scholarly journals The Atlas of the In Vivo HIV CD4 T Cell Reservoir

2020 ◽  
Author(s):  
Jason Neidleman ◽  
Xiaoyu Luo ◽  
Julie Frouard ◽  
Guorui Xie ◽  
Feng Hsiao ◽  
...  
Keyword(s):  
Reservoir Characterization ◽  
Ex Vivo ◽  
Cd4 T Cell ◽  
High Dimensional ◽  
Latent Reservoir ◽  
Hiv Persistence ◽  
Latently Infected ◽  
Infected Cells ◽  
Main Barrier

ABSTRACTThe latent reservoir is a main barrier for curing HIV. But because latently-infected cells cannot be phenotyped directly, the features of the in vivo reservoir have remained elusive. Here, we describe a method that leverages high-dimensional phenotyping using CyTOF to trace latently-infected cells reactivated ex vivo to their original pre-activation states. Our results suggest that contrary to common assumptions, the reservoir is not randomly distributed among cell subsets, and is remarkably conserved between individuals. However, reservoir composition differs between tissues and blood, as do cells successfully reactivated by different latency reversing agents. Most importantly, by selecting 8-10 of our 39 original CyTOF markers, we were able to isolate highly purified populations of unstimulated in vivo latent cells, thereby validating the PP-SLIDE approach for reservoir characterization. These purified populations were highly enriched for replication-competent and intact provirus, transcribed HIV, and displayed clonal expansion. The ability to isolate unstimulated latent cells from infected individuals enables previously impossible studies of HIV persistence.

scholarly journals Phenotypic analysis of the unstimulated in vivo HIV CD4 T cell reservoir

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jason Neidleman ◽  
Xiaoyu Luo ◽  
Julie Frouard ◽  
Guorui Xie ◽  
Feng Hsiao ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Cd4 T Cell ◽  
Latent Reservoir ◽  
Hiv Cure ◽  
Phenotypic Analysis ◽  
Major Barrier ◽  
Hiv Persistence ◽  
Latently Infected ◽  
Infected Cells

The latent reservoir is a major barrier to HIV cure. As latently infected cells cannot be phenotyped directly, the features of the in vivo reservoir have remained elusive. Here, we describe a method that leverages high-dimensional phenotyping using CyTOF to trace latently infected cells reactivated ex vivo to their original pre-activation states. Our results suggest that, contrary to common assumptions, the reservoir is not randomly distributed among cell subsets, and is remarkably conserved between individuals. However, reservoir composition differs between tissues and blood, as do cells successfully reactivated by different latency reversing agents. By selecting 8–10 of our 39 original CyTOF markers, we were able to isolate highly purified populations of unstimulated in vivo latent cells. These purified populations were highly enriched for replication-competent and intact provirus, transcribed HIV, and displayed clonal expansion. The ability to isolate unstimulated latent cells from infected individuals enables previously impossible studies on HIV persistence.

scholarly journals Humanized Mouse Model of HIV-1 Latency with Enrichment of Latent Virus in PD-1+and TIGIT+CD4 T Cells

2019 ◽  
Vol 93 (10) ◽  
Author(s):  
George N. Llewellyn ◽  
Eduardo Seclén ◽  
Stephen Wietgrefe ◽  
Siyu Liu ◽  
Morgan Chateau ◽  
...  
Keyword(s):  
T Cells ◽  
Mouse Model ◽  
Ex Vivo ◽  
Latent Reservoir ◽  
Humanized Mouse ◽  
Humanized Mouse Model ◽  
Latently Infected ◽  
Infected Cells ◽  
Latent Hiv ◽  
Hiv 1

