HIV-infected Cells Research Articles

Profound phenotypic and epigenetic heterogeneity of the HIV-1-infected CD4+ T cell reservoir

Understanding the complexity of the long-lived HIV reservoir during antiretroviral therapy (ART) remains a considerable impediment in research towards a cure for HIV. To address this, we developed a single-cell strategy to precisely define the unperturbed peripheral blood HIV-infected memory CD4+ T cell reservoir from ART-treated people living with HIV (ART-PLWH) via the presence of integrated accessible proviral DNA in concert with epigenetic and cell surface protein profiling. We identified profound reservoir heterogeneity within and between ART-PLWH, characterized by new and known surface markers within total and individual memory CD4+ T cell subsets. We further uncovered new epigenetic profiles and transcription factor motifs enriched in HIV-infected cells that suggest infected cells with accessible provirus, irrespective of reservoir distribution, are poised for reactivation during ART treatment. Together, our findings reveal the extensive inter- and intrapersonal cellular heterogeneity of the HIV reservoir, and establish an initial multiomic atlas to develop targeted reservoir elimination strategies.

Comparative HIV-1 Proviral Dynamics in Two Individuals That Maintained Viral Replication Control with or without Antiretroviral Therapy following Superinfection.

The analysis of the HIV-1 proviral dynamics after superinfection in the context of both natural and antiretroviral therapy (ART)-mediated suppression could yield unique insights into understanding the persistence of viral variants that seeded the infected cells at different times. In this study, we performed a longitudinal analysis of the env diversity of PBMC-associated HIV DNA quasispecies in two HIV controllers (EEC09 and VC32) that were superinfected with subtype F1 viruses several years after primoinfection with subtype B viruses. Patient EEC09 started ART soon after superinfection, while patient VC32 maintained a natural control of virus replication for at least six years following the superinfection. Our analysis revealed no significant temporal changes in the overall proportion of primo-infecting and superinfecting proviral variants over 2-3 years after superinfection in both HIV controllers. Upon the introduction of ART, individual EEC09 displayed no evidence of HIV-infected cell turnover or viral evolution, while subject VC32 displayed some level of HIV-infected cell reseeding and detectable evolution (divergence) of both viral variants. These results confirm that proviral variants that seeded the reservoir at different times throughout infection could persist for long periods under fully suppressive ART or natural viremic control, but the HIV-1 proviral dynamics could be different in both settings.

4-phenylquinoline-8-amine induces HIV-1 reactivation and apoptosis in latently HIV-1 infected cells

Combinational antiretroviral therapy (cART) dramatically suppresses the viral load to undetectable levels in human immunodeficiency virus (HIV)-infected patients. However, HIV-1 reservoirs in CD4+T cells and myeloid cells, which can evade cART and host antiviral immune systems, are still significant obstacles to HIV-1 eradication. The “Shock and Kill” approach using latently-reversing agents (LRAs) is therefore currently developing strategies for effective HIV-1 reactivation from latency and inducing cell death. Here, we performed small-molecular chemical library screening with monocytic HIV-1 latently-infected model cells, THP-1 Nluc #225, and identified 4-phenylquinoline-8-amine (PQA) as a novel LRA candidate. PQA induced efficient HIV-1 reactivation in combination with PKC agonists including Prostratin and showed a similar tendency for HIV-1 activation in primary HIV-1 reservoirs. Furthermore, PQA induced killing of HIV-1 latently-infected cells. RNA-sequencing analysis revealed PQA had different functional mechanisms from PKC agonists, and oxidative stress-inducible genes including DDIT3 or CTSD were only involved in PQA-mediated cell death. In summary, PQA is a potential LRA lead compound that exerts novel functions related to HIV-1 activation and apoptosis-mediated cell death to eliminate HIV-1 reservoirs.

Stem cells in HIV infection.

To date, four cases illustrate that stem cell transplantation (SCT) remains an option for cure of HIV in only few cases. Long-term follow-up data on viral shedding of reservoirs are still needed. So far, interruption of ART is the only way to validate long-term HIV remission.

