scholarly journals Inter-Laboratory Reproducibility of Inducible HIV-1 Reservoir Quantification by TILDA

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 973
Author(s):  
Cynthia Lungu ◽  
Francesco A. Procopio ◽  
Ronald J. Overmars ◽  
Rob J. J. Beerkens ◽  
Jolanda J. C. Voermans ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Concordance Correlation Coefficient ◽  
Concordance Correlation ◽  
Dilution Assay ◽  
Assay Performance ◽  
Latent Hiv ◽  
Hiv 1 ◽  
Limiting Dilution ◽  
Viral Outgrowth Assay

Substantial efforts to eliminate or reduce latent HIV-1 reservoirs are underway in clinical trials and have created a critical demand for sensitive, accurate, and reproducible tools to evaluate the efficacy of these strategies. Alternative reservoir quantification assays have been developed to circumvent limitations of the quantitative viral outgrowth assay. One such assay is tat/rev induced limiting dilution assay (TILDA), which measures the frequency of CD4+ T cells harboring inducible latent HIV-1 provirus. We modified pre-amplification reagents and conditions (TILDA v2.0) to improve assay execution and first internally validated assay performance using CD4+ T cells obtained from cART-suppressed HIV-1-infected individuals. Detection of tat/rev multiply spliced RNA was not altered by modifying pre-amplification conditions, confirming the robustness of the assay, and supporting the technique’s amenability to limited modifications to ensure better implementation for routine use in clinical studies of latent HIV-1 reservoirs. Furthermore, we cross-validated results of TILDA v2.0 and the original assay performed in two separate laboratories using samples from 15 HIV-1-infected individuals. TILDA and TILDA v2.0 showed a strong correlation (Lin’s Concordance Correlation Coefficient = 0.86). The low inter-laboratory variability between TILDAs performed at different institutes further supports use of TILDA for reservoir quantitation in multi-center interventional HIV-1 Cure trials.

scholarly journals Inducible HIV-1 Reservoir Quantification: Clinical Relevance, Applications and Advancements of TILDA

2021 ◽  
Vol 12 ◽  
Author(s):  
Cynthia Lungu ◽  
Riddhima Banga ◽  
Rob A. Gruters ◽  
Francesco A. Procopio
Keyword(s):  
Gold Standard ◽  
Clinical Relevance ◽  
Cost Effective ◽  
Viral Reservoir ◽  
Dilution Assay ◽  
Latent Hiv ◽  
Essential Prerequisite ◽  
Hiv 1 ◽  
Limiting Dilution ◽  
Viral Outgrowth Assay

The presence of a stable HIV-1 reservoir persisting over time despite effective antiretroviral suppression therapy precludes a cure for HIV-1. Characterizing and quantifying this residual reservoir is considered an essential prerequisite to develop and validate curative strategies. However, a sensitive, reproducible, cost-effective, and easily executable test is still needed. The quantitative viral outgrowth assay is considered the gold standard approach to quantify the reservoir in HIV-1-infected patients on suppressive ART, but it has several limitations. An alternative method to quantify the viral reservoir following the reactivation of latent HIV-1 provirus detects multiply-spliced tat/rev RNA (msRNA) molecules by real-time PCR [tat/rev induced limiting dilution assay (TILDA)]. This article provides a perspective overview of the clinical relevance, various applications, recent advancements of TILDA, and how the assay has contributed to our understanding of the HIV-1 reservoir.

Measuring the Frequency of Latent HIV-1 in Resting CD4+ T Cells Using a Limiting Dilution Coculture Assay

2016 ◽  
pp. 239-253 ◽  
Author(s):  
Gregory M. Laird ◽  
Daniel I. S. Rosenbloom ◽  
Jun Lai ◽  
Robert F. Siliciano ◽  
Janet D. Siliciano
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Latent Hiv ◽  
Hiv 1 ◽  
Limiting Dilution

scholarly journals Reactivation of latent HIV-1 in central memory CD4+ T cells through TLR-1/2 stimulation

Retrovirology ◽  
2013 ◽  
Vol 10 (1) ◽  
pp. 119 ◽  
Author(s):  
Camille L Novis ◽  
Nancie M Archin ◽  
Maria J Buzon ◽  
Eric Verdin ◽  
June L Round ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Central Memory ◽  
Latent Hiv ◽  
Memory Cd4 T Cells ◽  
Hiv 1

scholarly journals Endothelial Cell Stimulation Overcomes Restriction and Promotes Productive and Latent HIV-1 Infection of Resting CD4+ T Cells

2013 ◽  
Vol 87 (17) ◽  
pp. 9768-9779 ◽  
Author(s):  
A. Shen ◽  
J. J. Baker ◽  
G. L. Scott ◽  
Y. P. Davis ◽  
Y.-Y. Ho ◽  
...  
Keyword(s):  
T Cells ◽  
Endothelial Cell ◽  
Cd4 T Cells ◽  
Cell Stimulation ◽  
Latent Hiv ◽  
Hiv 1

scholarly journals An In-Depth Comparison of Latent HIV-1 Reactivation in Multiple Cell Model Systems and Resting CD4+ T Cells from Aviremic Patients

