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Approximately 70% of the HIV-1 latent reservoir originates from infections of CD4 T cells that occur in the months near the time of ART initiation, raising the possibility that interventions during this period might prevent reservoir seeding and reduce reservoir size. We identify class 1 histone deacetylase inhibitors (HDACi) as potent agents of latency prevention. Inhibiting HDACs in productively infected cells caused extended maintenance of HIV expression and this activity was associated with persistently elevated H3K9 acetylation and reduced H3K9 methylation at the viral LTR promoter region. HDAC inhibition in HIV-infected CD4 T cells during effector-to-memory transition led to striking changes in the memory phenotype of infected cells. Proviral silencing is accomplished through distinct activities of HDAC1/2 and HDAC3. Thus HDACs regulate a critical gateway process for HIV latency establishment and are required for the development of CD4 T-cell memory subsets that preferentially harbor long-lived, latent provirus.

MitoTracker ® dyes are fluorescent compounds that allow cellular mitochondrial content to be measured semi‐quantitatively by flow cytometry and have been used extensively in immunology publications. However, the parameters commonly reported, mean or median fluorescence intensity and percentage of cells that are MitoTracker® “high”, can be influenced by variability in cytometer setup, dye stability, and operator subjectivity, making it difficult to compare data between experiments. Here, we describe a method to identify MitoTracker® “high” populations in an objective manner. When analyzing data, we first removed outliers using a pre‐specified threshold, determined the fluorescence intensity of the brightest and dimmest events to obtain the fluorescence range and then gated cells within the top 90% of this range. This strategy substantially reduced variability between technical replicates and produced consistent results when data were analyzed by different operators. Consistent with previous reports and other analysis strategies, this analysis method demonstrated that within an individual, CD4+ T cells exhibit significantly higher mitochondrial mass than CD8+ T cells. Objective gating increases the reliability and utility of data generated using MitoTracker® dyes. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

HIV infection depletes CD4 T cells and dysregulates formation of long-lived memory CD4 T cells. Studies show that antiretroviral therapy (ART) in people with HIV (PWH) more effectively restores CD4 T cell memory phenotypes when initiated early (< Fiebig V). We used a 31 marker mass cytometry panel to assess CD4 T cell phenotype in PWH treated in acute infection (AHI) (median 2.3 years post ART initiation) and chronic infection (CHI) (median 5.8 years post ART initiation) compared to healthy donors (HD) (n=10 per group). Markers of activation (HLA-DR, CD25), exhaustion (PD-1), survival (Bcl-2) and memory (CD127) were examined. CD4 T cell memory phenotypes of AHI clustered closely with HD whereas CHI had fewer central memory CD4 T cells. We also examined IL-7 signaling in CD4 T cells, measuring STAT5 phosphorylation (pSTAT5) in response to IL-7. CHI exhibited significantly lower pSTAT5 than HD in contrast to AHI who exhibited restored IL-7 signaling. This suggests ongoing and underappreciated functional defects in CD4 T cells in CHI. To examine this further, we are completing detailed analysis of CHI before and after ART initiation (pre-ART, day 2, 7, 10, 14, 28, month 5, 9 and 18). Data will be compared to our recent work showing that CD4 T cell memory subsets, activation and exhaustion markers are highly stable in durably suppressed CHI on ART over 19–27 months. New work is examining CD4 T cell function in immunological non-responders (INR) who fail to restore CD4 counts following ART. Altogether these studies will inform how HIV impacts formation of both CD4 memory and the HIV reservoir (which is largely harbored in long lived memory CD4 T cells) following ART initiation, potentially identifying novel therapeutic targets to accelerate HIV cure in CHI. This work was made possible by the National Institute of Allergy and Infectious Diseases U01 AI131310, Collaboratory of AIDS Researchers for Eradication (UM1 AI164567) and UNC WIHS (U01 AI103390).

