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Author Notes:

Yury V. Kuzmichev, Email: yury.v.kuzmichev@emory.edu

Y.V.K. contributed to conceptualization, formal analysis, investigation, methodology, writing (original draft), and visualization. E.R.W. contributed to conceptualization, formal analysis, methodology, project administration, supervision, visualization, and writing (original draft). D.A.K. contributed to conceptualization, formal analysis, funding acquisition, methodology, project administration, supervision, and writing (original draft). C.L.-S. contributed to data curation, formal analysis, and methodology. A.W., M.J.B., and A.M. performed single-genome sequencing analysis and visualization. R.G.P. contributed to funding acquisition, methodology, and project administration. M.S. and S.B. performed HIV-1 RNA quantification on the Panther system. N.A. and J.A. contributed to investigation and provided participant samples from RV130/514 trial. W.B. and S.T. contributed to investigation. S.T. performed total and integrated HIV DNA measurement. M.F.K. contributed to methodology and supervision. M.P.B. provided participant samples from RAVEN cohort. All authors contributed to the critical review and editing of the manuscript.

This work was supported with federal funds from the NIH/NIAID/DAIDS under contract HHSN272201500017C entitled “Quantitative Viral Outgrowth Assay (QVOA) Service Resource”-Dr. Betty Poon (COR). This work was further supported by intramural funds from NIH/NCI to Dr. Mary F. Kearney. Hologic provided reagents and support for HIV RNA viral load testing of the QVOA culture supernatants using the Aptima assay on the Panther platform. This work was supported with funds from the Bill and Melinda Gates Foundation award INV-008500 (to M.P.B.). The RV130 study was executed by the Infectious Disease Clinical Research Program (IDCRP), a Department of Defense (DoD) program executed by the Uniformed Services University of the Health Sciences (USUHS) through a cooperative agreement by the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. (HJF). RV130 has been funded with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), under Inter-Agency Agreement Y1-AI-5072. RV514 study was supported through Military HIV Research Program. Furthermore, this work was supported by UM1AI164562, co-funded by National Heart, Lung and Blood Institute, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Neurological Disorders and Stroke, National Institute on Drug Abuse and the National Institute of Allergy and Infectious Diseases (to D.A.K. and Y.V.K.). We thank Drs. Betty Poon and Roger Miller for their intellectual input and discussions throughout this project. We are grateful to Krupa Subramanian, Christine Raney, Hayley P. Blankenship, Chunsheng Huang, and Jiayi Wei for technical expertise, and Ellen R. Nordgren and Rachel L. Jarret for their support in providing clinical sample management.

The authors have declared that no competing interests exist. The views expressed are those of the authors and should not be construed to represent the positions of the Uniformed Services University, the Department of Defense, US Army, or the United States Government. The investigators have adhered to the policies for protection of human subjects as prescribed in Army Regulation 70-25.

Subjects:

Keywords:

  • Science & Technology
  • Multidisciplinary Sciences
  • Science & Technology - Other Topics
  • IMMUNODEFICIENCY-VIRUS TYPE-1
  • CD4(+) T-CELLS
  • NUCLEIC-ACID AMPLIFICATION
  • LATENT RESERVOIR
  • PROVIRUSES PRODUCE
  • QUANTITATIVE ASSAY
  • DETECTION LIMITS
  • PROTEIN
  • IMMUNOASSAY
  • SENSITIVITY

Application of ultrasensitive digital ELISA for p24 enables improved evaluation of HIV-1 reservoir diversity and growth kinetics in viral outgrowth assays

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Journal Title:

SCIENTIFIC REPORTS

Volume:

Volume 13, Number 1

Publisher:

, Pages 10958-10958

Type of Work:

Article | Final Publisher PDF

Abstract:

The advent of combined antiretroviral therapy (cART) has been instrumental in controlling HIV-1 replication and transmission and decreasing associated morbidity and mortality. However, cART alone is not able to cure HIV-1 due to the presence of long-lived, latently infected immune cells, which re-seed plasma viremia when cART is interrupted. Assessment of HIV-cure strategies using ex vivo culture methods for further understanding of the diversity of reactivated HIV, viral outgrowth, and replication dynamics are enhanced using ultrasensitive digital ELISA based on single-molecule array (Simoa) technology to increase the sensitivity of endpoint detection. In viral outgrowth assays (VOA), exponential HIV-1 outgrowth has been shown to be dependent upon initial virus burst size surpassing a critical growth threshold of 5100 HIV-1 RNA copies. Here, we show an association between ultrasensitive HIV-1 Gag p24 concentrations and HIV-1 RNA copy number that characterize viral dynamics below the exponential replication threshold. Single-genome sequencing (SGS) revealed the presence of multiple identical HIV-1 sequences, indicative of low-level replication occurring below the threshold of exponential outgrowth early during a VOA. However, SGS further revealed diverse related HIV variants detectable by ultrasensitive methods that failed to establish exponential outgrowth. Overall, our data suggest that viral outgrowth occurring below the threshold necessary for establishing exponential growth in culture does not preclude replication competence of reactivated HIV, and ultrasensitive detection of HIV-1 p24 may provide a method to detect previously unquantifiable variants. These data strongly support the use of the Simoa platform in a multi-prong approach to measuring latent viral burden and efficacy of therapeutic interventions aimed at an HIV-1 cure.

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© The Author(s) 2023

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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