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Dr. Francesco Andrea Procopio
CHUV, Divison of Immunology and Allergy, Lausanne, Switzerland

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0 HIV
0 HIV vaccines
0 hiv cure
0 HIV reservoir
0 Quantification of HIV DNA and RNA

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Perspective article
Published: 15 June 2021 in Frontiers in Microbiology
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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.

ACS Style

Cynthia Lungu; Riddhima Banga; Rob A. Gruters; Francesco A. Procopio. Inducible HIV-1 Reservoir Quantification: Clinical Relevance, Applications and Advancements of TILDA. Frontiers in Microbiology 2021, 12, 1 .

AMA Style

Cynthia Lungu, Riddhima Banga, Rob A. Gruters, Francesco A. Procopio. Inducible HIV-1 Reservoir Quantification: Clinical Relevance, Applications and Advancements of TILDA. Frontiers in Microbiology. 2021; 12 ():1.

Chicago/Turabian Style

Cynthia Lungu; Riddhima Banga; Rob A. Gruters; Francesco A. Procopio. 2021. "Inducible HIV-1 Reservoir Quantification: Clinical Relevance, Applications and Advancements of TILDA." Frontiers in Microbiology 12, no. : 1.

Brief report
Published: 02 September 2020 in Viruses
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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.

ACS Style

Cynthia Lungu; Francesco A. Procopio; Ronald J. Overmars; Rob J. J. Beerkens; Jolanda J. C. Voermans; Shringar Rao; Henrieke A. B. Prins; Casper Rokx; Giuseppe Pantaleo; David A. M. C. Van De Vijver; Tokameh Mahmoudi; Charles A. B. Boucher; Rob A. Gruters; Jeroen J. A. Van Kampen. Inter-Laboratory Reproducibility of Inducible HIV-1 Reservoir Quantification by TILDA. Viruses 2020, 12, 973 .

AMA Style

Cynthia Lungu, Francesco A. Procopio, Ronald J. Overmars, Rob J. J. Beerkens, Jolanda J. C. Voermans, Shringar Rao, Henrieke A. B. Prins, Casper Rokx, Giuseppe Pantaleo, David A. M. C. Van De Vijver, Tokameh Mahmoudi, Charles A. B. Boucher, Rob A. Gruters, Jeroen J. A. Van Kampen. Inter-Laboratory Reproducibility of Inducible HIV-1 Reservoir Quantification by TILDA. Viruses. 2020; 12 (9):973.

Chicago/Turabian Style

Cynthia Lungu; Francesco A. Procopio; Ronald J. Overmars; Rob J. J. Beerkens; Jolanda J. C. Voermans; Shringar Rao; Henrieke A. B. Prins; Casper Rokx; Giuseppe Pantaleo; David A. M. C. Van De Vijver; Tokameh Mahmoudi; Charles A. B. Boucher; Rob A. Gruters; Jeroen J. A. Van Kampen. 2020. "Inter-Laboratory Reproducibility of Inducible HIV-1 Reservoir Quantification by TILDA." Viruses 12, no. 9: 973.

