Novel assay reveals a large, inducible, replication-competent HIV-1 reservoir in resting CD4+ T cells

Abstract

Although antiretroviral therapy can suppress HIV-1 infection to undetectable levels of plasma viremia, integrated latent HIV-1 genomes that encode replication competent virus persist in resting CD4+ T cells. This latent HIV-1 reservoir represents a major barrier to a cure. Currently, there are substantial ongoing efforts to identify therapeutic approaches that will eliminate or reduce the size of this latent HIV-1 reservoir. In this regard, a sensitive assay which can accurately and rapidly quantify inducible replication competent latent HIV-1 from resting CD4+ T cells is essential for HIV-1 eradication studies. Here we describe a reporter cell-based assay to quantify inducible replication competent latent HIV-1. This assay has several advantages over existing technology in that it: (i) is sensitive; (ii) requires only a small blood volume; (iii) is faster, less labor intensive, and less expensive, and (iv) can be readily adapted to a high-throughput format. Using this assay we show that the size of the inducible latent HIV-1 reservoir in aviremic participants on therapy is approximately 70-fold larger than previous estimates.

TZA: a novel assay for measuring the latent HIV-1 reservoir

“…Detecting a 70-fold larger reservoir size in the blood using the TZA compared to the Q-VOA method [32] suggests that latent HIV-1 may be present in 1 in 20,000 rCD4+ T cells than 1 in 1 million rCD4 as predicted earlier [11] These results also indicate that an additional 6% of latent HIV-1 has yet to be effectively targeted by LRAs. The level of fPVE determined by TZA can be used as an accurate measure of the size of the replication-competent reservoir.…”

“…We recently reported the development of a sensitive TZM-bl cell based assay (termed TZA) [32] to quantify the latent HIV-1 reservoir in blood. This assay utilizes the TZM-bl cell line, which stably expresses CD4, CCR5, and CXCR4, and carries an integrated copy of the β-galactosidase (β-gal) gene under control of an HIV-1 long terminal repeat (HIV-1 LTR) promoter that allows for the detection of inducible replication-competent HIV-1.…”

“…The TZA method involves two steps: (1) induction of latent HIV-1 production from rCD4+ T cells using a strong LRA, such as anti-CD3/CD28 activating antibodies and (2) quantification of induced replication-competent HIV-1 in the TZM-bl cells by measuring β-gal expression [32]. …”

“…We directly compared the TZA and Q-VOA methods for quantifying the size of the inducible replication-competent pool of latent HIV-1 in rCD4 isolated from 13 aviremic HIV-1 infected individuals on cART from the Pittsburgh Multicenter AIDS Cohort Study (MACS) [32]. For the TZA, the amount of replication-competent HIV-1 ranged from 1.2 to 141 (mean 46.4) IUPM.…”

“…In nine HIV-1 infected MACS participants, the fractional HIV-1 provirus expression (fPVE) was also evaluated. The fPVE as measured by the TZA ranged from 0.12% to 13.93% (mean 4.03%), while the fPVE as measured by the Q-VOA ranged from 0.02% to 0.9% (mean 0.14%) [32]. Control experiments showed that the addition of efavirenz, an HIV-1 RT inhibitor, during the induction of β-gal induction in TZM-bl cells decreased IUPM values, indicating that the IUPM values in the TZA were not due to other nonspecific factors, such as cytokines, released as a result of the activation of T cells by anti-CD3/CD28 monoclonal antibodies (mAbs).…”