Background The histone deacetylase (HDAC) inhibitor vorinostat (VOR) can increase HIV RNA expression in vivo within resting CD4+ T cells of aviremic HIV+ individuals. However, while studies of VOR or other HDAC inhibitors have reported reversal of latency, none has demonstrated clearance of latent infection. We sought to identify the optimal dosing of VOR for effective serial reversal of HIV latency. Methods In a study of 16 HIV-infected, aviremic individuals, we measured resting CD4+ T cell-associated HIV RNA ex vivo and in vivo following a single exposure to VOR, and then in vivo after a pair of doses separated by 48 or 72 hours, and finally following a series of 10 doses given at 72-hour intervals. Results Serial VOR exposures separated by 72 hours most often resulted in an increase in cell-associated HIV RNA within circulating resting CD4+ T cells. VOR was well tolerated by all participants. However, despite serial reversal of latency over 1 month of VOR dosing, we did not observe a measurable decrease (>0.3 log10) in the frequency of latent infection within resting CD4+ T cells. Conclusions These findings outline parameters for the experimental use of VOR to clear latent infection. Latency reversal can be achieved by VOR safely and repeatedly, but effective depletion of persistent HIV infection will require additional advances. In addition to improvements in latency reversal, these advances may include the sustained induction of potent antiviral immune responses capable of recognizing and clearing the rare cells in which HIV latency has been reversed. Trial registration Clinicaltrials.gov NCT01319383. Funding NIH grants U01 AI095052, AI50410, and P30 CA016086 and National Center for Advancing Translational Sciences grant KL2 TR001109.

HIV infection causes a rapid loss of CD4+ T cells that is attributed to both direct cytotoxicity of infected CD4+ T cells and bystander loss due to high immune activation. In addition to the absolute decrease of CD4+ T cells, a significant decrease in both frequency and number of long-lived memory CD4+ T cells is observed in untreated HIV infection. Antiretroviral therapies (ART) now reliably achieve viral control (

Antiretroviral therapy (ART) has transformed HIV from a fatal disease to a chronic condition. In recent years there has been considerable interest in strategies to enable HIV-infected individuals to cease ART without viral rebound, either by purging all cells infected harboring replication-competent virus (HIV eradication), or by boosting immune responses to allow durable suppression of virus without rebound (HIV remission). Both of these approaches may need to harness HIV-specific CD8+ T cells to eliminate infected cells and/or prevent viral spread. In untreated infection, both HIV-specific and total CD8+ T cells are dysfunctional. Here, we review our current understanding of both global and HIV-specific CD8+ T cell immunity in HIV-infected individuals with durably suppressed viral load under ART, and its implications for HIV cure, eradication or remission. Overall, the literature indicates significant normalization of global T cell parameters, including CD4/8 ratio, activation status, and telomere length. Global characteristics of CD8+ T cells from HIV+ART+ individuals align more closely with those of HIV-seronegative individuals than of viremic HIV-infected individuals. However, markers of senescence remain elevated, leading to the hypothesis that immune aging is accelerated in HIV-infected individuals on ART. This phenomenon could have implications for attempts to prime de novo, or boost existing HIV-specific CD8+ T cell responses. A major challenge for both HIV cure and remission strategies is to elicit HIV-specific CD8+ T cell responses superior to that elicited by natural infection in terms of response kinetics, magnitude, breadth, viral suppressive capacity, and tissue localization. Addressing these issues will be critical to the success of HIV cure and remission attempts.

Antiretroviral therapy (ART) regimens durably suppress HIV replication, but do not cure infection. This is partially attributable to the persistence of long-lived pools of resting CD4+ T-cells harboring latent replication-competent virus. Substantial clinical and pre-clinical research is currently being directed at purging this viral reservoir by combining pharmacological latency reversal with immune effectors, such as HIV-specific CD8+ T-cells, capable of eliminating reactivated targets – the so called ‘shock-and-kill’ approach. However, several studies indicate the latency reversing agents may affect CD8+ T-cell function. The current review aims to frame recent advances, and ongoing challenges, in implementing ‘shock-and-kill’ strategies from the perspective of effectively harnessing CD8+ T-cells. We review and contextualize findings indicating that latency reversing agents often have unintended impacts on CD8+ T-cell function, both detrimental and beneficial. We identify and attempt to bridge the gap between viral reactivation, as measured by the detection of RNA or protein, and bona fide presentation of viral antigens to CD8+ T-cells. Finally, we highlight factors on the effector (CD8+) and target (CD4+) cell sides that contribute to whether or not infected cell recognition results in killing/elimination. These perspectives may contribute to an integrated view of ‘shock-and-kill’, with implications for therapeutic development.