Other
Published: 24 May 2018
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A recent study conducted in blood has proposed CD32 as the marker identifying the ‘elusive’ HIV reservoir. We have investigated the distribution of CD32+CD4 T cells in blood and lymph nodes(LNs) of healthy HIV-1 uninfected, viremic untreated and long-term treated HIV-1 infected individuals and their relationship with PD-1+CD4 T cells. The frequency of CD32+CD4 T cells was increased in viremic as compared to treated individuals in LNs and a large proportion(up to 50%) of CD32+cells co-expressed PD-1 and were enriched within T follicular helper cells(Tfh) cells. We next investigated the role of LN CD32+CD4 T cells in the HIV reservoir. Total HIV DNA was enriched in CD32+and PD-1+CD4 T cells as compared to CD32-and PD-1-cells in both viremic and treated individuals but there was no difference between CD32+and PD-1+cells. There was not enrichment of latently infected cells with inducible HIV-1 in CD32+versus PD-1+cells in ART treated individuals. HIV-1 transcription was then analyzed in LN memory CD4 T cell populations sorted on the basis of CD32 and PD-1 expression. CD32+PD-1+CD4 T cells were significantly enriched in cell associated HIV RNA as compared to CD32-PD-1-(average 5.2 fold in treated and 86.6 fold in viremics), to CD32+PD-1-(2.2 fold in treated and 4.3 fold in viremics) and to CD32-PD-1+cell populations(2.2 fold in ART treated and 4.6 fold in viremics). Similar levels of HIV-1 transcription were found in CD32+PD-1-and CD32-PD-1+CD4 T cells. Interestingly, the proportion of CD32+and PD-1+CD4 T cells negatively correlated with CD4 T cell counts and length of therapy while positively correlated with viremia. Therefore, the expression of CD32 identifies, independently of PD-1, a CD4 T cell population with persistent HIV-1 transcription and CD32 and PD-1 co-expression the CD4 T cell population with the highest levels of HIV-1 transcription in both viremic and treated individuals.ImportanceThe existence of long-lived latently infected resting memory CD4 T cells represents a major obstacle to the eradication of HIV infection. Identifying cell markers defining latently infected cells containing replication competent virus is important in order to determine the mechanisms of HIV persistence and to develop novel therapeutic strategies to cure HIV infection. We provide evidence that PD-1 and CD32 may have a complementary role in better defining CD4 T cell populations infected with HIV-1. Furthermore, CD4 T cells co-expressing CD32 and PD-1 identify a CD4 T cell population with high levels of persistent HIV-1 transcription.

ACS Style

Alessandra Noto; Francesco A. Procopio; Riddhima Banga; Madeleine Suffiotti; Jean-Marc Corpataux; Matthias Cavassini; Craig Fenwick; Raphael Gottardo; Matthieu Perreau; Giuseppe Pantaleo. CD32+and PD-1+Lymph Node CD4 T Cells Support Persistent HIV-1 Transcription in Treated Aviremic Individuals. 2018, 329938 .

AMA Style

Alessandra Noto, Francesco A. Procopio, Riddhima Banga, Madeleine Suffiotti, Jean-Marc Corpataux, Matthias Cavassini, Craig Fenwick, Raphael Gottardo, Matthieu Perreau, Giuseppe Pantaleo. CD32+and PD-1+Lymph Node CD4 T Cells Support Persistent HIV-1 Transcription in Treated Aviremic Individuals. . 2018; ():329938.

Chicago/Turabian Style

Alessandra Noto; Francesco A. Procopio; Riddhima Banga; Madeleine Suffiotti; Jean-Marc Corpataux; Matthias Cavassini; Craig Fenwick; Raphael Gottardo; Matthieu Perreau; Giuseppe Pantaleo. 2018. "CD32+and PD-1+Lymph Node CD4 T Cells Support Persistent HIV-1 Transcription in Treated Aviremic Individuals." , no. : 329938.

Review
Published: 01 July 2016 in Current Opinion in HIV and AIDS
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The persistence of HIV within long-lived HIV-infected CD4 T cells is the primary obstacle towards HIV eradication and numerous strategies are currently being evaluated to target and kill HIV-infected cells to ultimately find a cure. HIV reservoirs are classically quantified by standard methods such as integrated HIV DNA (Alu PCR) and/or quantitative viral outgrowth assay; however, recent technical advances may offer new opportunities to comprehensively assess the impact of clinical interventions.Digital droplet PCR, tat/rev-induced limiting dilution analysis, enhanced quantitative viral outgrowth assay, and whole genome sequencing technologies offer increased precision and/or higher sensitivity to quantify and characterize HIV reservoirs in antiretroviral therapy-treated HIV-infected patients.The objective of this review is to highlight the characteristics and limits of recent technical advances that may help to monitor the impact of clinical interventions in antiretroviral therapy-treated patients.

ACS Style

Riddhima Banga; Francesco A. Procopio; Matthieu Perreau. Current approaches to assess HIV-1 persistence. Current Opinion in HIV and AIDS 2016, 11, 424 -431.

AMA Style

Riddhima Banga, Francesco A. Procopio, Matthieu Perreau. Current approaches to assess HIV-1 persistence. Current Opinion in HIV and AIDS. 2016; 11 (4):424-431.

Chicago/Turabian Style

Riddhima Banga; Francesco A. Procopio; Matthieu Perreau. 2016. "Current approaches to assess HIV-1 persistence." Current Opinion in HIV and AIDS 11, no. 4: 424-431.