ABSTRACTCombination anti-retroviral drug therapy (ART) potently suppresses HIV-1 replication but does not result in virus eradication or a cure. A major contributing factor is the long-term persistence of a reservoir of latently infected cells. To study this reservoir, we established a humanized mouse model of HIV-1 infection and ART suppression based on an oral ART regimen. Similar to humans, HIV-1 levels in the blood of ART-treated animals were frequently suppressed below the limits of detection. However, the limited timeframe of the mouse model and the small volume of available samples makes it a challenging model with which to achieve full viral suppression and to investigate the latent reservoir. We therefore used anex vivolatency reactivation assay that allows a semiquantitative measure of the latent reservoir that establishes in individual animals, regardless of whether they are treated with ART. Using this assay, we found that latently infected human CD4 T cells can be readily detected in mouse lymphoid tissues and that latent HIV-1 was enriched in populations expressing markers of T cell exhaustion, PD-1 and TIGIT. In addition, we were able to use theex vivolatency reactivation assay to demonstrate that HIV-specific TALENs can reduce the fraction of reactivatable virus in the latently infected cell population that establishesin vivo, supporting the use of targeted nuclease-based approaches for an HIV-1 cure.IMPORTANCEHIV-1 can establish latent infections that are not cleared by current antiretroviral drugs or the body’s immune responses and therefore represent a major barrier to curing HIV-infected individuals. However, the lack of expression of viral antigens on latently infected cells makes them difficult to identify or study. Here, we describe a humanized mouse model that can be used to detect latent but reactivatable HIV-1 in both untreated mice and those on ART and therefore provides a simple system with which to study the latent HIV-1 reservoir and the impact of interventions aimed at reducing it.

scholarly journals A Critical Review of the Evidence Concerning the HIV Latency Reversing Effect of Disulfiram, the Possible Explanations for Its Inability to Reduce the Size of the Latent Reservoir In Vivo, and the Caveats Associated with Its Use in Practice

2017 ◽  
Vol 2017 ◽  
pp. 1-7 ◽  
Author(s):  
Harry D. J. Knights
Keyword(s):  
Immune Deficiency Syndrome ◽  
Deficiency Syndrome ◽  
Latent Reservoir ◽  
Major Barrier ◽  
Latently Infected ◽  
Shock And Kill ◽  
Infected Cells ◽  
Hiv 1

Combination antiretroviral therapy (cART) effectively suppresses the replication of human immunodeficiency virus type 1 (HIV-1), improves immune function, and decreases the morbidity of acquired immune deficiency syndrome (AIDS). However, it is unable to eradicate the virus because it does not eliminate latently infected cells. The latent reservoir poses the major barrier to an HIV-1 cure. The “shock and kill” strategy aims to reactivate the virus and destroy latently infected cells. Many latency reversing agents (LRAs) reactivate HIV in vitro, but the absence of damaging side-effects and efficacy in vivo make disulfiram particularly promising. However, in clinical trials to date, disulfiram treatment has not resulted in a reduction in the size of the latent reservoir. In this article I will therefore discuss the evidence for the latency reversing effect of disulfiram, the possible explanations for its inability to reduce the size of the latent reservoir in vivo, and the caveats associated with its use in practice. These considerations will help to inform judgements about the prospect of an HIV cure from disulfiram based treatments.

scholarly journals Proliferation of latently infected CD4+ T cells carrying replication-competent HIV-1: Potential role in latent reservoir dynamics

2017 ◽  
Vol 214 (4) ◽  
pp. 959-972 ◽  
Author(s):  
Nina N. Hosmane ◽  
Kyungyoon J. Kwon ◽  
Katherine M. Bruner ◽  
Adam A. Capoferri ◽  
Subul Beg ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cell Activation ◽  
Cell Activation ◽  
Productive Infection ◽  
Latent Reservoir ◽  
Full Genome Sequencing ◽  
Latently Infected ◽  
Infected Cells ◽  
Hiv 1

A latent reservoir for HIV-1 in resting CD4+ T lymphocytes precludes cure. Mechanisms underlying reservoir stability are unclear. Recent studies suggest an unexpected degree of infected cell proliferation in vivo. T cell activation drives proliferation but also reverses latency, resulting in productive infection that generally leads to cell death. In this study, we show that latently infected cells can proliferate in response to mitogens without producing virus, generating progeny cells that can release infectious virus. Thus, assays relying on one round of activation underestimate reservoir size. Sequencing of independent clonal isolates of replication-competent virus revealed that 57% had env sequences identical to other isolates from the same patient. Identity was confirmed by full-genome sequencing and was not attributable to limited viral diversity. Phylogenetic and statistical analysis suggested that identical sequences arose from in vivo proliferation of infected cells, rather than infection of multiple cells by a dominant viral species. The possibility that much of the reservoir arises by cell proliferation presents challenges to cure.

scholarly journals The gammaherpesvirus 68 viral cyclin facilitates expression of LANA

