Replication competence of virions induced from CD4+ lymphocytes latently infected with HIV

Richman, Douglas D.; Huang, Karissa; Lada, Steven M.; Sun, Xiaoying; Jain, Sonia; Massanella, Marta; Menke, Bryson

doi:10.1186/s12977-019-0466-1

Cited by 8 publications

(10 citation statements)

References 17 publications

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“…Next-generation QVOAs reported 4.5–444-fold higher IUPM values. At these higher levels of detection, it is important to note the possibility of detecting p24 Ag or RNA produced by replication-defective virus [ 19 ], although approximately half of such cells producing RNA are replication competent [ 20 ].…”

Section: Resultsmentioning

confidence: 99%

Assessing the Suitability of Next-Generation Viral Outgrowth Assays to Measure Human Immunodeficiency Virus 1 Latent Reservoir Size

Stone

Rosenbloom

Bacchetti

et al. 2020

The Journal of Infectious Diseases

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Background Evaluations of human immunodeficiency virus (HIV) curative interventions require reliable and efficient quantification of replication-competent latent reservoirs. The “classic” quantitative viral outgrowth assay (QVOA) has been regarded as the reference standard, although prohibitively resource and labor intensive. We compared 6 “next-generation” viral outgrowth assays, using polymerase chain reaction or ultrasensitive p24 to assess their suitability as scalable proxies for QVOA. Methods Next-generation QVOAs were compared with classic QVOA using single leukapheresis-derived samples from 5 antiretroviral therapy–suppressed HIV-infected participants and 1 HIV-uninfected control; each laboratory tested blinded batches of 3 frozen and 1 fresh sample. Markov chain Monte Carlo methods estimated extra-Poisson variation at aliquot, batch, and laboratory levels. Models also estimated the effect of testing frozen versus fresh samples. Results Next-generation QVOAs had similar estimates of variation to QVOA. Assays with ultrasensitive readout reported higher infectious units per million values than classic QVOA. Within-batch testing had 2.5-fold extra-Poisson variation (95% credible interval [CI], 2.1–3.5-fold) for next-generation assays. Between-laboratory variation increased extra-Poisson variation to 3.4-fold (95% CI, 2.6–5.4-fold). Frozen storage did not substantially alter infectious units per million values (−18%; 95% CI, −52% to 39%). Conclusions The data offer cautious support for use of next-generation QVOAs as proxies for more laborious QVOA, while providing greater sensitivities and dynamic ranges. Measurement of latent reservoirs in eradication strategies would benefit from high throughput and scalable assays.

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Section: Resultsmentioning

confidence: 99%

Assessing the Suitability of Next-Generation Viral Outgrowth Assays to Measure Human Immunodeficiency Virus 1 Latent Reservoir Size

Stone

Rosenbloom

Bacchetti

et al. 2020

The Journal of Infectious Diseases

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“…To confirm and extend the results obtained measuring cf-RNA, we evaluated latency reversal in a second group of participants by measuring cell-associated (ca) HIV-1 mRNA using a ddPCR assay that detects multiply spliced Tat/Rev transcripts [ 27 ]. We stimulated nine million CD8 depleted PBMC from six participants with each peptide pool, again in the presence of 1 µM raltegravir to prevent viral spread.…”

Section: Resultsmentioning

confidence: 99%

Inconsistent reversal of HIV-1 latency ex vivo by antigens of HIV-1, CMV, and other infectious agents

