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Integrative assessment of total and intact HIV-1 reservoir by a 5-region multiplexed rainbow DNA digital PCR assay

Mareva Delporte (UGent) , Laurens Lambrechts, Evy Blomme (UGent) , Willem van Snippenberg, Sofie Rutsaert (UGent) , Maxime Verschoore (UGent) , Evelien De Smet (UGent) , Ytse Noppe (UGent) , Nele De Langhe (UGent) , Marie-Angélique De Scheerder (UGent) , et al.
(2025) CLINICAL CHEMISTRY. 71(1). p.203-214
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Abstract
Background Persistent latent reservoirs of intact HIV-1 proviruses, capable of rebounding despite suppressive antiretroviral therapy (ART), hinder efforts towards an HIV-1 cure. Hence, assays specifically quantifying intact proviruses are crucial to assess the impact of curative interventions. Two recent assays have been utilized in clinical trials: intact proviral DNA assay (IPDA) and quadruplex quantitative PCR (Q4PCR). While IPDA is more sensitive due to amplifying short fragments, it may overestimate intact fractions by relying only on quantification of 2 proviral regions. Q4PCR samples 4 proviral regions, yet is sequencing-based, favoring amplification of shorter, hence non-intact, proviral sequences.Methods Leveraging digital PCR (dPCR) advancements, we developed the "Rainbow" 5-plex proviral HIV-1 DNA assay. This first-in-its-kind assay was evaluated using standard materials and samples from 83 people living with HIV-1, enabling simultaneous quantification of both total and intact HIV-1 DNA levels. HIV proviral unique molecular identifier (UMI)-mediated long-read sequencing (HIV-PULSE) was used to validate the specificity of the Rainbow HIV-1 DNA assay.Results The Rainbow assay proved equally sensitive but more specific than IPDA and is not subjected to bias against full-length proviruses, enabling high-throughput quantification of total and intact reservoir size. The near full-length sequences allowed validation of the Rainbow specificity and the design of personalized Rainbow primer/probe sets, which enabled the detection of intact HIV-1 DNA.Conclusions This innovation offers potential for targeted evaluation and monitoring of potential rebound-competent reservoirs, contributing to HIV-1 management and cure strategies. ClinicalTrials.gov Registration Numbers: NCT04553081, NCT04305665.
Keywords
REAL-TIME PCR, PROVIRAL DNA, QUANTIFICATION, VISUALIZATION

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MLA
Delporte, Mareva, et al. “Integrative Assessment of Total and Intact HIV-1 Reservoir by a 5-Region Multiplexed Rainbow DNA Digital PCR Assay.” CLINICAL CHEMISTRY, vol. 71, no. 1, 2025, pp. 203–14, doi:10.1093/clinchem/hvae192.
APA
Delporte, M., Lambrechts, L., Blomme, E., van Snippenberg, W., Rutsaert, S., Verschoore, M., … Trypsteen, W. (2025). Integrative assessment of total and intact HIV-1 reservoir by a 5-region multiplexed rainbow DNA digital PCR assay. CLINICAL CHEMISTRY, 71(1), 203–214. https://doi.org/10.1093/clinchem/hvae192
Chicago author-date
Delporte, Mareva, Laurens Lambrechts, Evy Blomme, Willem van Snippenberg, Sofie Rutsaert, Maxime Verschoore, Evelien De Smet, et al. 2025. “Integrative Assessment of Total and Intact HIV-1 Reservoir by a 5-Region Multiplexed Rainbow DNA Digital PCR Assay.” CLINICAL CHEMISTRY 71 (1): 203–14. https://doi.org/10.1093/clinchem/hvae192.
Chicago author-date (all authors)
Delporte, Mareva, Laurens Lambrechts, Evy Blomme, Willem van Snippenberg, Sofie Rutsaert, Maxime Verschoore, Evelien De Smet, Ytse Noppe, Nele De Langhe, Marie-Angélique De Scheerder, Sarah Gerlo, Linos Vandekerckhove, and Wim Trypsteen. 2025. “Integrative Assessment of Total and Intact HIV-1 Reservoir by a 5-Region Multiplexed Rainbow DNA Digital PCR Assay.” CLINICAL CHEMISTRY 71 (1): 203–214. doi:10.1093/clinchem/hvae192.
Vancouver
1.
Delporte M, Lambrechts L, Blomme E, van Snippenberg W, Rutsaert S, Verschoore M, et al. Integrative assessment of total and intact HIV-1 reservoir by a 5-region multiplexed rainbow DNA digital PCR assay. CLINICAL CHEMISTRY. 2025;71(1):203–14.
IEEE
[1]
M. Delporte et al., “Integrative assessment of total and intact HIV-1 reservoir by a 5-region multiplexed rainbow DNA digital PCR assay,” CLINICAL CHEMISTRY, vol. 71, no. 1, pp. 203–214, 2025.
@article{01JMF7NJQ3AVQ5J8PRY43PCZ6R,
  abstract     = {{Background Persistent latent reservoirs of intact HIV-1 proviruses, capable of rebounding despite suppressive antiretroviral therapy (ART), hinder efforts towards an HIV-1 cure. Hence, assays specifically quantifying intact proviruses are crucial to assess the impact of curative interventions. Two recent assays have been utilized in clinical trials: intact proviral DNA assay (IPDA) and quadruplex quantitative PCR (Q4PCR). While IPDA is more sensitive due to amplifying short fragments, it may overestimate intact fractions by relying only on quantification of 2 proviral regions. Q4PCR samples 4 proviral regions, yet is sequencing-based, favoring amplification of shorter, hence non-intact, proviral sequences.Methods Leveraging digital PCR (dPCR) advancements, we developed the "Rainbow" 5-plex proviral HIV-1 DNA assay. This first-in-its-kind assay was evaluated using standard materials and samples from 83 people living with HIV-1, enabling simultaneous quantification of both total and intact HIV-1 DNA levels. HIV proviral unique molecular identifier (UMI)-mediated long-read sequencing (HIV-PULSE) was used to validate the specificity of the Rainbow HIV-1 DNA assay.Results The Rainbow assay proved equally sensitive but more specific than IPDA and is not subjected to bias against full-length proviruses, enabling high-throughput quantification of total and intact reservoir size. The near full-length sequences allowed validation of the Rainbow specificity and the design of personalized Rainbow primer/probe sets, which enabled the detection of intact HIV-1 DNA.Conclusions This innovation offers potential for targeted evaluation and monitoring of potential rebound-competent reservoirs, contributing to HIV-1 management and cure strategies. ClinicalTrials.gov Registration Numbers: NCT04553081, NCT04305665.}},
  author       = {{Delporte, Mareva and Lambrechts, Laurens and Blomme, Evy and van Snippenberg, Willem and Rutsaert, Sofie and Verschoore, Maxime and De Smet, Evelien and Noppe, Ytse and De Langhe, Nele and De Scheerder, Marie-Angélique and Gerlo, Sarah and Vandekerckhove, Linos and Trypsteen, Wim}},
  issn         = {{0009-9147}},
  journal      = {{CLINICAL CHEMISTRY}},
  keywords     = {{REAL-TIME PCR,PROVIRAL DNA,QUANTIFICATION,VISUALIZATION}},
  language     = {{eng}},
  number       = {{1}},
  pages        = {{203--214}},
  title        = {{Integrative assessment of total and intact HIV-1 reservoir by a 5-region multiplexed rainbow DNA digital PCR assay}},
  url          = {{http://doi.org/10.1093/clinchem/hvae192}},
  volume       = {{71}},
  year         = {{2025}},
}
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