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10.1186/1742-4690-10-129Retrovirology10112

Latent HIV-1 Infection of Resting CD4+ T Cells in the Humanized Rag2-/- c-/- Mouse

Persistent human immunodeficiency virus type 1 (HIV-1) infection of resting CD4+ T cells, unaffected by antiretroviral therapy (ART), provides a long-lived reservoir of HIV infection. Therapies that target this viral reservoir are needed to eradicate HIV-1 infection. A small-animal model that recapitulates HIV-1 latency in resting CD4+ T cells may accelerate drug discovery and allow the rational design of nonhuman primate (NHP) or human studies. We report that in humanized Rag2−/− γc−/− (hu-Rag2−/− γc−/−) mice, as in humans, resting CD4+ T cell infection (RCI) can be quantitated in pooled samples of circulating cells and tissue reservoirs (e.g., lymph node, spleen, bone marrow) following HIV-1 infection with the CCR5-tropic JR-CSF strain and suppression of viremia by ART. Replication-competent virus was recovered from pooled resting CD4+ T cells in 7 of 16 mice, with a median frequency of 8 (range, 2 to 12) infected cells per million T cells, demonstrating that HIV-1 infection can persist despite ART in the resting CD4+ T cell reservoir of hu-Rag2−/− γc−/− mice. This model will allow rapid preliminary assessments of novel eradication approaches and combinatorial strategies that may be challenging to perform in the NHP model or in humans, as well as a rigorous analysis of the effect of these interventions in specific anatomical compartments

Quantitation of Replication-Competent HIV-1 in Populations of Resting CD4+ T Cells

Central memory (TCM) CD4+ T cells are the principal reservoir of latent HIV-1 infection that persists despite durable, successful antiretroviral therapy (ART). In a study that measured HIV DNA in 17 patients and replication-competent HIV in 4 patients, pools of resting and activated transitional memory (TTM) CD4+ T cells were found to be a reservoir for HIV infection. As defective viruses account for the majority of integrated HIV DNA and do not reflect the actual frequency of latent, replication-competent proviral infection, we assessed the specific contribution of resting TTM cells to latent HIV infection. We measured the frequency of replication-competent HIV in purified resting memory cell subpopulations by a limiting-dilution, quantitative viral outgrowth assay (QVOA). HIV was routinely detected within the resting central memory compartment but was infrequently detected within the resting TTM compartment. These observations suggest that prolonged ART may limit persistent latent infection in the TTM compartment. Our results confirm the importance of latent infection within the TCM compartment and again focus attention on these cells as the most important latent viral reservoir. While proliferation may drive expansion of detectable viral genomes in cells, the frequency of replication-competent HIV must be carefully assessed. Latent infection appears to wane within the transitional memory compartment in patients who have sustained successful viral suppression via ART or were treated very early in infection

Peripheral Vγ9Vδ2 T Cells Are a Novel Reservoir of Latent HIV Infection

Eradication of HIV infection will require the identification of all cellular reservoirs that harbor latent infection. Despite low or lack of CD4 receptor expression on Vδ2 T cells, infection of these cells has previously been reported. We found that upregulation of the CD4 receptor may render primary Vδ2 cells target for HIV infection in vitro and we propose that HIV-induced immune activation may allow infection of γδ T cells in vivo. We assessed the presence of latent HIV infection by measurements of DNA and outgrowth assays within Vδ2 cells in 18 aviremic patients on long-standing antiretroviral therapy. In 14 patients we recovered latent but replication-competent HIV from highly purified Vδ2 cells demonstrating that peripheral Vδ2 T cells are a previously unrecognized reservoir in which latent HIV infection is unexpectedly frequent

In-vivo administration of histone deacetylase inhibitors does not impair natural killer cell function in HIV+ individuals