et al. 2020

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Background A reservoir of replication-competent but latent virus is the main obstacle to a cure for HIV-1 infection. Much of this reservoir resides in memory CD4 T cells. We hypothesized that these cells can be reactivated with antigens from HIV-1 and other common pathogens to reverse latency. Results We obtained mononuclear cells from the peripheral blood of antiretroviral-treated patients with suppressed viremia. We tested pools of peptides and proteins derived from HIV-1 and from other pathogens including CMV for their ability to reverse latency ex vivo by activation of memory responses. We assessed activation of the CD4 T cells by measuring the up-regulation of cell-surface CD69. We assessed HIV-1 expression using two assays: a real-time PCR assay for virion-associated viral RNA and a droplet digital PCR assay for cell-associated, multiply spliced viral mRNA. Reversal of latency occurred in a minority of cells from some participants, but no single antigen induced HIV-1 expression ex vivo consistently. When reversal of latency was induced by a specific peptide pool or protein, the extent was proportionally greater than that of T cell activation. Conclusions In this group of patients in whom antiretroviral therapy was started during chronic infection, the latent reservoir does not appear to consistently reside in CD4 T cells of a predominant antigen-specificity. Peptide-antigens reversed HIV-1 latency ex vivo with modest and variable activity. When latency was reversed by specific peptides or proteins, it was proportionally greater than the extent of T cell activation, suggesting partial enrichment of the latent reservoir in cells of specific antigen-reactivity.

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“…Further, we observed enhanced naive cell subsets with the α4β7 + fraction of CD4 + T cells that were accompanied by increased reactivation of the viral reservoir, emphasizing that the need for latency [ 42 ] central memory cells as the viral reservoir could be heterogeneously distributed within the diverse CD4 + T cell subsets [ 65 ]. This finding is supported by Zerbato et al, who recently reported that indeed naive CD4 + T cells harbor replication-competent virus, albeit at levels lower than central memory CD4 + T cells [ 66 ].…”

Section: Discussionmentioning

confidence: 99%

Retinoic Acid Improves the Recovery of Replication-Competent Virus from Latent SIV Infected Cells

Olwenyi

Acharya

Routhu

et al. 2020

Cells

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The accurate estimation and eradication of Human Immunodeficiency Virus (HIV) viral reservoirs is limited by the incomplete reactivation of cells harboring the latent replication-competent virus. We investigated whether the in vitro and in vivo addition of retinoic acid (RA) enhances virus replication and improves the detection of latent virus. Peripheral blood mononuclear cells (PBMCs) from naive and anti-retroviral therapy (ART)-treated SIV-infected rhesus macaques (RMs) were cultured in vitro with anti-CD3/CD28 + IL-2 in the presence/absence of RA. Viral RNA and p27 levels were quantified using RT-qPCR and ELISA, respectively. Viral reservoirs were estimated using the Tat/Rev-Induced Limited Dilution Assay (TILDA) and Quantitative Viral Outgrowth Assay (QVOA). In vitro and in vivo measures revealed that there was also an increase in viral replication in RA-treated versus without RA conditions. In parallel, the addition of RA to either CD3/CD28 or phorbol myristate acetate (PMA)/ionomycin during QVOA and TILDA, respectively, was shown to augment reactivation of the replication-competent viral reservoir in anti-retroviral therapy (ART)-suppressed RMs as shown by a greater than 2.3-fold increase for QVOA and 1 to 2-fold increments for multi-spliced RNA per million CD4+ T cells. The use of RA can be a useful approach to enhance the efficiency of current protocols used for in vitro and potentially in vivo estimates of CD4+ T cell latent reservoirs. In addition, flow cytometry analysis revealed that RA improved estimates of various viral reservoir assays by eliciting broad CD4 T-cell activation as demonstrated by elevated CD25 and CD38 but reduced CD69 and PD-1 expressing cells.

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Replication competence of virions induced from CD4+ lymphocytes latently infected with HIV

Cited by 8 publications

References 17 publications

Assessing the Suitability of Next-Generation Viral Outgrowth Assays to Measure Human Immunodeficiency Virus 1 Latent Reservoir Size

Assessing the Suitability of Next-Generation Viral Outgrowth Assays to Measure Human Immunodeficiency Virus 1 Latent Reservoir Size

Inconsistent reversal of HIV-1 latency ex vivo by antigens of HIV-1, CMV, and other infectious agents

Retinoic Acid Improves the Recovery of Replication-Competent Virus from Latent SIV Infected Cells

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