An Optimized Tat/Rev Induced Limiting Dilution Assay for the Characterization of HIV-1 Latent Reservoirs

Mishra, Swarnima; Gohil, Yuvraj; Mehta, Kavita; D'silva, Anish; Amanullah, Afzal; Selvam, Deepak; Pargain, Neelam; Nala, Narendra; Sanjeeva, G N; Ranga, Udaykumar

doi:10.21769/bioprotoc.4391

Cited by 3 publications

(3 citation statements)

References 17 publications

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“…Human in vitro models can be used for the investigation of individual-specific immune responses. A wide range of techniques has been developed to measure the persistent HIV reservoir in virally suppressed subjects [ 5 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 ]. These assays can be classified into two groups: (1) PCR/sequencing-based and (2) cell-culture-based assays.…”

Section: Discussionmentioning

confidence: 99%

“…PCR/sequencing methods [ 34 , 35 , 36 , 37 , 38 ] include molecular assays recently developed for profiling the transcriptional activity and the chromosomal locations of individual proviruses [ 38 ]. Focusing on cell culture assays, many studies of HIV latency and reactivation rely on the evaluation of viral reservoir after in vitro stimulation of PBMCs or CD4 + T cells cultured in RPMI (e.g., QVOA, TILDA) [ 31 , 33 , 39 , 40 ]. However, a growing body of evidence indicates that different cell culture matrices strongly influence cell behavior in in vitro systems.…”

Section: Discussionmentioning

confidence: 99%

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How CD4+ T Cells Transcriptional Profile Is Affected by Culture Conditions: Towards the Design of Optimal In Vitro HIV Reactivation Assays

et al. 2023

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Most of the current assays directed at the investigation of HIV reactivation are based on cultures of infected cells such as Peripheral Blood Mononuclear Cells (PBMCs) or isolated CD4+ T cells, stimulated in vitro with different activator molecules. The culture media in these in vitro tests lack many age- and donor-specific immunomodulatory components normally found within the autologous plasma. This triggered our interest in understanding the impact that different matrices and cell types have on T cell transcriptional profiles following in vitro culture and stimulation. Methods: Unstimulated or stimulated CD4+ T cells of three young adults with perinatal HIV-infection were isolated from PBMCs before or after culture in RPMI medium or autologous plasma. Transcriptomes were sequenced using Oxford Nanopore technologies. Results: Transcriptional profiles revealed the activation of similar pathways upon stimulation in both media with a higher magnitude of TCR cascade activation in CD4+ lymphocytes cultured in RPMI. Conclusions: These results suggest that for studies aiming at quantifying the magnitude of biological mechanisms under T cell activation, the autologous plasma could better approximate the in vivo environment. Conversely, if the study aims at defining qualitative aspects, then RPMI culture could provide more evident results.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

How CD4+ T Cells Transcriptional Profile Is Affected by Culture Conditions: Towards the Design of Optimal In Vitro HIV Reactivation Assays

et al. 2023

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“…Subsequently, subtype-specific assays have emerged [39]. A variant assay, called U-TILDA, attempted to circumvent the challenges of viral genetic diversity and amplify the various HIV-1 genetic subtypes with uniform efficiency [40 ▪ ,41]. Using a panel of viral molecular clones representing eight HIV-1 subtypes, a droplet-digital PCR identified that gag and pol regions, compared to ltr , are better suited to quantify HIV DNA [42].…”

Section: Subtypes and The Latent Viral Reservoirmentioning

confidence: 99%

HIV-1 subtypes and latent reservoirs

Ranga,

Panchapakesan,

Saini

2023

Current Opinion in HIV and AIDS

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Purpose of review We explore the current status of research on HIV-1 subtype-specific variations and their impact on HIV-1 latency. We also briefly address the controversy surrounding the decision-making process governing the ON/OFF states of HIV-1 transcription, specifically focusing on the regulatory elements, the long terminal repeat (LTR), and Tat. Understanding the decision-making process is crucial for developing effective intervention strategies, such as the 'shock-and-kill’ approach, to reactivate latent HIV-1. Recent findings Attention has been drawn to subtype-specific transcription factor binding site (TFBS) variations and the possible impact of these variations on viral latency. Further, diverse subtype-specific assays have been developed to quantify the latent viral reservoirs. One interesting observation is the relatively larger latent reservoirs in HIV-1B infection than those of other viral subtypes, which needs rigorous validation. The emergence of LTR-variant viral strains in HIV-1C demonstrating significantly higher levels of latency reversal has been reported. Summary Despite persistent and substantial efforts, latent HIV-1 remains a formidable challenge to a functional cure. Determined and continued commitment is needed to understand the ON/OFF decision-making process of HIV-1 latency, develop rigorous assays for accurately quantifying the latent reservoirs, and identify potent latency-reversing agents and cocktails targeting multiple latency stages. The review emphasizes the importance of including diverse viral subtypes in future latency research.

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The latent HIV reservoir: current advances in genetic sequencing approaches

Moar,

Premeaux,

Atkins

et al. 2023

mBio

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Multiple cellular HIV reservoirs in diverse anatomical sites can undergo clonal expansion and persist for years despite suppressive antiretroviral therapy, posing a major barrier toward an HIV cure. Commonly adopted assays to assess HIV reservoir size mainly consist of PCR-based measures of cell-associated total proviral DNA, intact proviruses and transcriptionally competent provirus (viral RNA), flow cytometry and microscopy-based methods to measure translationally competent provirus (viral protein), and quantitative viral outgrowth assay, the gold standard to measure replication-competent provirus; yet no assay alone can provide a comprehensive view of the total HIV reservoir or its dynamics. Furthermore, the detection of extant provirus by these measures does not preclude defects affecting replication competence. An accurate measure of the latent reservoir is essential for evaluating the efficacy of HIV cure strategies. Recent approaches have been developed, which generate proviral sequence data to create a more detailed profile of the latent reservoir. These sequencing approaches are valuable tools to understand the complex multicellular processes in a diverse range of tissues and cell types and have provided insights into the mechanisms of HIV establishment and persistence. These advancements over previous sequencing methods have allowed multiplexing and new assays have emerged, which can document transcriptional activity, chromosome accessibility, and in-depth cellular phenotypes harboring latent HIV, enabling the characterization of rare infected cells across restrictive sites such as the brain. In this manuscript, we provide a review of HIV sequencing-based assays adopted to address challenges in quantifying and characterizing the latent HIV reservoir.

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An Optimized Tat/Rev Induced Limiting Dilution Assay for the Characterization of HIV-1 Latent Reservoirs

Cited by 3 publications

References 17 publications

How CD4+ T Cells Transcriptional Profile Is Affected by Culture Conditions: Towards the Design of Optimal In Vitro HIV Reactivation Assays

How CD4+ T Cells Transcriptional Profile Is Affected by Culture Conditions: Towards the Design of Optimal In Vitro HIV Reactivation Assays

HIV-1 subtypes and latent reservoirs

The latent HIV reservoir: current advances in genetic sequencing approaches

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