promising step toward HIV eradication by demonstrating the potential of innate immune activation and epigenetic modulation for reducing the viral reservoir and inducing specific death of HIV-infected cells.IMPORTANCE One of the challenges associated with HIV-1 infection is that despite antiretroviral therapies that reduce HIV-1 loads to undetectable levels, proviral DNA remains dormant in a subpopulation of T lymphocytes. Numerous strategies to clear residual virus by reactivating latent virus and eliminating the reservoir of HIV-1 (so-called "shock-and-kill" strategies) have been proposed. In the present study, we use a combination of small molecules that activate the cGAS-STING antiviral innate immune response (the di-cyclic nucleotide cGAMP) and epigenetic modulators (histone deacetylase inhibitors) that induce reactivation and HIV-infected T cell killing in cell lines, primary T lymphocytes, and patient samples. These studies represent a novel strategy for HIV eradication by reducing the viral reservoir and inducing specific death of HIV-infected cells.

Targeting Immune Checkpoint Molecules to Eliminate Latent HIV.

The advent of antiretroviral therapy (ART) has seen a dramatic decrease in the morbidity and mortality of individuals infected with human immunodeficiency virus (HIV). However, ART is not curative and HIV persists in treated individuals within a pool of infected CD4+ memory T cells. The targeting and elimination of these cells, termed the latent HIV reservoir, may be essential in establishing a cure for HIV. Current HIV reservoir research is focused on identifying cells that harbor latent, replication-competent, HIV provirus using specific cell surface markers. Recently, studies have turned to immune checkpoint (IC) molecules, such as programmed cell death protein 1 (PD-1). IC molecules are regulators of the immune system and have previously been linked to HIV infection. Furthermore, cells isolated from treated individuals co-expressing PD-1 alongside other IC molecules are enriched for HIV DNA. Administration of a IC blocking antibodies resulted in an increase of cell-associated HIV RNA within an individual, indicating the potential for this therapeutic to be utilized as a latency reversing agent. IC inhibitors could target CD4+ T cells expressing IC molecules and possibly enhance HIV transcription, allowing for the elimination of these cells by either ART or the immune system. However, treatment with IC inhibitors has been associated with toxicities such as immune-related adverse events and therefore future studies should proceed with caution.

Cell-Associated Simian Immunodeficiency Virus Accelerates Initial Virus Spread and CD4+ T-Cell Depletion in the Intestinal Mucosa.

Cell-free and cell-associated human immunodeficiency virus (HIV) may differently affect the immune system and the efficacy of prevention strategies. Here we examined mucosal events in simian immunodeficiency virus (SIV) infection, using infected cells together with cell-free virus and cell-free virus alone. Intravenously inoculated SIV-infected cells disseminated virus to the intestine within 16 hours. Infection with both virus forms accelerated viral dissemination in the intestinal mucosa and the loss of mucosal CD4+ T cells as compared to infection with cell-free virus only. As all natural sources of HIV infection contain both virus forms, future prevention studies should focus on efficacy against both cell-free and cell-associated virus.

Human Immunodeficiency Virus Persistence and T-Cell Activation in Blood, Rectal, and Lymph Node Tissue in Human Immunodeficiency Virus-Infected Individuals Receiving Suppressive Antiretroviral Therapy.

Immune activation and inflammation remain elevated in human immunodeficiency virus (HIV)-infected individuals receiving antiretroviral therapy (ART) and may contribute to HIV persistence. Using flow cytometry expression of CD38, HLA-DR and PD-1 were measured in blood (n = 48), lymph node (LN; n = 9), and rectal tissue (n = 17) from virally suppressed individuals. Total and integrated HIV DNA, 2-LTR circles, and cell-associated unspliced HIV RNA were quantified. CD4+ T cells from rectal tissue had a higher frequency of integrated HIV DNA compared with blood (4.26 fold-change in DNA; 95% confidence interval [CI] = 2.61-7.00; P < .001) and LN (2.32 fold-change in DNA; 95% CI = 1.22-4.41; P = .01). In rectal tissue, there were positive associations between integrated HIV DNA with PD-1+ CD4+ T-cells (1.44 fold-change in integrated HIV DNA per 10-unit increase in PD-1+ CD4+ T cells; 95% CI = 1.01-2.05; P = .045) and CD38+HLA-DR+ CD8+ T cells (1.40 fold-change in integrated HIV DNA per 1-unit increase in CD38+HLA-DR+ CD8+ T cells; 95% CI = 1.05-1.86; P = .02). Both associations were independent of current and nadir CD4+ T-cell counts. During ART, rectal tissue is an important reservoir for HIV persistence with a high frequency of activated CD4+ and CD8+ T cells. PD-1 may represent a marker of HIV persistence in rectal tissue.

