Quantification of the Latent HIV-1 Reservoir Using Ultra Deep Sequencing and Primer ID in a Viral Outgrowth Assay

Abstract

Background In this study, we measured the latent HIV-1 reservoir harboring replication-competent HIV-1 in resting CD4+ T cells in participants on highly active antiretroviral therapy (HAART), quantitating the frequency of latent infection through the use of a Primer ID-based Ultra Deep Sequencing Assay (UDSA), in comparison to the readout of the quantitative viral outgrowth assay (QVOA). Methods Viral RNA derived from culture wells of QVOA that scored as HIV-1 p24 capsid (CA) antigen-positive were tagged with a specific barcode during cDNA synthesis, and the sequences within the V1–V3 region of the HIV-1 env gene were analyzed for diversity using the Primer ID-based paired-end MiSeq platform. We analyzed samples from a total of 19 participants, 2 initially treated with HAART in acute infection and 17 treated during chronic infection. Phylogenetic trees were generated with all viral lineages detected from culture wells derived from each participant to determine the number of distinct viral lineages (DVLs) growing out in each well, thus capturing another level of information beyond the well being positive for viral antigen. The infectious units per million cells (IUPM) values estimated using a maximum likelihood approach, based on the number of DVLs detected (VOA-UDSA), were compared with those obtained from QVOA measured using limiting dilution. Results IUPM estimates determined by VOA-UDSA ranged from 0.14 to 3.66 and strongly correlated with the IUPM estimates determined by QVOA (r=0.94; p<0.0001). Conclusions VOA-UDSA may be an alternative readout for that currently used for QVOA.

Paired quantitative and qualitative assessment of the replication-competent HIV-1 reservoir and comparison with integrated proviral DNA

“…Phylogenetic analyses of the replication-competent reservoir are usually limited because bulk outgrowth cultures produce species with little diversity, even when analyzed by ultra-deep sequencing (17)(18)(19)(20).…”

HIV Rebound Is Predominantly Fueled by Genetically Identical Viral Expansions from Diverse Reservoirs

“…the central research question (Wang and Palmer, 2018), and we are confident that the strategy we choose is a valuable surrogate for this extensive analysis of the viral reservoir and above all has little impact on our overall conclusion of heterogeneous viral rebound. However, we acknowledge that sequencing a subgenomic region overestimates the number of intact proviruses (Lee et al, 2017b). Furthermore, without integration site analysis, we are unable to determine if identical V1-V3 env sequences are derived from clonal expansions of the same cell (Cohn et al, 2015).…”

“…Although different CD4+ T cell subsets have been identified as major contributors to the HIV-1 reservoir (Chomont et al, 2009;Hiener et al, 2017;Lee et al, 2017b) and different surface markers (CXCR3, CD30, and CD32a) (Banga et al, 2018;Descours et al, 2017;Henrich et al, 2017) and immune exhaustion markers (PD-1, TIGIT, and LAG-3) (Khoury et al, 2017;McGary et al, 2017) have emerged as potential biomarkers for latently infected cells, previous studies have not converged on a clear pattern or predominant subsets responsible for HIV persistence.…”

Antiretroviral Therapy Interruption (ATI) in HIV-1 Infected Patients Participating in Therapeutic Vaccine Trials: Surrogate Markers of Virological Response

“…Another uncertainty is that measuring methods are not well standardized even though several different tests have been developed. The quantitative viral outgrowth assay (QVOA), which measures infectious units per million (UIPM) in resting CD4+ T cells is considered the gold standard for measuring latent HIV reservoir [ 58 , 59 ]; other measurements obtained by PCR have been proposed as surrogate biomarkers such as total DNA, also called cell-associated DNA (CA-DNA) [ 60 ], proviral, or integrated DNA [ 61 , 62 ] and cell-associated RNA (CA-RNA) [ 63 ].…”