Viral vector vaccines designed to elicit CD8(+) T cells in non-human primates exert potent control of immunodeficiency virus infections; however, similar approaches have been unsuccessful in humans. Adenoviral vectors elicit potent T cell responses but also induce production of immunosuppressive interleukin-10 (IL-10), which can limit the expansion of T cell responses. We investigated whether inhibiting IL-10 signaling prior to immunization with a candidate adenovirus vectored-HIV-1 vaccine, ChAdV63.HIVconsv, could modulate innate and adaptive immune responses in BALB/c mice. Transient IL-10 receptor blockade led to a modest but significant increase in the total magnitude CD8(+) T cell response to HIVconsv, but did not affect T cell responses to immunodominant epitopes. Anti-IL-10R-treated animals also exhibited greater expression of CD86 on CD11c(+) dendritic cells. Our data support further investigation and optimization of IL-10 blocking strategies to improve the immunogenicity of vaccines based on replication-defective adenoviruses.

Interleukin-10 (IL-10) plays a key role in regulating proinflammatory immune responses to infection but can interfere with pathogen clearance. Although IL-10 is upregulated throughout HIV-1 infection in multiple cell subsets, whether this is a viral immune evasion strategy or an appropriate response to immune activation is unresolved. Analysis of IL-10 production at the single cell level in 51 chronically infected subjects (31 antiretroviral (ART) naive and 20 ART treated) showed that a subset of CD8(+) T cells with a CD25(neg) FoxP3(neg) phenotype contributes substantially to IL-10 production in response to HIV-1 gag stimulation. The frequencies of gag-specific IL-10- and IFN-γ-producing T cells in ART-naive subjects were strongly correlated and the majority of these IL-10(+) CD8(+) T cells co-produced IFN-γ; however, patients with a predominant IL-10(+) /IFN-γ(neg) profile showed better control of viraemia. Depletion of HIV-specific CD8(+) IL-10(+) cells from PBMCs led to upregulation of CD38 on CD14(+) monocytes together with increased IL-6 production, in response to gag stimulation. Increased CD38 expression was positively correlated with the frequency of the IL-10(+) population and was also induced by exposure of monocytes to HIV-1 in vitro. Production of IL-10 by HIV-specific CD8(+) T cells may represent an adaptive regulatory response to monocyte activation during chronic infection.

The capacity of CD8+ T cells to inhibit HIV-1 replication in vitro strongly correlates with virus control in vivo. Post-hoc evaluations of HIV-1 vaccine candidates suggest that this immunological parameter is a promising benchmark of vaccine efficacy. Large-scale analysis of CD8+ T cell antiviral activity requires a rapid, robust and economical assay for accurate quantification of HIV-1 infection in primary CD4+ T cells. Detection of intracellular HIV-1 p24 antigen (p24 Ag) by flow cytometry is one such method but it is thought to be less sensitive and quantitative than p24 Ag ELISA. We report that fixation and permeabilisation of HIV-infected cells using paraformaldehyde/50% methanol/Nonidet P-40 instead of a conventional paraformaldehyde/saponin-based protocol improved their detection across multiplicities of infection (MOI) ranging from 10-2 to 8×10-5, and by nearly two-fold (p

