Elimination of SHIV Infected Cells by Combinations of Bispecific HIVxCD3 DART® Molecules

Bispecific HIVxCD3 DART molecules that co-engage the viral envelope glycoprotein (Env) on HIV-1-infected cells and the CD3 receptor on CD3+ T cells are designed to mediate the cytolysis of HIV-1-infected, Env-expressing cells. Using a novel ex vivo system with cells from rhesus macaques (RMs) infected with a chimeric Simian-Human Immunodeficiency Virus (SHIV) CH505 and maintained on ART, we tested the ability of HIVxCD3 DART molecules to mediate elimination of in vitro-reactivated CD4+ T cells in the absence or presence of autologous CD8+ T cells. HIVxCD3 DART molecules with the anti-HIV-1 Env specificities of A32 or 7B2 (non-neutralizing antibodies) or PGT145 (broadly neutralizing antibody) were evaluated individually or combined. DART molecule-mediated antiviral activity increased significantly in the presence of autologous CD8+ T cells. In this ex vivo system, the PGT145 DART molecule was more active than the 7B2 DART molecule, which was more active than the A32 DART molecule. A triple combination of the DART molecules exceeded the activity of the individual PGT145 DART molecule. Modified quantitative virus outgrowth assays confirmed the ability of the DART molecules to redirect RM CD3+ T cells to eliminate SHIV-infected RM CD4+ T cells as demonstrated by the decreased propagation of in vitro infection by the infected cells pre-incubated with DART molecules in presence of effector CD8+ T cells. While mediating cytotoxic activity, DART molecules did not increase proinflammatory cytokine production. In summary, combination of HIVxCD3 DART molecules that have broadly-neutralizing and non-neutralizing anti-HIV-1 Env specificities can leverage the host immune system for treatment of HIV-1 infection but will require appropriate reactivation of the latent reservoir.

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Suppression of HIV replication in vitro by CD8+ T-cells from HIV-infected and HIV-seronegative individuals

International Journal of STD & AIDS ◽

10.1258/0956462971920145 ◽

1997 ◽

Vol 8 (5) ◽

pp. 307-310 ◽

Cited By ~ 3

Author(s):

H Ichimura ◽

J M Dwyer ◽

H Tsuchie ◽

M A Detorio ◽

M M Hossain ◽

...

Keyword(s):

T Cells ◽

Nucleotide Sequence ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Inhibitory Effect ◽

Hiv Replication ◽

Anti Hiv ◽

Hiv 1 ◽

New Strategies

The inhibitory effect of CD8+ T-cells from HIV-infected or HIVseronegative individuals on HIV replication in the naturally-infected CD4+ T-cells in vitro was examined. Not only autologous CD8+ T-cells from HIV-infected individuals but also allogeneic CD8+ T-cells from HIV-seronegative individuals prevented or delayed HIV replication, even in transwell cocultures using a semipermeable 0.45 micron filter. The level of the inhibitory effect of allogeneic CD8+ Tcells from the HIV-seronegative individuals on the HIV replication was varied among CD4+ T-cells obtained from HIV-infected individuals used. The results suggested that CD8+ T-cells from HIV-seronegative individuals as well as HIVinfected individuals could produce some cytokine(s) which suppress HIV replication in vitro . The sensitivity to the cytokine(s) might be variable among HIV strains, depending on differences in the nucleotide sequence of different HIV-1 strains. Further studies of control of HIV replication by CD8+ anti-HIV cytokine(s) should provide new strategies for the therapy of HIV infection.

