Development of a highly sensitive and specific intact proviral DNA assay for HIV-1 subtype B and C.

Autor: Buchholtz NVEJ; Translational Virology, Department of Medical Microbiology, University Medical Center Utrecht, Heidelberglaan 100, 3584C, Utrecht, The Netherlands., Nühn MM; Translational Virology, Department of Medical Microbiology, University Medical Center Utrecht, Heidelberglaan 100, 3584C, Utrecht, The Netherlands., de Jong TCM; Translational Virology, Department of Medical Microbiology, University Medical Center Utrecht, Heidelberglaan 100, 3584C, Utrecht, The Netherlands., Stienstra TAT; Translational Virology, Department of Medical Microbiology, University Medical Center Utrecht, Heidelberglaan 100, 3584C, Utrecht, The Netherlands., Reddy K; Africa Health Research Institute (AHRI), Durban, South Africa., Ndung'u T; Africa Health Research Institute (AHRI), Durban, South Africa.; HIV Pathogenesis Programme, The Doris Duke Medical Research Institute, University of KwaZulu-Natal, Durban, South Africa.; The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, Harvard University, Cambridge, MA, 01238, USA.; Division of Infection and Immunity, University College London, London, UK., Ndhlovu ZM; Africa Health Research Institute (AHRI), Durban, South Africa., Fisher K; Centre for Virus Research, The Westmead Institute for Medical Research, The University of Sydney, Sydney, NSW, Australia., Palmer S; Centre for Virus Research, The Westmead Institute for Medical Research, The University of Sydney, Sydney, NSW, Australia., Wensing AMJ; Translational Virology, Department of Medical Microbiology, University Medical Center Utrecht, Heidelberglaan 100, 3584C, Utrecht, The Netherlands.; ha, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa., Symons J; Translational Virology, Department of Medical Microbiology, University Medical Center Utrecht, Heidelberglaan 100, 3584C, Utrecht, The Netherlands., Nijhuis M; Translational Virology, Department of Medical Microbiology, University Medical Center Utrecht, Heidelberglaan 100, 3584C, Utrecht, The Netherlands. m.nijhuis@umcutrecht.nl.; HIV Pathogenesis Research Unit, Faculty of Health Sciences, University of Witwatersrand, Johannesburg, South Africa. m.nijhuis@umcutrecht.nl.
Jazyk: angličtina
Zdroj: Virology journal [Virol J] 2024 Jan 31; Vol. 21 (1), pp. 36. Date of Electronic Publication: 2024 Jan 31.
DOI: 10.1186/s12985-024-02300-6
Abstrakt: Introduction: HIV reservoir quantification is essential for evaluation of HIV curative strategies and may provide valuable insights about reservoir dynamics during antiretroviral therapy. The Intact Proviral DNA Assay (IPDA) provides the unique opportunity to quantify the intact and defective reservoir. The current IPDA is optimized for HIV-1 subtype B, the dominant subtype in resource-rich settings. However, subtype C is dominant in Sub-Saharan Africa, jointly accounting for around 60% of the pandemic. We developed an assay capable of quantifying intact and defective proviral HIV-1 DNA of subtype B and C.
Methods: Primer and probe sequences were strategically positioned at conserved regions in psi and env and adapted to subtype B&C. In silico analysis of 752 subtype B and 697 subtype C near-full length genome sequences (nFGS) was performed to predict  the specificity and sensitivity. Gblocks were used to determine the limit of blank (LoB), limit of detection (LoD), and different annealing temperatures were tested to address impact of sequence variability.
Results: The in silico analysis showed that the HIV-1 B&C IPDA correctly identified 100% of the intact subtype B, and 86% of the subtype C sequences. In contrast, the original IPDA identified 86% and 12% of these subtype B and C sequences as intact. Furthermore, the HIV-1 B&C IPDA correctly identified hypermutated (87% and 88%) and other defective sequences (73% and 66%) for subtype B and C with comparable specificity as the original IPDA for subtype B (59% and 63%). Subtype B cis-acting sequences were more frequently identified as intact by the HIV-1 B&C IPDA compared to the original IPDA (39% and 2%). The LoB for intact proviral DNA copies was 0, and the LoD for intact proviral DNA copies was 6 (> 95% certainty) at 60 °C. Quantification of 2-6 copies can be performed with > 80% certainty. Lowering the annealing temperature to 55 °C slightly lowered the specificity but prevented exclusion of samples with single mutations in the primer/probe region.
Conclusions: We developed a robust and sensitive assay for the quantification of intact and defective HIV-1 subtype B and C proviral DNA, making this a suitable tool to monitor the impact of (large-scale) curative interventions.
(© 2024. The Author(s).)
Databáze: MEDLINE
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