Histone deacetylase inhibitors induce complex host responses that contribute to differential potencies of these compounds in HIV reactivation.

Histone deacetylase (HDAC) inhibitors (HDACis) have been widely tested in clinical trials for their ability to reverse HIV latency but have yielded only limited success. One HDACi, suberoylanilide hydroxamic acid (SAHA), exhibits off-target effects on host gene expression predicted to interfere with induction of HIV transcription. Romidepsin (RMD) has higher potency and specificity for class I HDACs implicated in maintaining HIV provirus in the latent state. More robust HIV reactivation has indeed been achieved with RMD use ex vivo than with SAHA; however, reduction of viral reservoir size has not been observed in clinical trials. Therefore, using RNA-Seq, we sought to compare the effects of SAHA and RMD on gene expression in primary CD4+ T cells. Among the genes whose expression was modulated by both HDACi agents, we identified genes previously implicated in HIV latency. Two genes, SMARCB1 and PARP1, whose modulation by SAHA and RMD is predicted to inhibit HIV reactivation, were evaluated in the major maturation subsets of CD4+ T cells and were consistently either up- or down-regulated by both HDACi compounds. Our results indicate that despite having different potencies and HDAC specificities, SAHA and RMD modulate an overlapping set of genes, implicated in HIV latency regulation. Some of these genes merit exploration as additional targets to improve the therapeutic outcomes of "shock and kill" strategies. The overall complexity of HDACi-induced responses among host genes with predicted stimulatory or inhibitory effects on HIV expression likely contributes to differential HDACi potencies and dictates the outcome of HIV reactivation.

Subclinical Cytomegalovirus DNA Is Associated with CD4 T Cell Activation and Impaired CD8 T Cell CD107a Expression in People Living with HIV despite Early Antiretroviral Therapy.

Most people living with HIV (PLWH) are coinfected with cytomegalovirus (CMV). Subclinical CMV replication is associated with immune dysfunction and with increased HIV DNA in antiretroviral therapy (ART)-naive and -suppressed PLWH. To identify immunological mechanisms by which CMV could favor HIV persistence, we analyzed 181 peripheral blood mononuclear cell (PBMC) samples from 64 PLWH starting ART during early HIV infection with subsequent virologic suppression up to 58 months. In each sample, we measured levels of CMV and Epstein-Barr virus (EBV) DNA by droplet digital PCR (ddPCR). We also measured expression of immunological markers for activation (HLA-DR+ CD38+), cycling (Ki-67+), degranulation (CD107a+), and the immune checkpoint protein PD-1 on CD4+ and CD8+ T cell memory subsets. Significant differences in percentages of lymphocyte markers by CMV/EBV shedding were identified using generalized linear mixed-effects models. Overall, CMV DNA was detected at 60/181 time points. At the time of ART initiation, the presence of detectable CMV DNA was associated with increased CD4+ T cell activation and CD107a expression and with increased CD8+ T cellular cycling and reduced CD107a expression on CD8+ T cells. While some effects disappeared during ART, greater CD4+ T cell activation and reduced CD107a expression on CD8+ T cells persisted when CMV was present (P < 0.01). In contrast, EBV was not associated with any immunological differences. Among the covariates, peak HIV RNA and CD4/CD8 ratio had the most significant effect on the immune system. In conclusion, our study identified immune differences in PLWH with detectable CMV starting early ART, which may represent an additional hurdle for HIV cure efforts.IMPORTANCE Chronic viral infections such as with HIV and CMV last a lifetime and can continually antagonize the immune system. Both viruses are associated with higher expression of inflammation markers, and recent evidence suggests that CMV may complicate efforts to deplete HIV reservoirs. Our group and others have shown that CMV shedding is associated with a larger HIV reservoir. Subclinical CMV replication could favor HIV persistence via bystander effects on our immune system. In this study, we collected longitudinal PBMC samples from people starting ART and measured immune changes associated with detectable CMV. We found that when CMV was detectable, CD4+ T cell activation was higher and CD8+ T cell degranulation was lower. Both results may contribute to the slower decay of the size of the reservoir during CMV replication, since activated CD4+ T cells are more vulnerable to HIV infection, while the loss of CD8+ T cell degranulation may impede the proper killing of infected cells.