2021 ◽  
Vol 17 (11) ◽  
pp. e1010019
Author(s):  
Brian F. Niemeyer ◽  
Bridget Sanford ◽  
Joy E. Gibson ◽  
Jennifer N. Berger ◽  
Lauren M. Oko ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Critical Role ◽  
Productive Infection ◽  
Latent Reservoir ◽  
Cellular Distribution ◽  
Viral Cyclin ◽  
Latently Infected ◽  
Infected Cells ◽  
Gammaherpesvirus 68 ◽  
The Impact

Gammaherpesviruses establish life-long infections within their host and have been shown to be the causative agents of devastating malignancies. Chronic infection within the host is mediated through cycles of transcriptionally quiescent stages of latency with periods of reactivation into detectable lytic and productive infection. The mechanisms that regulate reactivation from latency remain poorly understood. Previously, we defined a critical role for the viral cyclin in promoting reactivation from latency. Disruption of the viral cyclin had no impact on the frequency of cells containing viral genome during latency, yet it remains unclear whether the viral cyclin influences latently infected cells in a qualitative manner. To define the impact of the viral cyclin on properties of latent infection, we utilized a viral cyclin deficient variant expressing a LANA-beta-lactamase fusion protein (LANA::βla), to enumerate both the cellular distribution and frequency of LANA gene expression. Disruption of the viral cyclin did not affect the cellular distribution of latently infected cells, but did result in a significant decrease in the frequency of cells that expressed LANA::βla across multiple tissues and in both immunocompetent and immunodeficient hosts. Strikingly, whereas the cyclin-deficient virus had a reactivation defect in bulk culture, sort purified cyclin-deficient LANA::βla expressing cells were fully capable of reactivation. These data emphasize that the γHV68 latent reservoir is comprised of at least two distinct stages of infection characterized by differential LANA expression, and that a primary function of the viral cyclin is to promote LANA expression during latency, a state associated with ex vivo reactivation competence.

scholarly journals New Latency Reversing Agents for HIV-1 Cure: Insights from Nonhuman Primate Models

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1560
Author(s):  
Katherine M. Bricker ◽  
Ann Chahroudi ◽  
Maud Mavigner
Keyword(s):  
Nonhuman Primate ◽  
Immune Checkpoint Blockade ◽  
Tlr Agonists ◽  
Latently Infected ◽  
Shock And Kill ◽  
Infected Cells ◽  
Main Barrier ◽  
Hiv 1 ◽  
Reversing Agents

Antiretroviral therapy (ART) controls human immunodeficiency virus 1 (HIV-1) replication and prevents disease progression but does not eradicate HIV-1. The persistence of a reservoir of latently infected cells represents the main barrier to a cure. “Shock and kill” is a promising strategy involving latency reversing agents (LRAs) to reactivate HIV-1 from latently infected cells, thus exposing the infected cells to killing by the immune system or clearance agents. Here, we review advances to the “shock and kill” strategy made through the nonhuman primate (NHP) model, highlighting recently identified latency reversing agents and approaches such as mimetics of the second mitochondrial activator of caspase (SMACm), experimental CD8+ T cell depletion, immune checkpoint blockade (ICI), and toll-like receptor (TLR) agonists. We also discuss the advantages and limits of the NHP model for HIV cure research and methods developed to evaluate the efficacy of in vivo treatment with LRAs in NHPs.

scholarly journals Pharmacological Modulation of the Wnt/β-Catenin Pathway Inhibits Proliferation and Promotes Differentiation of Long-Lived Memory CD4+ T Cells in Antiretroviral Therapy-Suppressed Simian Immunodeficiency Virus-Infected Macaques

2019 ◽  
Vol 94 (1) ◽  
Author(s):  
M. Mavigner ◽  
M. Zanoni ◽  
G. K. Tharp ◽  
J. Habib ◽  
C. R. Mattingly ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Antiretroviral Therapy ◽  
Viral Reservoir ◽  
Self Renewal ◽  
Hiv Persistence ◽  
Latently Infected ◽  
Infected Cells