CD8+ T-cell–based vaccines against human immunodeficiency virus type 1 (HIV-1) aim to induce responses that abort or limit early viral replication and thus delay disease progression and reduce transmission risk. Recombinant DNA and attenuated viral vector vaccines showed promise because of their capacity to induce high frequencies of interferon γ (IFN-γ)–secreting and/or polyfunctional T cells in healthy volunteers [1, 2]. However, the disappointing results of the Step Study, in which there was no effect of vaccine-induced T-cell responses on viral replication after seroconversion, highlighted the need for a reliable immunological correlate of virus control [3]. CD8+ T-cell depletion experiments in macaques with simian immunodeficiency virus (SIV) infection and vaccine strategies to induce T-cell responses against SIV have demonstrated unequivocally that CD8+ T cells suppress AIDS virus replication, with the most promising results to date being achieved by vaccination with a rhesus cytomegalovirus–vectored vaccine [4–8]. CD8+ T cells are induced early during primary HIV-1 infection, in tandem with peak viremia and prior to the appearance of neutralizing antibodies, but in the majority of cases these early CD8+ T cells fail to attenuate viral replication [9, 10]. Patients who spontaneously control HIV-1 (ie, long-term nonprogressors [LTNPs] and/or controllers), who represent

OBJECTIVES:: Protection against HIV type 1 (HIV-1) infection/AIDS will likely require concerted actions of protective CD8 killer T cells and protective antibodies. The challenges in inducing such effectors by active immunization are such that the T-cell and antibody vaccine components require separate development. Here, a rational attempt is taken to combine two separately optimized heterologous regimens into a single T-cell-inducing and antibody-inducing vaccination schedule with minimal induction of unprotective Env-specific T cells. DESIGN:: Clade A BG505 Env-derived uncleaved gp140 (BG505u) and conserved region tHIVc immunogens were utilized and presented to the immune system using non-replicating simian (chimpanzee) adenovirus ChAdV-63 (C) and poxvirus modified vaccinia virus Ankara MVA (M). In addition, purified BG505 gp120 (P) was used for antibody induction. METHODS:: BALB/c mice were vaccinated to elicit Env antibodies alone using ChAdV63.BG505u. MVA.BG505u and BG505 gp120 in regimens CMP, CPP and PPP, and in combination with the ChAdV63.tHIVc and MVA.tHIVc components in regimens CMP+CMM, CPP+CMM and PPP+CMM. Antibody and T-cell responses to BG505 Env and conserved regions of the HIV-1 proteome were determined. RESULTS:: Although all three regimens delivering BG505 Env induced similar levels of antibodies, BG505-specific T cells were induced in the CMP>CPP>PPP hierarchy, which was maintained during coinduction of tHIVc-specific T cells. Adjuvanted BG505 PPP decreased induction of tHIVc-specific T cells and tHIVc T-cell induction decreased induction of BG505 Ab. As expected, the antibodies that were induced neutralized tier 1 HIV-1 strains. CONCLUSION:: These results inform designs of initial human studies combining separately optimized T-cell and B-cell HIV-1 vaccines into a single regimen.

Background— Patients with treated HIV infection have clear survival benefits although with increased cardiac morbidity and mortality. Mechanisms of heart disease may be partly related to untreated chronic inflammation. Cardiovascular magnetic resonance imaging allows a comprehensive assessment of myocardial structure, function, and tissue characterization. We investigated, using cardiovascular magnetic resonance, subclinical inflammation and myocardial disease in asymptomatic HIV-infected individuals. Methods and Results— Myocardial structure and function were assessed using cardiovascular magnetic resonance at 1.5-T in treated HIV-infected individuals without known cardiovascular disease (n=103; mean age, 45±10 years) compared with healthy controls (n=92; mean age, 44±10 years). Assessments included left ventricular volumes, ejection fraction, strain, regional systolic, diastolic function, native T1 mapping, edema, and gadolinium enhancement. Compared with controls, subjects with HIV infection had 6% lower left ventricular ejection fraction ( P P =0.02), 29% lower peak diastolic strain rate ( P P =0.02), and higher native T1 values (969 versus 956 ms in controls; P =0.01). Pericardial effusions and myocardial fibrosis were 3 and 4× more common, respectively, in subjects with HIV infection (both P Conclusions— Treated HIV infection is associated with changes in myocardial structure and function in addition to higher rates of subclinical myocardial edema and fibrosis and frequent pericardial effusions. Chronic systemic inflammation in HIV, which involves the myocardium and pericardium, may explain the high rate of myocardial fibrosis and increased cardiac dysfunction in people living with HIV.