Histone deacetylase inhibitors (HDACi) have proven to induce HIV-RNA and antigen expression in resting CD4 T cells of antiretroviral therapy (ART)-treated HIV-infected individuals. However, to achieve viral eradication, immune clearance must follow latency reversal, and thus it is essential to understand the impact of latency reversal agents on immune function.Design:Here we evaluate the impact of in-vivo administration of vorinostat (VOR) and panobinostat (PNB) during clinical trials on natural killer (NK) cell function and phenotype.Methods:Cryopreserved peripheral blood mononuclear cells from HIV-positive participants receiving VOR (NCT01319383) or PNB (NCT01680094) were selected to assess the impact of the drugs on cell composition, activation, NK cell phenotype (CD16, NKG2D, NKp30, NKp46 and DNAM-1), cytotoxic activity (CD107a), and interferon (IFN)-γ production.Results:No impairment of NK cell function was observed during treatment with either VOR or PNB. An increase in the frequency of CD3-CD56 NK cells was consistently observed. Interestingly, after VOR administration, NK cells increased expression of NKp46 and CD16, and showed improved degranulation and IFN-γ production capacity. Moreover, taking together VOR and PNB samples, HIV DNA levels in CD4 cells were negatively correlated with NK cell frequency and NK cell expression of CD16.Conclusions:In-vivo treatment with HDACi does not have measurable negative effects on NK cell function, with some evidence of improved function in vitro. These results have important implications for potential combinatorial approaches to target HIV reservoirs, suggesting that the use of HDACis as a latency reversal agent could be paired with interventions to enhance NK cell activity or recruitment

Machine learning approaches identify immunologic signatures of total and intact HIV DNA during long-term antiretroviral therapy

Understanding the interplay between the HIV reservoir and the host immune system may yield insights into HIV persistence during antiretroviral therapy (ART) and inform strategies for a cure. Here, we applied machine learning (ML) approaches to cross-sectional high-parameter HIV reservoir and immunology data in order to characterize host-reservoir associations and generate new hypotheses about HIV reservoir biology. High-dimensional immunophenotyping, quantification of HIV-specific T cell responses, and measurement of genetically intact and total HIV proviral DNA frequencies were performed on peripheral blood samples from 115 people with HIV (PWH) on long-term ART. Analysis demonstrated that both intact and total proviral DNA frequencies were positively correlated with T cell activation and exhaustion. Years of ART and select bifunctional HIV-specific CD4 T cell responses were negatively correlated with the percentage of intact proviruses. A leave-one-covariate-out inference approach identified specific HIV reservoir and clinical-demographic parameters, such as age and biological sex, that were particularly important in predicting immunophenotypes. Overall, immune parameters were more strongly associated with total HIV proviral frequencies than intact proviral frequencies. Uniquely, however, expression of the IL-7 receptor alpha chain (CD127) on CD4 T cells was more strongly correlated with the intact reservoir. Unsupervised dimension reduction analysis identified two main clusters of PWH with distinct immune and reservoir characteristics. Using reservoir correlates identified in these initial analyses, decision tree methods were employed to visualize relationships among multiple immune and clinical-demographic parameters and the HIV reservoir. Finally, using random splits of our data as training-test sets, ML algorithms predicted with approximately 70% accuracy whether a given participant had qualitatively high or low levels of total or intact HIV DNA . The techniques described here may be useful for assessing global patterns within the increasingly high-dimensional data used in HIV reservoir and other studies of complex biology

Epigenetic Silencing of HIV-1 by the Histone H3 Lysine 27 Methyltransferase Enhancer of Zeste 2