MIV-150/zinc acetate gel inhibits cell-associated simian-human immunodeficiency virus reverse transcriptase infection in a macaque vaginal explant model.

The transmission of both cell-free and cell-associated immunodeficiency viruses has been demonstrated directly in multiple animal species and possibly occurs in humans, as suggested by genotyping of the infecting human immunodeficiency virus (HIV) in acutely infected women and in semen from their partners. Therefore, a microbicide may need to block both mechanisms of HIV transmission to achieve maximum efficacy. To date, most of the preclinical evaluation of candidate microbicides has been performed using cell-free HIV. New models of mucosal transmission of cell-associated HIV are needed to evaluate candidate microbicide performance. The MIV-150/zinc acetate/carrageenan (MZC) gel protects Depo-Provera-treated macaques against cell-free simian-human immunodeficiency virus reverse transcriptase (SHIV-RT) infection when applied vaginally up to 8 h before challenge. We recently demonstrated the potent activity of MZC gel against cell-free SHIV-RT in macaque vaginal explants. In the current study, we established a cell-associated SHIV-RT infection model of macaque vaginal tissues and tested the activity of MZC gel in this model. MZC gel protected tissues against cell-associated SHIV-RT infection when present at the time of viral exposure or when applied up to 4 days prior to viral challenge. These data support clinical testing of the MZC gel. Overall, our ex vivo model of cell-associated SHIV-RT infection in macaque vaginal mucosa complements the cell-free infection models, providing tools for prioritization of products that block both modes of HIV transmission.

Early Antiretroviral Therapy in South African Children Reduces HIV-1-Infected Cells and Cell-Associated HIV-1 RNA in Blood Mononuclear Cells.

We measured cell-associated human immunodeficiency virus (HIV)-1 DNA (CAD) and RNA (CAR) and plasma HIV-1 RNA in blood samples from 20 children in the Children with HIV Early Antiretroviral (CHER) cohort after 7-8 years of suppressive combination antiretroviral therapy (cART). Children who initiated cART early (<2 months; n = 12) had lower HIV-1 CAD (median, 48 vs 216; P < .01) and CAR (median, 5 vs 436; P < .01) per million peripheral blood mononuclear cells than children who started later (≥ 2 months; n = 8). Plasma HIV-1 RNA levels were not significantly lower in early-treated children (0.5 vs 1.2 copies/mL; P = .16). Early treatment at <2 months of age reduces the number of HIV-infected cells and HIV CAR.

HIV infection en route to endogenization: two cases

The long-term spontaneous evolution of humans and the human immunodeficiency virus (HIV) is not well characterized; many vertebrate species, including humans, exhibit remnants of other retroviruses in their genomes that question such possible endogenization of HIV. We investigated two HIV-infected patients with no HIV-related disease and no detection with routine tests of plasma HIV RNA or cell-associated HIV DNA. We used Sanger and deep sequencing to retrieve HIV DNA sequences integrated in the human genome and tested the host humoral and cellular immune responses. We noticed that viruses from both patients were inactivated by the high prevalence of the transformation of tryptophan codons into stop codons (25% overall (3–100% per gene) and 24% overall (0–50% per gene)). In contrast, the humoral and/or cellular responses were strong for one patient and moderate for the other, indicating that a productive infection occurred at one stage of the infection. We speculate that the stimulation of APOBEC, the enzyme group that exchanges G for A in viral nucleic acids and is usually inhibited by the HIV protein Vif, has been amplified and made effective from the initial stage of the infection. Furthermore, we propose that a cure for HIV may occur through HIV endogenization in humans, as observed for many other retroviruses in mammals, rather than clearance of all traces of HIV from human cells, which defines viral eradication.