An effective human immunodeficiency virus type 1 (HIV-1) vaccine is the best solution for halting the acquired immune deficiency syndrome epidemic. Here, we describe the design and preclinical immunogenicity of T-cell vaccine expressing novel immunogens tHIVconsvX, vectored by DNA, simian (chimpanzee) adenovirus, and poxvirus modified vaccinia virus Ankara (MVA), a combination highly immunogenic in humans. The tHIVconsvX immunogens combine the three leading strategies for elicitation of effective CD8(+) T cells: use of regions of HIV-1 proteins functionally conserved across all M group viruses (to make HIV-1 escape costly on viral fitness), inclusion of bivalent complementary mosaic immunogens (to maximize global epitope matching and breadth of responses, and block common escape paths), and inclusion of epitopes known to be associated with low viral load in infected untreated people (to induce field-proven protective responses). tHIVconsvX was highly immunogenic in two strains of mice. Furthermore, the magnitude and breadth of CD8(+) T-cell responses to tHIVconsvX-derived peptides in treatment-naive HIV-1(+) patients significantly correlated with high CD4(+) T-cell count and low viral load. Overall, the tHIVconsvX design, combining the mosaic and conserved-region approaches, provides an indisputably better coverage of global HIV-1 variants than previous T-cell vaccines. These immunogens delivered in a highly immunogenic framework of adenovirus prime and MVA boost are ready for clinical development.

CD8+ T cells contribute to the control of viral infections, including HIV-1. Previous studies have reported that CD8+-mediated virus inhibition is predictive of subsequent HIV plasma viremia and CD4+T cell decline in HIV-infected, antiretroviral naïve individuals; higher virus inhibition is associated with a better clinical outcomes. Ex vivo CD8+ T cell-mediated inhibition of HIV replication is measured after co-culture of CD8+ T cells with HIV-1 super-infected, autologous CD4+ T cells, at different effector to target ratios. We adapted and standardized the CD8+ T cell virus inhibition assay (CD8-VIA) for use as an endpoint assay in therapeutic testing of HIV-1 T cell vaccines. CD8+ T cells from HIV infected, durably (>2 years) antiretroviral suppressed participants (n=11) were co-cultured with JR-CSF infected autologous CD4+ T cells at E:T ratios ranging from 1/2:1 to 1/160:1, generating CD8+ T cell virus inhibition curves for each participant. IC50 values varied by 100-fold across participants suggesting that CD8+ T cells harbor a broad range of virus inhibitory capacity even when viremia in participants is durably suppressed. Ongoing studies are comparing IC50 values to other measurements (magnitude, specificity, avidity, proliferation and production of cytokines and lytic molecules) of HIV-specific T cell immunity to understand the critical determinants of CD8+ T cell inhibitory capacity.

Human cytomegalovirus (HCMV)-specific CD8+ T cells are characterized by an unique, non-exhausting, effector memory phenotype. How HCMV induces this phenotype is poorly characterized. We hypothesized that HCMV may skew T cell priming producing functionally distinct CD8+ T cells. To investigate this question, we have established an autologous 3-cell culture system using human umbilical tissue in which naïve CD8+ T cells are isolated from cord blood, myeloid DCs (mDCs) are generated from cord blood CD34+ precursor cells and fibroblasts, which are a primary target of HCMV infection, are generated from matching umbilical cord (UC-F). Previous studies have reported IFN-γ treatment of fibroblasts induces MHC-II expression, leading to suggestions that fibroblasts can acquire APC-like activity. To investigate this, we used flow cytometry to examine the effects of HCMV infection +/− IFN-γ on MHC-II, CD40, CD80 and CD86 surface expression on fibroblasts. HCMV alone did not induce MHC-II or costimulatory molecules. HCMV infection +/− IFN-γ induced MHC-II but costimulatory molecules remained undetectable. This suggests HCMV-infected fibroblasts do not acquire APC-like phenotype and that fibroblasts mediate T cell priming through cross-presentation. Fibroblasts release extracellular vesicles (EVs), which are important mediators of cell signaling, including antigen cross-presentation. We used electron microscopy to confirm that fibroblasts release EVs. Following AD169-GFP infection, 5.5–8% of EV collected were GFP-positive by ImageStream. Future studies will investigate whether UC-F modulate mDC priming of CD8+ T cells through direct cell-cell contact and/or indirectly, including through the release of EVs.