ABSTRACT The major obstacle to human immunodeficiency type 1 virus (HIV-1) eradication is a reservoir of latently infected cells that persists despite long-term antiretroviral therapy (ART) and is maintained through cellular proliferation. Long-lived memory CD4+ T cells with high self-renewal capacity, such as central memory (CM) T cells and stem cell memory (SCM) T cells, are major contributors to the viral reservoir in HIV-infected individuals on ART. The Wnt/β-catenin signaling pathway regulates the balance between self-renewal and differentiation of SCM and CM T cells, and pharmacological manipulation of this pathway offers an opportunity to interfere with the proliferation of latently infected cells. Here, we evaluated in vivo a novel approach to inhibit self-renewal of SCM and CM CD4+ T cells in the rhesus macaque (RM) model of simian immunodeficiency (SIV) infection. We used an inhibitor of the Wnt/β-catenin pathway, PRI-724, that blocks the interaction between the coactivator CREB-binding protein (CBP) and β-catenin, resulting in the cell fate decision to differentiate rather than proliferate. Our study shows that PRI-724 treatment of ART-suppressed SIVmac251-infected RMs resulted in decreased proliferation of SCM and CM T cells and modified the SCM and CM CD4+ T cell transcriptome toward a profile of more differentiated memory T cells. However, short-term treatment with PRI-724 alone did not significantly reduce the size of the viral reservoir. This work demonstrates for the first time that stemness pathways of long-lived memory CD4+ T cells can be pharmacologically modulated in vivo, thus establishing a novel strategy to target HIV persistence. IMPORTANCE Long-lasting CD4+ T cell subsets, such as central memory and stem cell memory CD4+ T cells, represent critical reservoirs for human immunodeficiency virus (HIV) persistence despite suppressive antiretroviral therapy. These cells possess stem cell-like properties of enhanced self-renewal/proliferation, and proliferation of latently infected memory CD4+ T cells plays a key role in maintaining the reservoir over time. Here, we evaluated an innovative strategy targeting the proliferation of long-lived memory CD4+ T cells to reduce viral reservoir stability. Using the rhesus macaque model, we tested a pharmacological inhibitor of the Wnt/β-catenin signaling pathway that regulates T cell proliferation. Our study shows that administration of the inhibitor PRI-724 decreased the proliferation of SCM and CM CD4+ T cells and promoted a transcriptome enriched in differentiation genes. Although the viral reservoir size was not significantly reduced by PRI-724 treatment alone, we demonstrate the potential to pharmacologically modulate the proliferation of memory CD4+ T cells as a strategy to limit HIV persistence.

scholarly journals Targeting the latent human cytomegalovirus reservoir for T-cell-mediated killing with virus-specific nanobodies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Timo W. M. De Groof ◽  
Elizabeth G. Elder ◽  
Eleanor Y. Lim ◽  
Raimond Heukers ◽  
Nick D. Bergkamp ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Early Gene ◽  
Latent Reservoir ◽  
Solid Organ ◽  
Cell Transplant ◽  
Viral Receptor ◽  
Transplant Patients ◽  
Latently Infected ◽  
Infected Cells

AbstractLatent human cytomegalovirus (HCMV) infection is characterized by limited gene expression, making latent HCMV infections refractory to current treatments targeting viral replication. However, reactivation of latent HCMV in immunosuppressed solid organ and stem cell transplant patients often results in morbidity. Here, we report the killing of latently infected cells via a virus-specific nanobody (VUN100bv) that partially inhibits signaling of the viral receptor US28. VUN100bv reactivates immediate early gene expression in latently infected cells without inducing virus production. This allows recognition and killing of latently infected monocytes by autologous cytotoxic T lymphocytes from HCMV-seropositive individuals, which could serve as a therapy to reduce the HCMV latent reservoir of transplant patients.

scholarly journals Similar frequency and inducibility of intact HIV-1 proviruses in blood and lymph nodes

2020 ◽  
Author(s):  
Alyssa R Martin ◽  
Alexandra M Bender ◽  
Jada Hackman ◽  
Kyungyoon J Kwon ◽  
Briana A Lynch ◽  
...  
Keyword(s):  
T Cells ◽  
Lymph Nodes ◽  
Cd4 T Cells ◽  
Viral Rna ◽  
Latent Reservoir ◽  
Similar Frequency ◽  
Latently Infected ◽  
Infected Cells ◽  
Secondary Lymphoid Organs ◽  
Hiv 1