Transcriptomic Analysis Implicates the p53 Signaling Pathway in the Establishment of HIV-1 Latency in Central Memory CD4 T Cells in an In Vitro Model.

The search for an HIV-1 cure has been greatly hindered by the presence of a viral reservoir that persists despite antiretroviral therapy (ART). Studies of HIV-1 latency in vivo are also complicated by the low proportion of latently infected cells in HIV-1 infected individuals. A number of models of HIV-1 latency have been developed to examine the signaling pathways and viral determinants of latency and reactivation. A primary cell model of HIV-1 latency, which incorporates the generation of primary central memory CD4 T cells (TCM), full-length virus infection (HIVNL4-3) and ART to suppress virus replication, was used to investigate the establishment of HIV latency using RNA-Seq. Initially, an investigation of host and viral gene expression in the resting and activated states of this model indicated that the resting condition was reflective of a latent state. Then, a comparison of the host transcriptome between the uninfected and latently infected conditions of this model identified 826 differentially expressed genes, many of which were related to p53 signaling. Inhibition of the transcriptional activity of p53 by pifithrin-α during HIV-1 infection reduced the ability of HIV-1 to be reactivated from its latent state by an unknown mechanism. In conclusion, this model may be used to screen latency reversing agents utilized in shock and kill approaches to cure HIV, to search for cellular markers of latency, and to understand the mechanisms by which HIV-1 establishes latency.

Pharmacologic Inhibition of Nedd8 Activation Enzyme Exposes CD4-Induced Epitopes within Env on Cells Expressing HIV-1.

UNLABELLED: HIV-1 Vpu decreases the exposure of epitopes within the viral envelope glycoprotein (Env) on the surface of infected cells by downregulating both BST2 and CD4. To test the hypothesis that inhibiting Vpu activity would increase the exposure of these epitopes and sensitize infected cells to antibody-dependent cellular cytotoxicity (ADCC), we treated cells with the Nedd8 activation enzyme (NAE) inhibitor MLN4924, which inhibits the cullin1-based ubiquitin ligase complex coopted by Vpu to degrade cellular targets. Treatment of HeLa cells with MLN4924 or expression of a dominant negative mutant of cullin1 inhibited the Vpu-mediated downregulation of CD4 but not the downregulation of BST2. NAE inhibition also increased the surface exposure of CD4-induced epitopes within Env on HEK293 cells containing an inducible HIV genome, on infected CEM T cells, and on infected primary T cells. In contrast, the Vpu-mediated downregulation of BST2 was substantially inhibited by MLN4924 only when T cells were treated with alpha interferon (IFN-α) to induce high levels of BST2 expression. As reported previously, the absence of vpu or nef and even more so the combined absence of these two genes sensitized infected cells to ADCC. However, NAE inhibition affected ADCC minimally. Paradoxically, even in infected, IFN-treated cells in which NAE inhibition substantially rescued the surface level of BST2, the surface level of Env detected with an antibody recognizing a CD4-independent epitope (2G12) was minimally increased. Mutation of the C-terminal Vpu residue W76, which supports the ability of Vpu to stimulate virion release by displacing BST2 from assembly sites on the plasma membrane by a cullin1-independent mechanism, increased the exposure of Env detected by 2G12 on infected T cells. Thus, inhibiting the displacement function of Vpu together with its ability to degrade CD4 and BST2 may be required to sensitize infected cells to ADCC. IMPORTANCE: Pathogenic viruses encode gene products that enable evasion of host immune surveillance mechanisms. One such mechanism is antibody-dependent cellular cytotoxicity (ADCC), whereby host antibodies bind envelope glycoproteins of the virus that are inserted into the cellular membrane and direct the destruction of infected cells. Targeting pharmacologically the activity of HIV-1 Vpu, which contributes to evasion of ADCC, could potentially sensitize infected cells to this immune surveillance mechanism, an outcome that would have therapeutic implications with respect to the goal of curing HIV-1 infection. The Nedd8 activation enzyme inhibitor MLN4924 blocks the activity of the host ubiquitin ligase that Vpu coopts to direct the degradation of CD4 and BST2. We observed that while MLN4924 partially reverses the activity of Vpu and could become part of a therapeutic approach by virtue of CD4-induced epitope exposure, sufficient Vpu activity as an antagonist of BST2 persists despite this drug to allow escape from ADCC.

