A pinboard by
Michael Dapp

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.


Defective proviruses rapidly accumulate during acute HIV-1 infection

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