Latent HIV proviruses are silenced as the result of deacetylation and methylation of histones located at the viral long terminal repeat (LTR). Inhibition of histone deacetylases (HDACs) leads to the reemergence of HIV-1 from latency, but the contribution of histone lysine methyltransferases (HKMTs) to maintaining HIV latency remains uncertain. Chromatin immunoprecipitation experiments using latently infected Jurkat T-cell lines demonstrated that the HKMT enhancer of Zeste 2 (EZH2) was present at high levels at the LTR of silenced HIV proviruses and was rapidly displaced following proviral reactivation. Knockdown of EZH2, a key component of the Polycomb repressive complex 2 (PRC2) silencing machinery, and the enzyme which is required for trimethyl histone lysine 27 (H3K27me3) synthesis induced up to 40% of the latent HIV proviruses. In contrast, there was less than 5% induction of latent proviruses following knockdown of SUV39H1, which is required for H3K9me3 synthesis. Knockdown of EZH2 also sensitized latent proviruses to external stimuli, such as T-cell receptor stimulation, and slowed the reversion of reactivated proviruses to latency. Similarly, cell populations that responded poorly to external stimuli carried HIV proviruses that were enriched in H3K27me3 and relatively depleted in H3K9me3. Treating latently infected cells with the HKMT inhibitor 3-deazaneplanocin A, which targets EZH2, led to the reactivation of silenced proviruses, whereas chaetocin and BIX01294 showed only minimal reactivation activities. These findings suggest that PRC2-mediated silencing is an important feature of HIV latency and that inhibitors of histone methylation may play a useful role in induction strategies designed to eradicate latent HIV pools

Measuring the contribution of γδ T cells to the persistent HIV reservoir

Objective:To study the contribution of γδ T cells to the persistent HIV reservoir.Design:Fifteen HIV-seropositive individuals on suppressive ART were included. We performed parallel quantitative viral outgrowth assays (QVOA) of resting CD4+ T (rCD4) cells in the presence or absence of γδ T cells.Methods:Resting αβ+CD4+ T cells were magnetically isolated from PBMCs using two different custom cocktails, only one kit contained antibodies to deplete γδ T cells, resulting in two populations: rCD4 cells and rCD4 cells depleted of γδ cells. Frequency of infection was analyzed by QVOA and DNA measurements.Results:Recovery of replication-competent HIV from cultures of rCD4 cells was similar in 11 individuals despite the presence of γδ T cells. In four donors, HIV recovery was lower when γδ T cells were present. Expression of the cytotoxic marker CD16+ on Vδ2 cells was the only variable associated with the lower HIV recovery. Our results highlight the potency of those responses since a mean of 10000 γδ T cells were present within 2.5 million rCD4 cells. However, despite the low frequency of γδ T cells, the presence of cytotoxic Vδ2 cells correlated with lower HIV recovery from cultures of rCD4 cells.Conclusion:Results of this study show that quantification of the contribution of γδ T cells to the reservoir is challenging because of their low numbers compared with conventional rCD4 cells and highlights the potent antiviral function of γδ T cells and the impact of their presence on the frequency of latent HIV infection

Transient CD4+ T cell depletion during suppressive ART reduces the HIV reservoir in humanized mice

Lifelong treatment is required for people living with HIV as current antiretroviral therapy (ART) does not eradicate HIV infection. Latently infected cells are essentially indistinguishable from uninfected cells and cannot be depleted by currently available approaches. This study evaluated antibody mediated transient CD4+ T cell depletion as a strategy to reduce the latent HIV reservoir. Anti-CD4 antibodies effectively depleted CD4+ T cells in the peripheral blood and tissues of humanized mice. We then demonstrate that antibody-mediated CD4+ T cell depletion of HIV infected ART-suppressed animals results in substantial reductions in cell-associated viral RNA and DNA levels in peripheral blood cells over the course of anti-CD4 antibody treatment. Recovery of CD4+ T cells was observed in all tissues analyzed except for the lung 26 days after cessation of antibody treatment. After CD4+ T cell recovery, significantly lower levels of cell-associated viral RNA and DNA were detected in the tissues of anti-CD4 antibody-treated animals. Further, an 8.5-fold reduction in the levels of intact HIV proviral DNA and a 3.1-fold reduction in the number of latently infected cells were observed in anti-CD4-antibody-treated animals compared with controls. However, there was no delay in viral rebound when ART was discontinued in anti-CD4 antibody-treated animals following CD4+ T cell recovery compared with controls. Our results suggest that transient CD4+ T cell depletion, a long-standing clinical intervention that might have an acceptable safety profile, during suppressive ART can reduce the size of the HIV reservoir in humanized mice