Depletion of HIV reservoir by activation of ISR signaling in resting CD4+ T cells

HIV reservoirs are extremely stable and pose a tremendous challenge to clear HIV infection. Here, we demonstrate that activation of ISR/ATF4 signaling reverses HIV latency, which also selectively eliminates HIV+ cells in primary CD4+T cell model of latency without effect on HIV-negative CD4+T cells. The reduction of HIV+ cells is associated with apoptosis enhancement, but surprisingly is largely seen in HIV-infected cells in which gag-pol RNA transcripts are detected in HIV RNA-induced ATF4/IFIT signaling. In resting CD4+ (rCD4+) Tcells isolated from people living with HIV on antiretroviral therapy, induction of ISR/ATF4 signaling reduced HIV reservoirs by depletion of replication-competent HIV without global reduction in the rCD4+ Tcell population. These findings suggest that compromised ISR/ATF4 signaling maintains stable and quiescent HIV reservoirs whereas activation of ISR/ATF4 signaling results in the disruption of latent HIV and clearance of persistently infected CD4+T cells.

HIV-Infected Cell

HIV-Infected Cell

PP 1.35 – 00172 A histone deacetylase network regulates epigenetic reprogramming and viral silencing in HIV infected cells

PP 1.35 – 00172 A histone deacetylase network regulates epigenetic reprogramming and viral silencing in HIV infected cells

OP 4.5 – 00101 Comparative single-cell transcriptome and TCR profiling of HIV infected cells in the blood and cerebrospinal fluid of PLWH before and after ART

OP 4.5 – 00101 Comparative single-cell transcriptome and TCR profiling of HIV infected cells in the blood and cerebrospinal fluid of PLWH before and after ART

PP 8.8 – 00129 Antibody mediated killing of HIV-1 infected cells with glycoengineered broadly neutralizing antibodies

PP 8.8 – 00129 Antibody mediated killing of HIV-1 infected cells with glycoengineered broadly neutralizing antibodies

PP 3.16 – 00209 Prolonged persistence of HIV-infected cells in tissues after allogeneic hematopoietic transplant

PP 3.16 – 00209 Prolonged persistence of HIV-infected cells in tissues after allogeneic hematopoietic transplant

Human galectin-9 promotes the expansion of HIV reservoirs in vivo in humanized mice.

The human endogenous protein galectin-9 (Gal-9) reactivates latently HIV-infected cells in vitro and ex vivo , which may allow for immune-mediated clearance of these cells. However, Gal-9 also activates several immune cells, which could negatively affect HIV persistence by promoting chronic activation/exhaustion. This potential 'double-edged sword' effect of Gal-9 raises the question of the overall impact of Gal-9 on HIV persistence in vivo . We used the BLT (bone marrow, liver, thymus) humanized mouse model to evaluate the impact of Gal-9 on HIV persistence in vivo during antiretroviral therapy (ART). Two independent cohorts of ART-suppressed HIV-infected BLT mice were treated with either recombinant Gal-9 or phosphate-buffered saline control. Plasma viral loads and levels of tissue-associated HIV DNA and RNA were measured by qPCR. Immunohistochemistry and HIV RNAscope were used to quantify CD4 + T, myeloid, and HIV RNA+ cells in tissues. T cell activation and exhaustion were measured by flow cytometry, and plasma markers of inflammation were measured by multiplex cytokine arrays. Gal-9 did not induce plasma markers of inflammation or T cell markers of activation/exhaustion in vivo . However, the treatment significantly increased levels of tissue-associated HIV DNA and RNA compared to controls ( P = 0.0007 and P = 0.011, respectively, for cohort I and P = 0.002 and P = 0.005, respectively, for cohort II). RNAscope validated the Gal-9 mediated induction of HIV RNA in tissue-associated myeloid cells, but not T cells. Our study highlights the overall adverse effects of Gal-9 on HIV persistence and the potential need to block Gal-9 interactions during ART-suppressed HIV infection.

