Adaptation of Droplet Digital PCR-Based HIV Transcription Profiling to Digital PCR and Association of HIV Transcription and Total or Intact HIV DNA
Carolin Tumpach,
Ajantha Rhodes,
Youry Kim,
Jesslyn Ong,
Haoming Liu,
Doris Chibo,
Julian Druce,
Deborah Williamson,
Rebecca Hoh,
Steven G. Deeks,
Steven A. Yukl,
Michael Roche,
Sharon R. Lewin,
Sushama Telwatte
Affiliations
Carolin Tumpach
Department of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia
Ajantha Rhodes
Department of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia
Youry Kim
Department of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia
Jesslyn Ong
Department of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia
Haoming Liu
Department of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia
Doris Chibo
Victorian Infectious Diseases Reference Laboratory, The Royal Melbourne Hospital at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia
Julian Druce
Victorian Infectious Diseases Reference Laboratory, The Royal Melbourne Hospital at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia
Deborah Williamson
Department of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia
Rebecca Hoh
Department of Medicine, University of California San Francisco (UCSF), San Francisco, CA 94143, USA
Steven G. Deeks
Department of Medicine, University of California San Francisco (UCSF), San Francisco, CA 94143, USA
Steven A. Yukl
Department of Medicine, University of California San Francisco (UCSF), San Francisco, CA 94143, USA
Michael Roche
Department of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia
Sharon R. Lewin
Department of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia
Sushama Telwatte
Department of Infectious Diseases, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia
In most people living with HIV (PLWH) on effective antiretroviral therapy (ART), cell-associated viral transcripts are readily detectable in CD4+ T cells despite the absence of viremia. Quantification of HIV RNA species provides insights into the transcriptional activity of proviruses that persist in cells and tissues throughout the body during ART (‘HIV reservoir’). One such technique for HIV RNA quantitation, ‘HIV transcription profiling’, developed in the Yukl laboratory, measures a series of HIV RNA species using droplet digital PCR. To take advantage of advances in digital (d)PCR, we adapted the ‘HIV transcription profiling’ technique to Qiagen’s dPCR platform (QIAcuity) and compared its performance to droplet digital (dd)PCR (Bio-Rad QX200 system). Using RNA standards, the two technologies were tested in parallel and assessed for multiple parameters including sensitivity, specificity, linearity, and intra- and inter-assay variability. The newly validated dPCR assays were then applied to samples from PLWH to determine HIV transcriptional activity relative to HIV reservoir size. We report that HIV transcriptional profiling was readily adapted to dPCR and assays performed similarly to ddPCR, with no differences in assay characteristics. We applied these assays in a cohort of 23 PLWH and found that HIV reservoir size, based on genetically intact proviral DNA, does not predict HIV transcriptional activity. In contrast, levels of total DNA correlated with levels of most HIV transcripts (initiated, proximally and distally elongated, unspliced, and completed, but not multiply spliced), suggesting that a considerable proportion of HIV transcripts likely originate from defective proviruses. These findings may have implications for measuring and assessing curative strategies and clinical trial outcomes.
