Senior Fellow, University of Washington
The standard assay to estimate latent HIV reservoirs in PBMC is the quantitative viral outgrowth assay (QVOA). QVOA underestimates reservoir size due to the presence of intact proviruses (IPs) resistant to activation by one or more mitogens and latency reversal agents. In contrast, viral DNA load assays overestimate reservoir size, as most infected cells harbor defective proviruses. IPs have been variously estimated to correspond to 3-11% of provirus populations, however, we argue that these are overestimates due to the PCR methods employed. More accurate measurements are therefore needed to define reservoirs and to assess the efficacy of cure strategies. We used two newly developed assays to assess the composition and functionality of HIV reservoirs – the infected cell expansion (ICE) assay, that employs culturing infected cells at limiting dilution with PHA, IL2 and antiretroviral drugs to block virus spread, followed a 3- or 6-probe viral ORF detection assay (VODA). These assays permit an accurate view of complete proviral structures and integration sites, although they too introduce potential bias by requiring cell outgrowth and survival in cell culture prior to analysis. We found that <1% of infected cells from 4 individuals that initiated long-term ART early in infection harbor IPs. Proviruses from ICE cultures derived from the same proliferating cell population in vivo had the same genome structure, and similar transcription and DNA methylation patterns, indicating provirus stability in culture. The frequencies of clonal cell populations detected by ICE are similar to the frequencies observed in vivo, suggesting a lack of selective loss of these cells during ICE. Indeed, cells found to be proliferating in vivo were often overrepresented relative to uninfected cells in ICE cultures, suggesting that cells found to be proliferating in vivo have a growth advantage in vitro. The vast majority of cells we examined harbored non-infectious proviruses, most commonly resulting from deletions between the 5’LTR and gag. Targeted analysis of RNA transcripts at proviral loci showed that deletions in the 5’LTR was associated with 3’LTR-initiated transcription and read-through into cellular sequences. ICE and VODA are likely to broadly assist further investigation and understanding of latent reservoirs as well as the possible role of defective proviruses influencing T-cell proliferation and functionality.
Abstract: Antiretroviral treatment (ART) of HIV infection suppresses viral replication. Yet if ART is stopped, virus reemerges because of the persistence of infected cells. We evaluated the contribution of infected-cell proliferation and sites of proviral integration to HIV persistence. A total of 534 HIV integration sites (IS) and 63 adjacent HIV env sequences were derived from three study participants over 11.3 to 12.7 years of ART. Each participant had identical viral sequences integrated at the same position in multiple cells, demonstrating infected-cell proliferation. Integrations were overrepresented in genes associated with cancer and favored in 12 genes across multiple participants. Over time on ART, a greater proportion of persisting proviruses were in proliferating cells. HIV integration into specific genes may promote proliferation of HIV-infected cells, slowing viral decay during ART.
Pub.: 12 Jul '14, Pinned: 16 Jun '17
Abstract: Although antiretroviral therapy (ART) suppresses viral replication to clinically undetectable levels, human immunodeficiency virus type 1 (HIV-1) persists in CD4+ T cells in a latent form that is not targeted by the immune system or by ART1, 2, 3, 4, 5. This latent reservoir is a major barrier to curing individuals of HIV-1 infection. Many individuals initiate ART during chronic infection, and in this setting, most proviruses are defective6. However, the dynamics of the accumulation and the persistence of defective proviruses during acute HIV-1 infection are largely unknown. Here we show that defective proviruses accumulate rapidly within the first few weeks of infection to make up over 93% of all proviruses, regardless of how early ART is initiated. By using an unbiased method to amplify near-full-length proviral genomes from HIV-1-infected adults treated at different stages of infection, we demonstrate that early initiation of ART limits the size of the reservoir but does not profoundly affect the proviral landscape. This analysis allows us to revise our understanding of the composition of proviral populations and estimate the true reservoir size in individuals who were treated early versus late in infection. Additionally, we demonstrate that common assays for measuring the reservoir do not correlate with reservoir size, as determined by the number of genetically intact proviruses. These findings reveal hurdles that must be overcome to successfully analyze future HIV-1 cure strategies.
Pub.: 08 Aug '16, Pinned: 16 Jun '17
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 efforts to identify therapeutic approaches that will eliminate or reduce the size of this latent HIV-1 reservoir. In this regard, a sensitive assay that 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 into 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.
Pub.: 30 May '17, Pinned: 16 Jun '17