Integrative assessment of total and intact HIV-1 reservoir by a five-region multiplexed Rainbow digital PCR assay

Persistent latent reservoirs of intact HIV-1 proviruses, capable of rebounding despite suppressive ART, hinder efforts towards an HIV-1 cure. Hence, assays specifically quantifying intact proviruses are crucial to assess the impact of curative interventions. Clinical trials have utilized two recent assays: intact proviral DNA assay (IPDA) and Q4PCR. While IPDA is more sensitive due to amplifying short fragments, it may overestimate intact fractions by relying only on two small regions. Q4PCR is sequencing-based and its performance might be subjected to bias against full-length proviruses. Leveraging digital PCR (dPCR) advancements, we developed the ‘Rainbow’ 5-plex proviral HIV-1 DNA assay, assessing it with standard materials and samples from 69 PLWH. The Rainbow assay proved equally sensitive but more specific than IPDA, is not subjected to bias against full-length proviruses, enabling high-throughput quantification of total and intact reservoir size. This innovation offers potential for targeted evaluation and monitoring of rebound-competent reservoirs, contributing to HIV-1 management and cure strategies. Teaser The 5 color ‘HIV-1 Rainbow’ digital PCR assay offers a multi-level view on the HIV reservoir in one snapshot reaction. ### Competing Interest Statement Wim Trypsteen is a member of the Scientific Advisory Board for dPCR at Qiagen (Hilden, Germany). All other authors declare they have no competing interests.