Histone deacetylase inhibitor romidepsin induces HIV expression in CD4 T cells from patients on suppressive antiretroviral therapy at concentrations achieved by clinical dosing.

Persistent latent reservoir of replication-competent proviruses in memory CD4 T cells is a major obstacle to curing HIV infection. Pharmacological activation of HIV expression in latently infected cells is being explored as one of the strategies to deplete the latent HIV reservoir. In this study, we characterized the ability of romidepsin (RMD), a histone deacetylase inhibitor approved for the treatment of T-cell lymphomas, to activate the expression of latent HIV. In an in vitro T-cell model of HIV latency, RMD was the most potent inducer of HIV (EC50 = 4.5 nM) compared with vorinostat (VOR; EC50 = 3,950 nM) and other histone deacetylase (HDAC) inhibitors in clinical development including panobinostat (PNB; EC50 = 10 nM). The HIV induction potencies of RMD, VOR, and PNB paralleled their inhibitory activities against multiple human HDAC isoenzymes. In both resting and memory CD4 T cells isolated from HIV-infected patients on suppressive combination antiretroviral therapy (cART), a 4-hour exposure to 40 nM RMD induced a mean 6-fold increase in intracellular HIV RNA levels, whereas a 24-hour treatment with 1 µM VOR resulted in 2- to 3-fold increases. RMD-induced intracellular HIV RNA expression persisted for 48 hours and correlated with sustained inhibition of cell-associated HDAC activity. By comparison, the induction of HIV RNA by VOR and PNB was transient and diminished after 24 hours. RMD also increased levels of extracellular HIV RNA and virions from both memory and resting CD4 T-cell cultures. The activation of HIV expression was observed at RMD concentrations below the drug plasma levels achieved by doses used in patients treated for T-cell lymphomas. In conclusion, RMD induces HIV expression ex vivo at concentrations that can be achieved clinically, indicating that the drug may reactivate latent HIV in patients on suppressive cART.

Cytomegalovirus replication in semen is associated with higher levels of proviral HIV DNA and CD4+ T cell activation during antiretroviral treatment.

Asymptomatic cytomegalovirus (CMV) replication occurs frequently in the genital tract in untreated HIV-infected men and is associated with increased immune activation and HIV disease progression. To determine the connections between CMV-associated immune activation and the size of the viral reservoir, we evaluated the interactions between (i) asymptomatic seminal CMV replication, (ii) levels of T cell activation and proliferation in blood, and (iii) the size and transcriptional activity of the HIV DNA reservoir in blood from 53 HIV-infected men on long-term antiretroviral therapy (ART) with suppressed HIV RNA in blood plasma. We found that asymptomatic CMV shedding in semen was associated with significantly higher levels of proliferating and activated CD4(+) T cells in blood (P < 0.01). Subjects with detectable CMV in semen had approximately five times higher average levels of HIV DNA in blood CD4(+) T cells than subjects with no CMV. There was also a trend for CMV shedders to have increased cellular (multiply spliced) HIV RNA transcription (P = 0.068) compared to participants without CMV, but it is unclear if this transcription pattern is associated with residual HIV replication. In multivariate analysis, the presence of seminal plasma CMV (P = 0.04), detectable 2-long terminal repeat (2-LTR), and lower nadir CD4(+) (P < 0.01) were independent predictors of higher levels of proviral HIV DNA in blood. Interventions aimed at reducing seminal CMV and associated immune activation may be important for HIV curative strategies. Future studies of anti-CMV therapeutics will help to establish causality and determine the mechanisms underlying these described associations. Importance: Almost all individuals infected with HIV are also infected with cytomegalovirus (CMV), and the replication dynamics of the two viruses likely influence each other. This study investigated interactions between asymptomatic CMV replication within the male genital tract, levels of inflammation in blood, and the size of the HIV DNA reservoir in 53 HIV-infected men on long-term antiretroviral therapy (ART) with suppressed HIV RNA in blood plasma. In support of our primary hypothesis, shedding of CMV DNA in semen was associated with increased activation and proliferation of T cells in blood and also significantly higher levels of HIV DNA in blood cells. These results suggest that CMV reactivation might play a role in the maintenance of the HIV DNA reservoir during suppressive ART and that it could be a target of pharmacologic intervention in future studies.

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

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.