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In VitroReactivation of Replication-Competent and Infectious HIV-1 by Histone Deacetylase Inhibitors

Journal of Virology ◽

10.1128/jvi.02359-15 ◽

2015 ◽

Vol 90 (4) ◽

pp. 1858-1871 ◽

Cited By ~ 22

Author(s):

Riddhima Banga ◽

Francesco Andrea Procopio ◽

Matthias Cavassini ◽

Matthieu Perreau

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Repeated Treatment ◽

Major Barrier ◽

Infected Cells ◽

Memory Cd4 T Cells ◽

Hiv 1

ABSTRACTThe existence of long-lived HIV-1-infected resting memory CD4 T cells is thought to be the primary obstacle to HIV-1 eradication. In the search for novel therapeutic approaches that may reverse HIV-1 latency, inhibitors of histone deacetylases (HDACis) have been tested to reactivate HIV-1 replication with the objective of rendering HIV-1-infected cells susceptible to elimination either by HIV-specific CD8 T cells or through virus-mediated cytopathicity. In the present study, we evaluated the efficiency of HDACis to reactivate HIV-1 replication from resting memory CD4 T cells isolated from aviremic long-term-treated HIV-1-infected subjects. We demonstrate that following prolonged/repeated treatment of resting memory CD4 T cells with HDACis, HIV-1 replication may be induced from primary resting memory CD4 T cells isolated from aviremic long-term-treated HIV-1-infected subjects. More importantly, we demonstrate that HIV-1 reactivated in the cell cultures was not only replication competent but also infectious. Interestingly, givinostat, an HDACi that has not been investigated in clinical trials, was more efficient than vorinostat, panobinostat, and romidepsin in reversing HIV-1 latencyin vitro. Taken together, these results support further evaluation of givinostat as a latency-reversing agent (LRA) in aviremic long-term-treated HIV-1-infected subjects.IMPORTANCEThe major barrier to HIV cure is the existence of long-lived latently HIV-1-infected resting memory CD4 T cells. Latently HIV-1-infected CD4 T cells are transcriptionally silent and are therefore not targeted by conventional antiretroviral therapy (ART) or the immune system. In this context, one strategy to target latently infected cells is based on pharmacological molecules that may force the virus to replicate and would therefore render HIV-1-infected cells susceptible to elimination either by HIV-specific CD8 T cells or through virus-mediated cytopathicity. In this context, we developed an experimental strategy that would allow the evaluation of latency-reversing agent (LRA) efficiencyin vitrousing primary CD4 T cells. In the present study, we demonstrate that HDACis are potent inducers of replication-competent and infectious HIV-1 in resting memory CD4 T cells of long-term ART-treated patients and identify givinostat as the most efficient LRA tested.

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Infection of CD8+CD45RO+ Memory T-Cells by HIV-1 and Their Proliferative Response

The Open AIDS Journal ◽

10.2174/1874613600802010043 ◽

2008 ◽

Vol 2 (1) ◽

pp. 43-57 ◽

Cited By ~ 7

Author(s):

Naveed Gulzar ◽

Sowyma Balasubramanian ◽

Greg Harris ◽

Jaime Sanchez-Dardon ◽

Karen F.T. Copeland

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Proliferative Response ◽

Cell Infection ◽

Cellular Target ◽

Potential Reservoir ◽

Infected Cells ◽

Hiv 1

CD8+ T-cells are involved in controlling HIV-1 infection by eliminating infected cells and secreting soluble factors that inhibit viral replication. To investigate the mechanism and significance of infection of CD8+ T-cells by HIV-1in vitro, we examined the susceptibility of these cells and their subsets to infection. CD8+ T-cells supported greater levels of replication with T-cell tropic strains of HIV-1, though viral production was lower than that observed in CD4+ T-cells. CD8+ T-cell infection was found to be productive through ELISA, RT-PCR and flow cytometric analyses. In addition, the CD8+CD45RO+ memory T-cell population supported higher levels of HIV-1 replication than CD8+CD45RA+ naïve T-cells. However, infection of CD8+CD45RO+ T-cells did not affect their proliferative response to the majority of mitogens tested. We conclude, with numerous lines of evidence detecting and measuring infection of CD8+ T-cells and their subsets, that this cellular target and potential reservoir may be central to HIV-1 pathogenesis.

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Evaluation of HIV-1 latency reversal and antibody-dependent viral clearance by quantification of singly spliced HIV-1 vpu/env mRNA

Journal of Virology ◽

10.1128/jvi.02124-20 ◽

2021 ◽

Author(s):

Hongbo Gao ◽

Ayşe N. Ozantürk ◽

Qiankun Wang ◽

Gray H. Harlan ◽

Aaron J. Schmitz ◽

...

