Delporte Mareva, Lambrechts Laurens, Blomme Evy E, van Snippenberg Willem, Rutsaert Sofie, Verschoore Maxime, De Smet Evelien, Noppe Ytse, De Langhe Nele, De Scheerder Marie-Angélique, Gerlo Sarah, Vandekerckhove Linos, Trypsteen Wim
Department of Internal Medicine and Pediatrics, HIV Cure Research Center, Ghent University Hospital, Ghent University Ghent, Belgium.
BioBix, Department of Data Analysis and Mathematical Modelling, Faculty of Bioscience Engineering, Ghent University Ghent, Belgium.
Clin Chem. 2025 Jan 3;71(1):203-214. doi: 10.1093/clinchem/hvae192.
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.
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.
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.
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.
完整的HIV-1前病毒持续潜伏库，即使在抗逆转录病毒疗法（ART）抑制病毒的情况下仍能反弹，阻碍了实现HIV-1治愈的努力。因此，专门用于定量完整前病毒的检测方法对于评估治愈性干预措施的效果至关重要。最近的两项检测方法已用于临床试验：完整前病毒DNA检测（IPDA）和四重定量PCR（Q4PCR）。虽然IPDA由于扩增短片段而更敏感，但它可能仅通过对两个前病毒区域进行定量来高估完整部分。Q4PCR对4个前病毒区域进行采样，但基于测序，更有利于较短（因此不完整）的前病毒序列的扩增。
利用数字PCR（dPCR）技术进展，我们开发了“彩虹”五重前病毒HIV-1 DNA检测方法。使用标准材料和来自83名HIV-1感染者的样本对这种首创的检测方法进行了评估，能够同时定量总HIV-1 DNA水平和完整HIV-1 DNA水平。HIV前病毒独特分子标识符（UMI）介导的长读长测序（HIV-PULSE）用于验证彩虹HIV-1 DNA检测方法的特异性。
彩虹检测方法证明与IPDA同样敏感，但特异性更高，并且不会对全长前病毒产生偏差，能够高通量定量总潜伏库大小和完整潜伏库大小。近乎全长的序列使彩虹检测方法的特异性得以验证，并设计了个性化的彩虹引物/探针组，从而能够检测完整HIV-1 DNA。
这一创新为有针对性地评估和监测具有潜在反弹能力的潜伏库提供了可能，有助于HIV-1的管理和治愈策略。临床试验.gov注册号：NCT04553081、NCT04305665