Cell-associated transmission of HIV type 1 and other lentiviruses in small-animal models.

Small-animal models of lentivirus transmission have repeatedly demonstrated transmission by cell-associated virus via vaginal, rectal, and oral routes. The earliest experiments were in the cat/feline immunodeficiency virus model, followed a decade later by successful vaginal transmission of cell-associated human immunodeficiency virus (HIV) in mice bearing transplanted human immune cells. After early unsuccessful attempts at cell-associated transmission in nonhuman primates, renewed investigation in diverse primate models has now confirmed the findings from the cat and humanized mouse models. Improvements in humanized mouse models have made them the preferred small-animal models to study HIV mucosal transmission. They provide complementary systems to nonhuman primate models to aid in the elucidation of the many remaining questions on the mechanism of and means to prevent both cell-associated and cell-free HIV transmission across mucosal barriers.

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

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.

Strategies for preventing mucosal cell-associated HIV transmission.

Human immunodeficiency virus (HIV) may be transmitted through either cell-free virions or leukocytes harboring intracellular HIV in bodily fluids. In recent years, the early initiation of combination antiretroviral therapy leading to virological suppression has resulted in decreased HIV transmission to uninfected partners. Additionally, the efficacy of primary chemoprophylaxis with oral or topical antiretroviral regimens containing tenofovir (with or without emtricitabine) has been demonstrated. However, the efficacy of these approaches may be compromised by suboptimal adherence, decreased drug concentrations in mucosal compartments in women, and genital inflammation. Furthermore, in vitro studies on the effects of tenofovir on cell-associated HIV transmission have produced conflicting results. Preclinical studies suggest that combination preventive approaches may be most effective in stopping the transmission of HIV after mucosal exposure. Since the development of antibodies were found to correlate with protection in the only effective HIV vaccine trial, the administration of preformed mucosal and systemic antibodies may inform the development of safe and effective antibody-based oral, topical, and/or systemic preexposure prophylaxis agents and provide guidance in the development of HIV vaccines that effectively block cell-associated HIV transmission.

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

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.

Cell-associated HIV mucosal transmission: the neglected pathway.

This supplement to The Journal of Infectious Diseases is devoted to the important and understudied topic of cell-associated human immunodeficiency virus Type 1 (HIV) mucosal transmission. It stems from a workshop held in Boston, Massachusetts, in October 2013, in which scientists discussed their research and insights regarding cell-associated HIV mucosal transmission. The 10 articles in this supplement present the case for cell-associated HIV transmission as an important element contributing to the HIV epidemic, review evidence for the efficacy of current HIV prevention strategies against cell-associated HIV transmission and opportunities for further development, and describe in vitro, ex vivo, and animal cell-associated transmission models that can be used to further elucidate the molecular mechanisms of cell-associated HIV mucosal transmission and test HIV prevention strategies. We hope that these articles will help to inform and invigorate the HIV prevention field and contribute to the development of more-effective vaccine, treatment, and microbicide strategies for HIV prevention.

Decreased HIV type 1 transcription in CCR5-Δ32 heterozygotes during suppressive antiretroviral therapy.

Individuals who are heterozygous for the CCR5-Δ32 mutation provide a natural model to examine the effects of reduced CCR5 expression on human immunodeficiency virus (HIV) persistence. We evaluated the HIV reservoir in 18 CCR5-Δ32 heterozygotes and 54 CCR5 wild-type individuals during suppressive antiretroviral therapy. Cell-associated HIV RNA levels (P=.035), RNA to DNA transcriptional ratios (P=.013), and frequency of detectable HIV 2-long terminal repeat circular DNA (P=.013) were significantly lower in CD4+ T cells from CCR5-Δ32 heterozygotes. Cell-associated HIV RNA was significantly correlated with CCR5 surface expression on CD4+ T cells (r2=0.136; P=.002). Our findings suggest that curative strategies should further explore manipulation of CCR5.