Keyword(s):

T Cells ◽

Neutralizing Antibodies ◽

Ex Vivo ◽

Viral Envelope ◽

Latent Reservoir ◽

Broadly Neutralizing Antibodies ◽

Major Barrier ◽

Hiv 1 ◽

Reversing Agents

The latent reservoir of HIV-1 is a major barrier for viral eradication. Potent HIV-1 broadly neutralizing antibodies (bNabs) have been used to prevent and treat HIV-1 infections in animal models and clinical trials. Combination of bNabs and latency-reversing agents (LRAs) is considered a promising approach for HIV-1 eradication. PCR-based assays that can rapidly and specifically measure singly spliced HIV-1 vpu/env mRNA are needed to evaluate the induction of the viral envelope production at the transcription level and bNab-mediated reservoir clearance. Here we reported a PCR-based method to accurately quantify the production of intracellular HIV-1 vpu/env mRNA. With the vpu/env assay, we determined the LRA combinations that could effectively induce vpu/env mRNA production in CD4+ T cells from ART-treated individuals. None of the tested LRAs were effective alone. A comparison between the quantitative viral outgrowth assay (Q-VOA) and the vpu/env assay showed that vpu/env mRNA production was closely associated with the reactivation of replication-competent HIV-1, suggesting that vpu/env mRNA was mainly produced by intact viruses. Finally, antibody-mediated in vitro killing in HIV-1-infected humanized mice demonstrated that the vpu/env assay could be used to measure the reduction of infected cells in tissues and was more accurate than the commonly used gag-based PCR assay which measured unspliced viral genomic RNA. In conclusion, the vpu/env assay allows convenient and accurate assessment of HIV-1 latency reversal and bNab-mediated therapeutic strategies. Importance HIV-1 persists in individuals on antiretroviral therapy (ART) due to the long-lived cellular reservoirs that contain dormant viruses. Recent discoveries of HIV-1-specific broadly neutralizing antibodies (bNabs) targeting HIV-1 Env protein rekindled the interest in antibody-mediated elimination of latent HIV-1. Latency-reversing agents (LRAs) together with HIV-1 bNabs is a possible strategy to clear residual viral reservoirs, which makes the evaluation of HIV-1 Env expression upon LRA treatment critical. We developed a PCR-based assay to quantify the production of intracellular HIV-1 vpu/env mRNA. Using patient CD4+ T cells, we found that induction of HIV-1 vpu/env mRNA required a combination of different LRAs. Using in vitro, ex vivo and humanized mouse models, we showed that the vpu/env assay could be used to measure antibody efficacy in clearing HIV-1 infection. These results suggest that the vpu/env assay can accurately evaluate HIV-1 reactivation and bNab-based therapeutic interventions.

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Antigen sensitivity is a major determinant of CD8+ T-cell polyfunctionality and HIV-suppressive activity

Blood ◽

10.1182/blood-2009-02-206557 ◽

2009 ◽

Vol 113 (25) ◽

pp. 6351-6360 ◽

Cited By ~ 162

Author(s):