In vivo killing of primary HIV-infected cells by peripheral-injected early memory-enriched anti-HIV duoCAR T cells.

HIV-specific chimeric antigen receptor-T cell (CAR T cell) therapies are candidates to functionally cure HIV infection in people with HIV (PWH) by eliminating reactivated HIV-infected cells derived from latently infected cells within the HIV reservoir. Paramount to translating such therapeutic candidates successfully into the clinic will require anti-HIV CAR T cells to localize to lymphoid tissues in the body and eliminate reactivated HIV-infected cells such as CD4+ T cells and monocytes/macrophages. Here we show that i.v. injected anti-HIV duoCAR T cells, generated using a clinical-grade anti-HIV duoCAR lentiviral vector, localized to the site of active HIV infection in the spleen of humanized mice and eliminated HIV-infected PBMCs. CyTOF analysis of preinfusion duoCAR T cells revealed an early memory phenotype composed predominantly of CCR7+ stem cell-like/central memory T cells (TSCM/TCM) with expression of some effector-like molecules. In addition, we show that anti-HIV duoCAR T cells effectively sense and kill HIV-infected CD4+ T cells and monocytes/macrophages. Furthermore, we demonstrate efficient genetic modification of T cells from PWH on suppressive ART into anti-HIV duoCAR T cells that subsequently kill autologous PBMCs superinfected with HIV. These studies support the safety and efficacy of anti-HIV duoCAR T cell therapy in our presently open phase I/IIa clinical trial (NCT04648046).

Relationships between the factors of innate immune system and results of neurocognitive function parameters in the persons living with HIV infection: interim results of the study

HIV infection is closely associated with damage to the nervous system, even despite usage of antiretroviral therapy. Thus, the patients are at a high risk for neurological complications. Due to limited permeability of the blood-brain barrier, the opportunity of its damage, both directly by the proteins of HIV-infected cells and indirectly, by immune system, e.g., inducing the production of inflammatory mediators, the involvement of the central nervous system in pathogenesis of HIV infection seems to be only a matter of time. HIV-associated neurocognitive disorders (HAND) comprise a poly-etiological pathological condition associated with direct and indirect damage to neurons in HIV infection. The role of interaction between the innate immune system and nervous system mediators in viral infections suggests that the specific changes in content and ratios of pro- and anti-inflammatory cytokines that are among the most polypotent mediators of immune regulation can be detectable in the persons prone to the development of HAND in HIV infection. The purpose of the present study was to characterize the features of serum content and the relationship between the indexes of innate immunity factors with the results of neurocognitive testing in the persons living with HIV.
 123 persons were included in the study, of which 93 subjects live with HIV infection. They were divided into 3 groups according to the stages of HIV infection, according to the Russian Classification of HIV infection. 30 persons represented a group of healthy volunteers, comparable in age and gender with the members of main groups, and served as a control group. Determination of IL-1 and IL-10 contents in blood serum was carried out by solid-phase enzyme immunoassay. The level of CD4+T lymphocytes was determined by flow cytometry. Neurocognitive testing using Schulte tables, the Munsterberg test was conducted by a medical psychologist, according to generally accepted methods.
 A significant increase in the serum IL-10 content in subjects from the main groups was revealed as compared with the control group, as well as in group III compared with the values groups I and II. The levels of IL- 1, CD4+T lymphocytes were significantly lower in group III, compared with all other groups. The personal performance index determined by testing with Schulte tables was reduced in all groups, relative to the controls, with a significant predominance of the results in groups I and II over the results of the participants from group III. The scores in Munsterberg test were equally diminished in all the main groups compared to the control values. When assessing the Spearman coefficient, the presence of various correlation profiles was established, depending on the stage of the disease.

CD38+CD39+ NK cells associate with HIV disease progression and negatively regulate T cell proliferation.