The effect of cell subset isolation method on gene expression in leukocytes.

Multiple scientific disciplines require the isolation of specific subsets of blood cells from patient samples for gene expression analysis by microarray or RNA-sequencing, preserving disease- or treatment-related signatures. However, little is known with respect to the impact of different cell isolation methods on gene expression and the effects of positive selection, negative selection, and fluorescence activated cell sorting (FACS) have not previously been assessed in parallel. To address this knowledge gap, CD4+ T cells, CD8+ T cells, B cells, and monocytes were isolated from blood samples from five independent donors using positive immunomagnetic selection, negative immunomagnetic selection, and FACS. We hypothesized that positive selection and FACS would yield higher purity but may have an impact on gene expression since both methods utilize antibodies that bind surface receptors of the cell type of interest. Moreover, FACS might upregulate stress response genes due to passage of the cells through the sorter. Microarray gene expression data were generated and subjected to unsupervised clustering and differential gene expression analysis. Surprisingly, these analyses revealed that gene expression signatures were more similar between cells isolated by negative selection and FACS compared to cells isolated by positive selection. Moreover, genes that are involved in the response to stress generally had the highest expression in cells isolated by negative or positive selection and not FACS. Thus, FACS is the recommended method for isolation of leukocyte subsets for gene expression studies since this method results in the purest subset populations and does not appear to induce a stress response.

Associations between virologic and immunologic dynamics in blood and in the male genital tract.

To determine the influence of asymptomatic genital viral infections on the cellular components of semen and blood, we evaluated the associations between the numbers and activation statuses of CD4+ and CD8+ T lymphocytes in both compartments and the seminal levels of cytomegalovirus (CMV), herpes simplex virus (HSV), and human immunodeficiency virus 1 (HIV). Paired blood and semen samples were collected from 36 HIV-infected antiretroviral-naïve individuals and from 40 HIV-uninfected participants. We performed multiparameter flow cytometry analysis (CD45, CD45RA, CD3, CD4, CD8, and CD38) of seminal and blood cellular components and measured HIV RNA and CMV and HSV DNA levels in seminal and blood plasma by real-time PCR. Compared to HIV-uninfected participants, in the seminal compartment HIV-infected participants had higher levels of CMV (P < 0.05), higher numbers of total CD3+ (P < 0.01) and CD8+ subset (P < 0.01) T lymphocytes, and higher CD4+ and CD8+ T lymphocyte activation (RA-CD38+) (P < 0.01). Seminal CMV levels positively correlated with absolute numbers of CD4+ and CD8+ T cells in semen (P < 0.05) and with the activation status of CD4+ T cells in semen and in blood (P < 0.01). HIV levels in semen (P < 0.05) and blood (P < 0.01) were positively associated with T-cell activation in blood. Activation of CD8+ T cells in blood remained an independent predictor of HIV levels in semen in multivariate analysis. The virologic milieu in the male genital tract strongly influences the recruitment and activation of immune cells in semen and may also modulate T-cell immune activation in blood. These factors likely influence replication dynamics, sexual transmission risk, and disease outcomes for all three viruses.

HIV-1 latency is established preferentially in minimally activated and non-dividing cells during productive infection of primary CD4 T cells.

Latently infected CD4 T cells form a stable reservoir of HIV that leads to life-long viral persistence; the mechanisms involved in establishment of this latency are not well understood. Three scenarios have been proposed: 1) an activated, proliferating cell becomes infected and reverts back to a resting state; 2) an activated cell becomes infected during its return to resting; or 3) infection is established directly in a resting cell. The aim of this study was, therefore, to investigate the relationship between T cell activation and proliferation and the establishment of HIV latency. Isolated primary CD4 cells were infected at different time points before or after TCR-induced stimulation. Cell proliferation within acutely infected cultures was tracked using CFSE viable dye over 14 days; and cell subsets that underwent varying degrees of proliferation were isolated at end of culture by flow cytometric sorting. Recovered cell subpopulations were analyzed for the amount of integrated HIV DNA, and the ability to produce virus, upon a second round of cell stimulation. We show that cell cultures exposed to virus, prior to stimulus addition, contained the highest levels of integrated and replication-competent provirus after returning to quiescence; whereas, cells infected during the height of cell proliferation retained the least. Cells that did not divide or exhibited limited division, following virus exposure and stimulation contained greater amounts of integrated and inducible HIV than did cells that had divided many times. Based on these results, co-culture experiments were conducted to demonstrate that latent infection could be established directly in non-dividing cells via cell-to-cell transmission from autologous productively infected cells. Together, the findings from our studies implicate the likely importance of direct infection of sub-optimally activated T cells in establishment of latently infected reservoirs in vivo, especially in CD4 lymphocytes that surround productive viral foci within immune tissue microenvironments.