Interleukin-10 (IL-10) plays a key role in regulating proinflammatory immune responses to infection but can interfere with pathogen clearance. Although IL-10 is upregulated throughout HIV-1 infection in multiple cell subsets, whether this is a viral immune evasion strategy or an appropriate response to immune activation is unresolved. Analysis of IL-10 production at the single cell level in 51 chronically infected subjects (31 antiretroviral (ART) naïve and 20 ART treated) showed that a subset of CD8(+) T cells with a CD25(neg) FoxP3(neg) phenotype contributes substantially to IL-10 production in response to HIV-1 gag stimulation. The frequencies of gag-specific IL-10- and IFN-γ-producing T cells in ART-naïve subjects were strongly correlated and the majority of these IL-10(+) CD8(+) T cells co-produced IFN-γ; however, patients with a predominant IL-10(+) /IFN-γ(neg) profile showed better control of viraemia. Depletion of HIV-specific CD8(+) IL-10(+) cells from PBMCs led to upregulation of CD38 on CD14(+) monocytes together with increased IL-6 production, in response to gag stimulation. Increased CD38 expression was positively correlated with the frequency of the IL-10(+) population and was also induced by exposure of monocytes to HIV-1 in vitro. Production of IL-10 by HIV-specific CD8(+) T cells may represent an adaptive regulatory response to monocyte activation during chronic infection.

Background Patients with treated Human Immunodeficiency Virus-1 (HIV) infection are at increased risk of cardiovascular events. Traditionally much of this risk has been attributed to metabolic and anthropometric abnormalities associated with HIV, which are similar to the metabolic syndrome (MS), an established risk factor for cardiovascular mortality. It remains unclear whether treated HIV infection is itself associated with increased risk, via increase vascular stiffness. Methods 226 subjects (90 with HIV) were divided into 4 groups based on HIV and MS status: 1) HIV-ve/MS-ve, 2) HIV-ve/MS + ve, 3) HIV + ve/MS-ve and 4)HIV + ve/MS + ve. CMR was used to determine aortic pulse wave velocity (PWV) and regional aortic distensibility (AD). Results PWV was 11% higher and regional AD up to 14% lower in the HIV + ve/MS-ve group when compared to HIV-ve/MS-ve (p 0.99 all analyses). The HIV + ve/MS + ve group had 32% higher PWV and 30-34% lower AD than the HIV-ve/MS-ve group (all p

Background— HIV infection continues to be endemic worldwide. Although treatments are successful, it remains controversial whether patients receiving optimal therapy have structural, functional, or biochemical cardiac abnormalities that may underlie their increased cardiac morbidity and mortality. The purpose of this study was to characterize myocardial abnormalities in a contemporary group of HIV-infected individuals undergoing combination antiretroviral therapy. Methods and Results— Volunteers with HIV who were undergoing combination antiretroviral therapy and age-matched control subjects without a history of cardiovascular disease underwent cardiac magnetic resonance imaging and spectroscopy for the determination of cardiac function, myocardial fibrosis, and myocardial lipid content. A total of 129 participants were included in this analysis. Compared with age-matched control subjects (n=39; 30.23%), HIV-infected subjects undergoing combination antiretroviral therapy (n=90; 69.77%) had 47% higher median myocardial lipid levels ( P P P Conclusions— Comprehensive cardiac imaging revealed cardiac steatosis, alterations in cardiac function, and a high prevalence of myocardial fibrosis in a contemporary group of asymptomatic HIV-infected subjects undergoing combination antiretroviral therapy. Cardiac steatosis and fibrosis may underlie cardiac dysfunction and increased cardiovascular morbidity and mortality in subjects with HIV.