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

Yury V. Kuzmichev*, Carol Lackman-Smith, Sonia Bakkour, Ann Wiegand, Michael J. Bale, Andrew Musick, Wendy Bernstein, Naomi Aronson, Julie Ake, Sodsai Tovanabutra, Mars Stone, Roger G. Ptak, Mary F. Kearney, Michael P. Busch, Elizabeth R. Wonderlich, Deanna A. Kulpa*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


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.

Original languageEnglish
Article number10958
JournalScientific Reports
Issue number1
StatePublished - Dec 2023
Externally publishedYes


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