2013 ◽  
Vol 9 (12) ◽  
pp. e1003834 ◽  
Author(s):  
Celsa A. Spina ◽  
Jenny Anderson ◽  
Nancie M. Archin ◽  
Alberto Bosque ◽  
Jonathan Chan ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Cell Model ◽  
Model Systems ◽  
Multiple Cell ◽  
Latent Hiv ◽  
Hiv 1

scholarly journals A two-color haploid genetic screen identifies novel host factors involved in HIV latency

2021 ◽  
Author(s):  
Michael D Röling ◽  
Mahsa Mollapour Sisakht ◽  
Enrico Ne ◽  
Panagiotis Moulos ◽  
Mateusz Stoszko ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Cd4 T Cells ◽  
Genetic Screen ◽  
Host Factors ◽  
Hiv Latency ◽  
Novel Host ◽  
Latent Hiv ◽  
Hiv 1

AbstractTo identify novel host factors as putative targets to reverse HIV latency, we performed an insertional mutagenesis genetic screen in a latently HIV-1-infected pseudo-haploid KBM7 cell line (Hap-Lat). Following mutagenesis, insertions were mapped to the genome and bioinformatic analysis resulted in the identification of 69 candidate host genes involved in maintaining HIV-1 latency. A select set of candidate genes was functionally validated using shRNA mediated depletion in latent HIV-1 infected J-Lat A2 and 11.1 T cell lines. We confirmed ADK, CHD9, CMSS1, EVI2B, EXOSC8, FAM19A, GRIK5, IRF2BP2, NF1, and USP15 as novel host factors involved in the maintenance of HIV latency. Chromatin immunoprecipitation assays indicated that CHD9, a Chromodomain Helicase DNA-binding protein, maintains HIV latency via direct association with the HIV 5’LTR, and its depletion results in increased histone acetylation at the HIV-1 promoter, concomitant with HIV-1 latency reversal. FDA-approved inhibitors 5-Iodotubercidin, Trametinib, and Topiramate, targeting ADK, NF1, and GRIK5, respectively were characterized for their latency reversal potential. While 5-Iodotubercidin exhibited significant cytotoxicity in both J-Lat and primary CD4+ T cells, Trametinib reversed latency in J-Lat cells but not in latently HIV-1-infected primary CD4+ T cells. Crucially, Topiramate reversed latency in cell-line models and latently infected primary CD4+ T cells, without inducing T cell activation or significant toxicity. Thus, using an adaptation of a haploid forward genetic screen, we identified novel and druggable host factors contributing to HIV-1 latency.ImportanceA reservoir of latent HIV-1-infected cells persists in the presence of combination antiretroviral therapy (cART), representing a major obstacle for viral eradication. Reactivation of the latent HIV-1 provirus is part of curative strategies which aim to promote clearance of the infected cells. Using a two-color haploid screen, we identified 69 candidate genes as latency maintaining host factors and functionally validated a subset of 10 of those in additional T-cell based cell line models of HIV-1 latency. We further demonstrated that CHD9 is associated with HIV-1’s promoter, the 5’LTR while this association is lost upon reactivation. Additionally, we characterized the latency reversal potential of FDA compounds targeting ADK, NF1, and GRIK5 and identify the GRIK5 inhibitor Topiramate as a viable latency reversal agent with clinical potential.

scholarly journals Activation of the Anti-Oxidative Stress Response Reactivates Latent HIV-1 Through the Mitochondrial Antiviral Signaling Protein Isoform MiniMAVS

2021 ◽  
Vol 12 ◽  
Author(s):  
Indra Sarabia ◽  
Camille L. Novis ◽  
Amanda B. Macedo ◽  
Hiroshi Takata ◽  
Racheal Nell ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
T Cells ◽  
Cd4 T Cells ◽  
Cellular Stress ◽  
Antiviral Signaling ◽  
Signaling Protein ◽  
Latent Hiv ◽  
Mitochondrial Antiviral Signaling ◽  
Hiv 1 ◽  
Mitochondrial Antiviral Signaling Protein

The mitochondrial antiviral signaling protein (MAVS) is part of the cell’s innate immune mechanism of defense. MAVS mRNA is bicistronic and can give rise to a full length-MAVS and a shorter isoform termed miniMAVS. In response to viral infections, viral RNA can be sensed by the cytosolic RNA sensors retinoic acid-inducible gene I (RIG-I) and/or melanoma differentiation-associated protein 5 (MDA5) and activate NF-κB through interaction with MAVS. MAVS can also sense cellular stress and activate an anti-oxidative stress (AOS) response through the activation of NF-κB. Because NF-κB is a main cellular transcription factor for HIV-1, we wanted to address what role MAVS plays in HIV-1 reactivation from latency in CD4 T cells. Our results indicate that RIG-I agonists required full length-MAVS whereas the AOS response induced by Dynasore through its catechol group can reactivate latent HIV-1 in a MAVS dependent manner through miniMAVS isoform. Furthermore, we uncover that PKC agonists, a class of latency-reversing agents, induce an AOS response in CD4 T cells and require miniMAVS to fully reactivate latent HIV-1. Our results indicate that the AOS response, through miniMAVS, can induce HIV-1 transcription in response to cellular stress and targeting this pathway adds to the repertoire of approaches to reactivate latent HIV-1 in ‘shock-and-kill’ strategies.

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