Jorge R. Almeida ◽

Delphine Sauce ◽

David A. Price ◽

Laura Papagno ◽

So Youn Shin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Ex Vivo ◽

Cd8 T Cell ◽

Basic Knowledge ◽

Suppressive Activity ◽

Antiviral Efficacy ◽

Hiv 1

Abstract CD8+ T cells are major players in the immune response against HIV. However, recent failures in the development of T cell–based vaccines against HIV-1 have emphasized the need to reassess our basic knowledge of T cell–mediated efficacy. CD8+ T cells from HIV-1–infected patients with slow disease progression exhibit potent polyfunctionality and HIV-suppressive activity, yet the factors that unify these properties are incompletely understood. We performed a detailed study of the interplay between T-cell functional attributes using a bank of HIV-specific CD8+ T-cell clones isolated in vitro; this approach enabled us to overcome inherent difficulties related to the in vivo heterogeneity of T-cell populations and address the underlying determinants that synthesize the qualities required for antiviral efficacy. Conclusions were supported by ex vivo analysis of HIV-specific CD8+ T cells from infected donors. We report that attributes of CD8+ T-cell efficacy against HIV are linked at the level of antigen sensitivity. Highly sensitive CD8+ T cells display polyfunctional profiles and potent HIV-suppressive activity. These data provide new insights into the mechanisms underlying CD8+ T-cell efficacy against HIV, and indicate that vaccine strategies should focus on the induction of HIV-specific T cells with high levels of antigen sensitivity to elicit potent antiviral efficacy.

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Th1/17 Polarization of CD4 T Cells Supports HIV-1 Persistence during Antiretroviral Therapy

Journal of Virology ◽

10.1128/jvi.01595-15 ◽

2015 ◽

Vol 89 (22) ◽

pp. 11284-11293 ◽

Cited By ~ 51

Author(s):

Hong Sun ◽

Dhohyung Kim ◽

Xiaodong Li ◽

Maja Kiselinova ◽

Zhengyu Ouyang ◽

...

Keyword(s):

T Cells ◽

Antiretroviral Therapy ◽

Cd4 T Cells ◽

Ex Vivo ◽

Memory Cell ◽

Viral Reservoir ◽

Target Cells ◽

Infected Cells ◽

Hiv 1

ABSTRACTThe ability to persist long term in latently infected CD4 T cells represents a characteristic feature of HIV-1 infection and the predominant barrier to efforts aiming at viral eradication and cure. Yet, increasing evidence suggests that only small subsets of CD4 T cells with specific developmental and maturational profiles are able to effectively support HIV-1 long-term persistence. Here, we analyzed how the functional polarization of CD4 T cells shapes and structures the reservoirs of HIV-1-infected cells. We found that CD4 T cells enriched for a Th1/17 polarization had elevated susceptibilities to HIV-1 infection inex vivoassays, harbored high levels of HIV-1 DNA in persons treated with antiretroviral therapy, and made a disproportionately increased contribution to the viral reservoir relative to their contribution to the CD4 T memory cell pool. Moreover, HIV-1 DNA levels in Th1/17 cells remained stable over many years of antiretroviral therapy, resulting in a progressively increasing contribution of these cells to the viral reservoir, and phylogenetic studies suggested preferential long-term persistence of identical viral sequences during prolonged antiretroviral treatment in this cell compartment. Together, these data suggest that Th1/17 CD4 T cells represent a preferred site for HIV-1 DNA long-term persistence in patients receiving antiretroviral therapy.IMPORTANCECurrent antiretroviral therapy is very effective in suppressing active HIV-1 replication but does not fully eliminate virally infected cells. The ability of HIV-1 to persist long term despite suppressive antiretroviral combination therapy represents a perplexing aspect of HIV-1 disease pathogenesis, since most HIV-1 target cells are activated, short-lived CD4 T cells. This study suggests that CD4 T helper cells with Th1/17 polarization have a preferential role as a long-term reservoir for HIV-1 infection during antiretroviral therapy, possibly because these cells may imitate some of the functional properties traditionally attributed to stem cells, such as the ability to persist for extremely long periods of time and to repopulate their own pool size through homeostatic self-renewal. These observations support the hypothesis that HIV-1 persistence is driven by small subsets of long-lasting stem cell-like CD4 T cells that may represent particularly promising targets for clinical strategies aiming at HIV-1 eradication and cure.