The ectonucleotidases CD38 and CD39 have a critical regulatory effect on tumors and viral infections via the adenosine axis. Natural killer (NK) cells produce cytokines, induce cytotoxic responses against viral infection, and acquire immunoregulatory properties. However, the roles of CD38 and CD39 expressed NK cells in HIV disease require elucidation. Our study showed that the proportions of CD38+CD39+ NK cells in HIV-infected individuals were positively associated with HIV viral loads and negatively associated with the CD4+ T cell count. Furthermore, CD38+CD39+ NK cells expressed additional inhibitory receptors, TIM-3 and LAG-3, and produced more TGF-β. Moreover, autologous NK cells suppressed the proliferation of CD8+ T and CD4+ T cells of HIV-infected individuals, and inhibiting CD38 and CD39 on NK cells restored CD8+ T and CD4+ T cell proliferation in vitro. In conclusion, these data support a critical role for CD38 and CD39 on NK cells in HIV infection and targeting CD38 and CD39 on NK cells may be a potential therapeutic strategy against HIV infection.

Cell-associated HIV cross-presentation by pDC is potentiated by non-cognate CD8+ T cell pre-activation

Antigens (Ag) from apoptotic HIV-infected cells can be cross-presented by human plasmacytoid dendritic cells (pDC) to CD8 + T cells 1 . We studied the mechanisms leading to this cross-presentation. For this, we generated HLA-A2-restricted HIV-Gag-specific CD8 + T cell clones. HLA-A2 negative, HIV-infected or uninfected, H9 cells were used as Ag donor cells. They were irradiated by UV-C to induce apoptosis. Direct viral presentation was prevented by using saquinavir. DC populations were immunomagnetically and FACS-sorted to >95% purity, from HLA-A2+ or -donor buffy coats. CD8 + T cells IFN-γ secretion or intracellular production were assessed by ELISA or flow cytometry, respectively. Apoptotic H9-HIV cell cross-presentation by pDC led to CD8 + T cells IFN-γ secretion, which was fully MHC-class I-restricted. Surprisingly, a lower level of non-MHC restricted intracellular IFN-γ production was also induced by HLA-A2-negative pDC incubated with HIV-infected cells. Both MHC-Class I-dependent and -independent IFN-γ intracellular productions were partly dependent on TLR-7 signalization, indicating a role for stimulation of TLR7 by HIV. Indeed, H9-HIV, and not H9 cells, induced maturation and production of cytokines by pDC. Among those cytokines, IFN-α or β induced intracellular IFN-γ production when added to the CD8 + T cell clones, in the absence of pDC or H9-HIV cells. To perform cognate activation of the CD8 + T cell clones without viral stimulation, we used CD3-and CD28-bound beads: IFN-γ secretion was stimulated, and was increased by adding these cytokines. Conversely, neutralizing antibodies against these cytokines abolished the non-MHC-restricted intracellular IFN-γ production triggered by pDC incubated with by HIV-infected cells. Compared to pDC, purified cDC1 or cDC2 required additional TLR-3 or -2/4 stimulation to cross-present antigens from H9-HIV cells. Indeed, H9-HIV apoptotic cells induced maturation of pDC, but not cDC. Our work demonstrates that pDC produce type I IFNs following HIV-infected cells detection, which pre-activate CD8+ T cells by making them produce, but not release, IFN-γ. Upon additional cognate interaction, IFN-γ. release is potentiated by this previous intracellular accumulation.

