New Frontiers in Measuring and Characterizing the HIV Reservoir

Abstract

A cure for HIV infection remains elusive due to the persistence of replication-competent HIV proviral DNA during suppressive antiretroviral therapy (ART). With the exception of rare elite or post-treatment controllers of viremia, withdrawal of ART invariably results in the rebound of viremia and progression of HIV disease. A thorough understanding of the reservoir is necessary to develop new strategies in order to reduce or eliminate the reservoir. However, there is significant heterogeneity in the sequence composition, genomic location, stability, and expression of the HIV reservoir both within and across individuals, and a majority of proviral sequences are replication-defective. These factors, and the low frequency of persistently infected cells in individuals on suppressive ART, make understanding the reservoir and its response to experimental reservoir reduction interventions challenging. Here, we review the characteristics of the HIV reservoir, stateof-the-art assays to measure and characterize the reservoir, and how these assays can be applied to accurately detect reductions in reservoir during efforts to develop a cure for HIV infection. In particular, we highlight recent advances in the development of direct measures of provirus, including intact proviral DNA assays and full-length HIV DNA sequencing with integration site analysis. We also focus on novel techniques to quantitate persistent and inducible HIV, including RNA sequencing and RNA/gag protein staining techniques, as well as modified viral outgrowth methods that seek to improve upon throughput, sensitivity and dynamic range.

Measuring the Inducible, Replication-Competent HIV Reservoir Using an Ultra-Sensitive p24 Readout, the Digital ELISA Viral Outgrowth Assay

“…However, the IPDA still over-estimates the frequency of true intact proviruses (6,7). Near full-length or full genome sequencing may provide better assessment of intact proviruses than standard DNA PCR, however, the inefficiency associated with long distance PCR for DNA sequencing, and the extreme costs and labor involved makes them difficult to apply on a large scale [reviewed in Falcinelli et al (13)]. The QVOA, though labor intensive, provides a minimal estimate of the frequency of latent HIV infection, and is still the gold standard for measuring true replication competent HIV.…”

“…Given the importance of the QVOA in assessing replication competent HIV, it is no surprise that efforts have been devoted to streamlining the assay (13). Increasing sensitivity to detect viral outgrowth while minimizing cell input has been a common goal in the field.…”

“…Despite its limitations, the QVOA remains the most reliable method to measure replication competent HIV (12). Thus several modifications of this assay have been made to improve throughput, sensitivity and increased its dynamic range [reviewed in Falcinelli et al (13)]. We report here a modified QVOA, the Digital ELISA Viral Outgrowth or DEVO assay which takes advantage of the Simoa platform (Quanterix Inc., Billerica, MA, United States) (14,15).…”

Lessons Learned From Failures and Success Stories of HIV Breakthroughs: Are We Getting Closer to an HIV Cure?

“…Last year, an assay known as the intact proviral DNA (IPDA) enabled the estimation of intact proviral DNA with as few as 5 million CD4 + T cells, simultaneously detecting several conserved regions of the HIV genome such as intact packaging signal (PS) and the Rev-responsive element (RRE) within env (Bruner et al, 2019). Some researchers cautioned that this assay can only detect a small sub-genomic region (2% of the HIV genome), resulting in the risk of overestimation of intact proviral DNA (Falcinelli et al, 2019). Furthermore, the assay may also possibly quantify non-inducible provirus that may remain permanently latent for the lifetime of an HIV-infected patient.…”

Measuring the Success of HIV-1 Cure Strategies

“…Critical to the elimination of HIV-1 is the elucidation of the specific anatomical and cellular reservoirs of HIV-1. Various differentiation states of CD4 T cells appear to play important roles in the establishment and maintenance of the LR as well as viral recrudescence following ART interruption (Buzon et al, 2014;Kulpa and Chomont, 2015;Banga et al, 2016;De Scheerder et al, 2019;Falcinelli et al, 2019). As discussed above, the LR is primarily hosted in memory CD4 T cells, specifically, central (T CM ), transitional (T TM ), effector memory (T EM ), and memory stem (T SCM ) cells, although the exact contribution of each cell type to the replication competent reservoir is still to be determined (Chomont et al, 2009;Buzon et al, 2014;Soriano-Sarabia et al, 2014;Banga et al, 2016Banga et al, , 2018Kwon et al, 2020).…”