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, 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 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 highly active antiretroviral therapy 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 growing out in each well, thus capturing another level of information beyond the well being positive for viral antigen. The infectious units per million (IUPM) cell values estimated using a maximum likelihood approach, based on the number of distinct viral lineages 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.

INTRODUCTION

Eradication of HIV-1 from infected individuals on suppressive therapy has been hampered due to HIV-1 persistence in viral reservoirs. Among potential cellular reservoirs contributing to HIV-1 persistence, 1–3 latent infection of resting memory CD4+ T cells is well demonstrated. 4–8 Assays have been developed to quantify resting CD4+ T cells that harbor replication-competent HIV-1 in people on highly active antiretroviral therapy (ART). The current most widely recognized culture assay, the quantitative viral outgrowth assay (QVOA), measures infectious units per million (IUPM) resting CD4+ T cells. Although QVOA is the current gold standard for measuring the latent HIV-1 reservoir, the assay has limited throughput capacity due to the need for large numbers of cells and the need to wait for the outgrowth of virus from single cells to assess the titer by limiting dilution. 9 Using QVOA, it has been possible to show that a small fraction of the resting CD4+ T cells harbor replication-competent HIV-1 (approximately 0.1–10 per million cells). 4, 10, 11 However, Ho et al 12 in a recent study, demonstrated that this number is likely an underestimate, as some cells that encode an HIV-1 provirus and can be induced to express HIV-1 RNA do not produce a replication-competent virus that can be recovered after a single activation event during the QVOA. A modified version of QVOA has recently been developed. In this modified assay, a CD4+/CCR5+ cell line (MOLT-4/CCR5) replaces primary cells to support virus outgrowth, and an HIV-specific reverse-transcription polymerase chain reaction (RT-PCR) assay replaces p24 enzyme-linked immunosorbent assay (ELISA) for virus detection in the supernatant, which allows earlier detection of virus outgrowth. 13 These modifications shorten the time required to complete the QVOA; however, the modified assay still requires large amounts of blood from a patient to allow limiting dilution of resting CD4+ T cells to measure an IUPM value.