Autophagy Receptors in the presentation of Viral antigens by MHC-II molecules

CD4+ T lymphocytes play a major role in the establishment and maintenance of antiviral immunity. CD4+ T cells are activated by antigenic peptides, derived from pathogens, presented by MHC-II molecules. It is still widely assumed that CD4+ T cells recognize epitopes derived solely from exogenous viral particles or proteins. However, alternative sources of MHC-II-restricted antigens have been described. We showed that HIV-infected dendritic cells present MHC-II–restricted epitopes derived from newly synthesized proteins to HIV-specific CD4+ T cells. In fact, we confirmed that multiple cellular degradation pathways lead to endogenous presentation of MHC-II-restricted viral antigens, the generation of some viral epitopes being dependent and/or -independent on macroautophagy (herein referred as autophagy). We are charactering further these MHC-II-restricted endogenous presentation pathways. In particular, using RNA silencing, we asked whether autophagy receptors/adaptors that contribute to selective autophagy but also regulate endosome and autophagosome maturation might play a role. Surprisingly, although these receptors share a high structural homology and similar functions, we identified a single autophagy adaptor that strongly influences the presentation of viral antigens by MHC-II molecules. Although silencing of this autophagy adaptor does not impact the expression levels and the recycling of MHC-II molecules, it has a major influence on the ligandome of MHC-II molecules. In the absence of this adaptor, MHC-II molecules accumulate at the proximity of the nucleus in CD63+Lamp1+ acidified compartments. In addition, the lack of this autophagy adaptor has a strong influence on the invariant chain degradation and expression levels, strongly suggesting that it regulates the activation or traffic of vesicles involved in the processing of newly synthesised antigens. Interestingly, some viruses target these autophagy adaptors to favour viral replication. Our work might reveal new escape mechanisms developed by viruses to damper MHC-II restricted antigen presentation and immune responses.

Cross presentation from live cells

Antigens (Ag) from apoptotic HIV-infected cells can be cross-presented by human plasmacytoid dendritic cells (pDC) to CD8 + T cells 1 . Antigens from live cells can also be cross-presented by human monocyte-derived DC 2 or by murine bone-marrow-derived DC 3 . We wondered whether DC isolated from human blood were able to cross-present antigens from live cells. We will present our results on cross-presentation from live infected cells. Our aim is to understand how to stimulate different types of DC to induce presentation of cellular antigens from HIV-1 reservoirs to cytotoxic CD8 + T lymphocytes, in order to decrease the size of these reservoirs during antiretroviral therapy.

Antiretroviral Drug Discovery Targeting the HIV-1 Nef Virulence Factor.

While antiretroviral drugs have transformed the lives of HIV-infected individuals, chronic treatment is required to prevent rebound from viral reservoir cells. People living with HIV also are at higher risk for cardiovascular and neurocognitive complications, as well as cancer. Finding a cure for HIV-1 infection is therefore an essential goal of current AIDS research. This review is focused on the discovery of pharmacological inhibitors of the HIV-1 Nef accessory protein. Nef is well known to enhance HIV-1 infectivity and replication, and to promote immune escape of HIV-infected cells by preventing cell surface MHC-I display of HIV-1 antigens. Recent progress shows that Nef inhibitors not only suppress HIV-1 replication, but also restore sufficient MHC-I to the surface of infected cells to trigger a cytotoxic T lymphocyte response. Combining Nef inhibitors with latency reversal agents and therapeutic vaccines may provide a path to clearance of viral reservoirs.

Potentiation of Drug Toxicity through Virus Latency Reversal Promotes Preferential Elimination of HIV Infected Cells

AbstractEliminating latently infected cells is a highly challenging, indispensable step toward the cure for HIV/AIDS. Hypothesis put forward herein is that the unique HIV protease cut site (Phe‐Pro) can be reconstructed using a potent inhibitor of tubulin polymerization, monomethyl auristatin F (MMAF), which features Phe at its C‐terminus. This presents opportunities to design prodrugs that are specifically activated by the HIV protease. To this end, a series of MMAF derivatives is synthesized and evaluated in cell culture using latently HIV‐infected cells. The cytotoxicity of compounds is indeed enhanced upon latency reversal by up to 11‐fold. As a result, in a mixed cell population, nanomolar concentrations of the lead compounds depletes predominantly the HIV‐infected cells and in doing so markedly enriches the pool of the uninfected cells. Affinity of the lead compounds to the viral protease is validated computationally and experimentally but despite expectations, the mechanism of action of the synthesized toxins is shown to be independent from the enzymatic activity of the HIV protease.

A Phenotypic Screen Identifies Potent DPP9 Inhibitors Capable of Killing HIV-1 Infected Cells.

Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 ("inducer of cell death-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.