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Dressed to kill? A review of why antiviral CD8 T lymphocytes fail to prevent progressive immunodeficiency in HIV-1 infection

Blood ◽

10.1182/blood.v98.6.1667 ◽

2001 ◽

Vol 98 (6) ◽

pp. 1667-1677 ◽

Cited By ~ 169

Author(s):

Judy Lieberman ◽

Premlata Shankar ◽

N. Manjunath ◽

Jan Andersson

Keyword(s):

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Cd8 T Cells ◽

Interleukin 2 ◽

Interferon Γ ◽

Short Term Exposure ◽

Infected Cells ◽

Hiv 1

Abstract CD8 T cells play an important role in protection and control of HIV-1 by direct cytolysis of infected cells and by suppression of viral replication by secreted factors. However, although HIV-1–infected individuals have a high frequency of HIV-1–specific CD8 T cells, viral reservoirs persist and progressive immunodeficiency generally ensues in the absence of continuous potent antiviral drugs. Freshly isolated HIV-specific CD8 T cells are often unable to lyse HIV-1–infected cells. Maturation into competent cytotoxic T lymphocytes may be blocked during the initial encounter with antigen because of defects in antigen presentation by interdigitating dendritic cells or HIV-infected macrophages. The molecular basis for impaired function is multifactorial, due to incomplete T-cell signaling and activation (in part related to CD3ζ and CD28 down-modulation), reduced perforin expression, and inefficient trafficking of HIV-specific CD8 T cells to lymphoid sites of infection. CD8 T-cell dysfunction can partially be corrected in vitro with short-term exposure to interleukin 2, suggesting that impaired HIV-specific CD4 T helper function may play a significant causal or exacerbating role. Functional defects are qualitatively different and more severe with advanced disease, when interferon γ production also becomes compromised.

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Chromatin maturation of the HIV-1 provirus in primary resting CD4+ T cells

10.1101/526053 ◽

2019 ◽

Author(s):

Birgitta Lindqvist ◽

Sara Svensson Akusjarvi ◽

Anders Sonnerborg ◽

Marios Dimitriou ◽

J. Peter Svensson

Keyword(s):

T Cells ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

Ex Vivo ◽

Immunodeficiency Virus ◽

Latently Infected ◽

Infected Cells ◽

Active Transcription ◽

Hiv 1

Human immunodeficiency virus type 1 (HIV-1) infection is a chronic condition, where viral DNA integrates into the genome. Latently infected cells form a persistent, heterogeneous reservoir. The reservoir that reinstates an active replication comprises only cells with intact provirus that can be reactivated. We confirmed that latently infected cells from patients exhibited active transcription throughout the provirus. To find transcriptional determinants, we characterized the establishment and maintenance of viral latency during proviral chromatin maturation in cultures of primary CD4+ T-cells for four months after ex vivo HIV-1 infection. As heterochromatin (marked with H3K9me3 or H3K27me3) gradually stabilized, the provirus became less accessible with reduced activation potential. In a subset of infected cells, active marks (i.e., H3K27ac) remained detectable, even after prolonged proviral silencing. After T-cell activation, the proviral activation occurred uniquely in cells with H3K27ac-marked proviruses. Our observations suggested that, after transient proviral activation, cells were actively returned to latency.

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Gliotoxin, identified from a screen of fungal metabolites, disrupts 7SK snRNP, releases P-TEFb and reverses HIV-1 latency

10.1101/848929 ◽

2019 ◽

Cited By ~ 1

Author(s):

Mateusz Stoszko ◽

Abdullah M.S. Al-Hatmi ◽

Anton Skriba ◽

Michael Roling ◽

Enrico Ne ◽

...

Keyword(s):

Gene Expression ◽

Mass Spectrometry ◽

T Cells ◽

Cd4 T Cells ◽

Ex Vivo ◽

Reversal Agents ◽

Latently Infected ◽

Infected Cells ◽

Therapeutic Compounds ◽

Hiv 1

AbstractA leading pharmacological strategy towards HIV cure requires “shock” or activation of HIV gene expression in latently infected cells with Latency Reversal Agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs we used fungal secondary metabolites (extrolites) as a source of bio-active molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the P-TEFb inhibitory 7SK snRNP complex to be significantly reduced upon GTX treatment of independent donor CD4+T cells. GTX disrupted 7SK snRNP, releasing active P-TEFb, which then phosphorylated RNA Pol II CTD, inducing HIV transcription. Our data highlight the power of combining a medium throughput bioassay, mycology and orthogonal mass spectrometry to identify novel potentially therapeutic compounds.

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