Integrated proteomics and transcriptomics analyses identify novel cell surface markers of HIV latency.

Elimination of the latent HIV cell reservoir may be possible, if the molecular identity of latently infected cells were fully elucidated. We conducted comprehensive molecular profiling, at the protein and RNA levels, of primary T cells latently infected with HIV in vitro. Isobaric labelling quantitative proteomics and RNA sequencing identified 1453 proteins and 618 genes, altered in latently infected cells compared to mock-infected controls (p < 0.05). Biomarker selection was based on results from integrated data analysis. Relative enrichment for latently infected cells was monitored using flow cytometric sorting and the HIV integrant assay. Antibodies against selected proteins, encoded by CEACAM1 and PLXNB2, enabled enrichment of latently infected cells from cell mixtures by 3-10 fold (5.8 average, p < 0.001), comparable to levels obtained with biomarkers reported previously. Individual biomarkers are likely linked to subsets of latently infected cells, and an extended antibody panel will be required to inclusively target the latent HIV reservoir.

Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections.

The viruses HIV-1, Epstein-Barr virus (EBV), cytomegalovirus (CMV) and hepatitis C virus (HCV) are characterized by the establishment of lifelong infection in the human host, where their replication is thought to be tightly controlled by virus-specific CD8+ T cells. Here we present detailed studies of the differentiation phenotype of these cells, which can be separated into three distinct subsets based on expression of the costimulatory receptors CD28 and CD27. Whereas CD8+ T cells specific for HIV, EBV and HCV exhibit similar characteristics during primary infection, there are significant enrichments at different stages of cellular differentiation in the chronic phase of persistent infection according to the viral specificity, which suggests that distinct memory T-cell populations are established in different virus infections. These findings challenge the current definitions of memory and effector subsets in humans, and suggest that ascribing effector and memory functions to subsets with different differentiation phenotypes is no longer appropriate.

Micro RNA Targets in HIV Latency: Insights into Novel Layers of Latency Control.

Despite the considerable progress that has been made in identifying cellular factors and pathways that contribute to establishment and maintenance of the latent HIV reservoir, it remains the major obstacle to eradicating this virus. Most recently, noncoding genes have been implicated in regulation of HIV expression. In this study, small RNA sequencing was used to profile expression of microRNAs (miRNAs) in a primary CD4+ T cell in vitro model of HIV latency. Previously, we have shown that protein-coding genes dysregulated in this model were enriched for the p53 signaling pathway, which was confirmed experimentally. We further found a link between p53 signaling and dysregulated long noncoding RNAs. In this study, we hypothesized that miRNAs may provide an additional level of regulation of the p53 signaling pathway during HIV latency. Twenty-six miRNAs were identified to be dysregulated in our latency model. A subset of these miRNAs was validated by real-time quantitative polymerase chain reaction. Predicted messenger RNA (mRNA) targets and cellular pathways enriched for mRNA targets were identified using several analytical methods. Our analyses showed that many protein-coding genes and pathways targeted by dysregulated miRNAs have relevance to regulation of HIV expression or establishment of HIV latency. The p53 signaling pathway was found among pathways that were targeted by dysregulated miRNAs at a greater level than expected by chance. This study provides a mechanistic insight into regulation of the p53 pathway through miRNAs that may contribute to the establishment of latency.

HIV-specific cytotoxic T cells from long-term survivors select a unique T cell receptor.