Abstract Background The HIV-1 latent reservoir (LR) in resting CD4 + T cells is a barrier to cure. LR measurements are commonly performed on blood samples and therefore may miss latently infected cells residing in tissues, including lymph nodes. Methods We determined the frequency of intact HIV-1 proviruses and proviral inducibility in matched peripheral blood (PB) and lymph node (LN) samples from ten HIV-1-infected patients on ART using the intact proviral DNA assay and a novel quantitative viral induction assay. Prominent viral sequences from induced viral RNA were characterized using a next-generation sequencing assay. Results The frequencies of CD4 + T cells with intact proviruses were not significantly different in PB vs LN (61vs104/10 6CD4 + cells), and were substantially lower than frequencies of CD4 + T cells with defective proviruses. The frequencies of CD4 + T cells induced to produce high levels of viral RNA were not significantly different in PB vs LN (4.3/10 6 vs 7.9/10 6), but were 14-fold lower than the frequencies of cells with intact proviruses. Sequencing of HIV-1 RNA from induced proviruses revealed comparable sequences in paired PB and LN samples. Conclusions These results further support the use of PB as an appropriate proxy for the HIV-1 LR in secondary lymphoid organs

scholarly journals Establishment of a Novel Humanized Mouse Model To Investigate In Vivo Activation and Depletion of Patient-Derived HIV Latent Reservoirs

2019 ◽  
Vol 93 (6) ◽  
Author(s):  
Nina C. Flerin ◽  
Ariola Bardhi ◽  
Jian Hua Zheng ◽  
Maria Korom ◽  
Joy Folkvord ◽  
...  
Keyword(s):  
Mouse Model ◽  
Hiv Infection ◽  
Mononuclear Cells ◽  
Systemic Infection ◽  
Humanized Mouse Model ◽  
Peripheral Blood Mononuclear ◽  
Immunodeficient Mice ◽  
Latently Infected ◽  
Infected Cells

ABSTRACT Curing HIV infection has been thwarted by the persistent reservoir of latently infected CD4+ T cells, which reinitiate systemic infection after antiretroviral therapy (ART) interruption. To evaluate reservoir depletion strategies, we developed a novel preclinical in vivo model consisting of immunodeficient mice intrasplenically injected with peripheral blood mononuclear cells (PBMC) from long-term ART-suppressed HIV-infected donors. In the absence of ART, these mice developed rebound viremia which, 2 weeks after PBMC injection, was 1,000-fold higher (mean = 9,229,281 HIV copies/ml) in mice injected intrasplenically than in mice injected intraperitoneally (mean = 6,838 HIV copies/ml) or intravenously (mean = 591 HIV copies/ml). One week after intrasplenic PBMC injection, in situ hybridization of the spleen demonstrated extensive disseminated HIV infection, likely initiated from in vivo-reactivated primary latently infected cells. The time to viremia was delayed significantly by treatment with a broadly neutralizing antibody, 10-1074, compared to treatment with 10-1074-FcRnull, suggesting that 10-1074 mobilized Fc-mediated effector mechanisms to deplete the replication-competent reservoir. This was supported by phylogenetic analysis of Env sequences from viral-outgrowth cultures and untreated, 10-1074-treated, or 10-1074-FcRnull-treated mice. The predominant sequence cluster detected in viral-outgrowth cultures and untreated mouse plasma was significantly reduced in the plasma of 10-1074-treated mice, whereas two new clusters emerged that were not detected in viral-outgrowth cultures or plasma from untreated mice. These new clusters lacked mutations associated with 10-1074 resistance. Taken together, these data indicated that 10-1074 treatment depletes the reservoir of latently infected cells harboring replication competent HIV. Furthermore, this mouse model represents a new in vivo approach for the preclinical evaluation of new HIV cure strategies. IMPORTANCE Sustained remission of HIV infection is prevented by a persistent reservoir of latently infected cells capable of reinitiating systemic infection and viremia. To evaluate strategies to reactivate and deplete this reservoir, we developed and characterized a new humanized mouse model consisting of highly immunodeficient mice intrasplenically injected with peripheral blood mononuclear cells from long-term ART-suppressed HIV-infected donors. Reactivation and dissemination of HIV infection was visualized in the mouse spleens in parallel with the onset of viremia. The applicability of this model for evaluating reservoir depletion treatments was demonstrated by establishing, through delayed time to viremia and phylogenetic analysis of plasma virus, that treatment of these humanized mice with a broadly neutralizing antibody, 10-1074, depleted the patient-derived population of latently infected cells. This mouse model represents a new in vivo approach for the preclinical evaluation of new HIV cure strategies.

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