HIVepsilon-seq-scalable characterization of intact persistent proviral HIV reservoirs in women

Abstract

The lack of a reliable method to accurately detect when replication-competent HIV has been cleared is a major challenge in developing a cure. This study introduces a new approach called the HIVepsilon-seq (HIVε-seq) assay, which uses long-read sequencing technology and bioinformatics to scrutinize the HIV genome at the nucleotide level, distinguishing between defective and intact HIV. This study included 30 participants on antiretroviral therapy, including 17 women, and was able to discriminate between defective and genetically intact viruses at the single DNA strand level. The HIVε-seq assay is an improvement over previous methods, as it requires minimal sample, less specialized lab equipment, and offers a shorter turnaround time. The HIVε-seq assay offers a promising new tool for researchers to measure the intact HIV reservoir, advancing efforts towards finding a cure for this devastating disease.

Keywords: HIV; HIV persistence; third-generation sequencing.

Fig 1

HIVε-seq pipeline. Illustration of HIV ε-seq workflow to detect intact HIV proviral genomes. DNA is extracted from either PBMCs or CD4⁺ T cells, and nearly full-length PCR is performed on the extracted DNA. The reads are aligned to the HXB2 reference HIV sequence. Intact reads are inferred through the bioinformatic removal of reads containing large deletions/inversions, premature stop codons, or regulatory region defects.

Fig 2

Consensus congruence of amplified HIV provirus. Percent identity of a consensus sequence generated from randomly selected groups of reads containing 1, 5, 10, or 100 reads amplified from an integrated HIV-1 genome (J89GFP cell line).

Fig 3

HIVe-seq characterization of proviral reservoir in PLWH on ART. Characterization of the persistent proviral reservoir in CD4⁺ T cells or PBMCs from women (A) and men (B) chronically infected with HIV-1. Amplification—reads with both PCR binding sites. Full-length—reads mapping to greater than 8,500 bp of HXB2 reference. No stops—full length reads with no stop codons detected in protein coding regions. Intact—no stop reads without known regulatory region defects.

Fig 4

HIVε-seq categories by viral levels measured with QVOA and IPDA. (A) Frequency of replication-competent persistent provirus in HIV-1 infected adults on ART with fully suppressed viremia on days 15 and 19 (B) measured by the QVOA assay or (C) frequency of total DNA and intact DNA (D) as measured by IPDA compared to HIVε-seq category. No amplification—no reads map to HXB2 reference. Amplification—reads that map to HXB2 and contain 5′ and 3′ PCR primer binding sites. Full-length—reads mapping to greater than 8,500 bp of HXB2 reference. No stops—full-length reads with no stop codons detected in protein-coding regions. Intact—no stop reads without known regulatory region defects. Colors denote individual participants. Asterisks indicate P-value less than 0.05.