HIV-specific cytotoxic T lymphocytes (CTL) are important in controlling HIV replication, but the magnitude of the CTL response does not predict clinical outcome. In four donors with delayed disease progression we identified Vbeta13.2 T cell receptors (TCRs) with very similar and unusually long beta-chain complementarity determining region 3 (CDR3) regions in CTL specific for the immunodominant human histocompatibility leukocyte antigens (HLA)-B8-restricted human immunodeficiency virus-1 (HIV-1) nef epitope, FLKEKGGL (FL8). CTL expressing Vbeta13.2 TCRs tolerate naturally arising viral variants in the FL8 epitope that escape recognition by other CTL. In addition, they expand efficiently in vitro and are resistant to apoptosis, in contrast to FL8-specific CTL using other TCRs. Selection of Vbeta13.2 TCRs by some patients early in the FL8-specific CTL response may be linked with better clinical outcome.

Flow cytometric screening for anti-leishmanials in a human macrophage cell line.

High-throughput drug screening methods against the intracellular stage of Leishmania have been facilitated by the development of in vitro models of infection. The use of cell lines rather than primary cells facilitates these methods. Peripheral blood mononuclear cell (PBMC) derived macrophages and THP-1 cells were infected with stationary phase egfp transfected Leishmania amazonensis parasites and then treated with anti-leishmanial compounds. Drug activity was measured using a flow cytometric approach, and toxicity was assessed using either the MTT assay or trypan blue dye exclusion. Calculated EC(50)'s for amphotericin B, sodium stibogluconate, and miltefosine were 0.1445±0.0005µg/ml, 0.1203±0.018mg/ml, and 26.71µM using THP-1 cells, and 0.179±0.035µg/ml, 0.1948±0.0364mg/ml, and 13.77±10.74µM using PBMC derived macrophages, respectively. We conclude that a flow cytometric approach using egfp transfected Leishmania species can be used to evaluate anti-leishmanial compounds against the amastigote stage of the parasite in THP-1 cells with excellent concordance to human PBMC derived macrophages.

Long non-coding RNAs and latent HIV - A search for novel targets for latency reversal.

The latent cellular reservoir of HIV is recognized as the major barrier to cure from HIV infection. Long non-coding RNAs (lncRNAs) are more tissue and cell type-specific than protein coding genes, and may represent targets of choice for HIV latency reversal. Using two in vitro primary T-cell models, we identified lncRNAs dysregulated in latency. PVT1 and RP11-347C18.3 were up-regulated in common between the two models, and RP11-539L10.2 was down-regulated. The major component of the latent HIV reservoir, memory CD4+ T-cells, had higher expression of these lncRNAs, compared to naïve T-cells. Guilt-by-association analysis demonstrated that lncRNAs dysregulated in latency were associated with several cellular pathways implicated in HIV latency establishment and maintenance: proteasome, spliceosome, p53 signaling, and mammalian target of rapamycin (MTOR). PVT1, RP11-347C18.3, and RP11-539L10.2 were down-regulated by latency reversing agents, suberoylanilide hydroxamic acid and Romidepsin, suggesting that modulation of lncRNAs is a possible secondary mechanism of action of these compounds. These results will facilitate prioritization of lncRNAs for evaluation as targets for HIV latency reversal. Importantly, our study provides insights into regulatory function of lncRNA during latent HIV infection.

Transcriptional Modulation of Human Endogenous Retroviruses in Primary CD4+ T Cells Following Vorinostat Treatment.

The greatest obstacle to a cure for HIV is the provirus that integrates into the genome of the infected cell and persists despite antiretroviral therapy. A "shock and kill" approach has been proposed as a strategy for an HIV cure whereby drugs and compounds referred to as latency-reversing agents (LRAs) are used to "shock" the silent provirus into active replication to permit "killing" by virus-induced pathology or immune recognition. The LRA most utilized to date in clinical trials has been the histone deacetylase (HDAC) inhibitor-vorinostat. Potentially, pathological off-target effects of vorinostat may result from the activation of human endogenous retroviruses (HERVs), which share common ancestry with exogenous retroviruses including HIV. To explore the effects of HDAC inhibition on HERV transcription, an unbiased pharmacogenomics approach (total RNA-Seq) was used to evaluate HERV expression following the exposure of primary CD4+ T cells to a high dose of vorinostat. Over 2,000 individual HERV elements were found to be significantly modulated by vorinostat, whereby elements belonging to the ERVL family (e.g., LTR16C and LTR33) were predominantly downregulated, in contrast to LTR12 elements of the HERV-9 family, which exhibited the greatest signal, with the upregulation of 140 distinct elements. The modulation of three different LTR12 elements by vorinostat was confirmed by droplet digital PCR along a dose-response curve. The monitoring of LTR12 expression during clinical trials with vorinostat may be indicated to assess the impact of this HERV on the human genome and host immunity.

Immune activation and CD8+ T-cell differentiation towards senescence in HIV-1 infection.

Progress in the fight against the HIV/AIDS epidemic is hindered by our failure to elucidate the precise reasons for the onset of immunodeficiency in HIV-1 infection. Increasing evidence suggests that elevated immune activation is associated with poor outcome in HIV-1 pathogenesis. However, the basis of this association remains unclear. Through ex vivo analysis of virus-specific CD8(+) T-cells and the use of an in vitro model of naïve CD8(+) T-cell priming, we show that the activation level and the differentiation state of T-cells are closely related. Acute HIV-1 infection induces massive activation of CD8(+) T-cells, affecting many cell populations, not only those specific for HIV-1, which results in further differentiation of these cells. HIV disease progression correlates with increased proportions of highly differentiated CD8(+) T-cells, which exhibit characteristics of replicative senescence and probably indicate a decline in T-cell competence of the infected person. The differentiation of CD8(+) and CD4(+) T-cells towards a state of replicative senescence is a natural process. It can be driven by excessive levels of immune stimulation. This may be part of the mechanism through which HIV-1-mediated immune activation exhausts the capacity of the immune system.

Relative efficacy of T cell stimuli as inducers of productive HIV-1 replication in latently infected CD4 lymphocytes from patients on suppressive cART.

Quantification of cell-associated replication-competent HIV, in blood samples from patients with undetectable plasma viremia, requires specialized culture conditions that include exogenous pan T cell stimulation. Different research groups have used several stimuli for this purpose; however, the relative efficacies of these T cell stimuli to induce productive HIV replication from latently infected cells ex vivo have not been systematically evaluated. To this end, we compared four commonly used T cell stimuli: 1) irradiated allogeneic cells plus phytohaemagglutinin (PHA); 2) PHA alone; 3) phorbol myristate acetate plus Ionomycin; and 4) immobilized αCD3 plus αCD28 antibodies. End-point dilutions of patient CD4 T cells were performed, using virion RNA production to quantify HIV induction. Our results demonstrated that these activation approaches were not equivalent and that antibody cross-linking of CD3 and CD28 membrane receptors was the most effective means to activate HIV replication from a resting cell state, closely followed by stimulation with irradiated allogeneic cells plus PHA.

HIV viral kinetics and T cell dynamics in antiretroviral naïve persons starting an integrase strand transfer inhibitor and protease inhibitor regimen.

BACKGROUND: Nucleos(t)ide reverse transcriptase inhibitor (NRTI)-sparing regimens may potentially minimize antiretroviral (ART) toxicities, but demonstrate mixed efficacy and toxicity results. The impact of an integrase strand transfer inhibitor (INSTI) and protease inhibitor (PI) regimen on HIV viral dynamics and T cell kinetics remains underdescribed. OBJECTIVE: To compare the effect of raltegravir + ritonavir boosted lopinavir (RAL + LPV/r) to efavirenz/tenofovir disoproxil fumarate/emtricitabine (EFV/TDF/FTC) on HIV kinetics and T cell dynamics. METHODS: Fifty participants naïve to ART underwent HIV viral kinetic sampling evaluated using biexponential mixed effects modeling. A subset of 28 subjects (with complete viral suppression) underwent flow cytometry and evaluation of soluble markers of inflammation at weeks 0, 4, and 48 of ART. RESULTS: RAL + LPV/r compared to EFV/TDF/FTC resulted in a prolonged first phase viral decay rate (18 vs. 13 days p < 0.01). From weeks 0 to 4, RAL + LPV/r was associated with a trend toward greater decreases in activated CD4+ T cells (-3.81 vs. -1.18 p = 0.09) and less decreases in activated effector memory CD4+ T cells (-0.63 vs. -2.69 p-0.07). These trends did not persist to week 48. No differences were noted at any time point for soluble markers of immune activation. CONCLUSIONS: The prolonged first phase viral decay observed with RAL + LPV/r in persons starting ART did not result in differences in viral suppression at week 48. We also observed trends in declines in certain cellular markers of immune activation but it remains unclear if this could translate to long-term immunologic benefits in persons on an INSTI + PI.