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. 2021 Jul 2;224(1):92-100.
doi: 10.1093/infdis/jiaa718.

Longitudinal Dynamics of Intact HIV Proviral DNA and Outgrowth Virus Frequencies in a Cohort of Individuals Receiving Antiretroviral Therapy

Shane D Falcinelli  1   2 , Kayla W Kilpatrick  3 , Jenna Read  1 , Ross Murtagh  1 , Brigitte Allard  1 , Simon Ghofrani  1 , Jennifer Kirchherr  1 , Katherine S James  1 , Erin Stuelke  1 , Caroline Baker  1   4 , JoAnn D Kuruc  1   4 , Joseph J Eron  4 , Michael G Hudgens  3 , Cynthia L Gay  1   4 , David M Margolis  1   2   4 , Nancie M Archin  1   4
Affiliations

Longitudinal Dynamics of Intact HIV Proviral DNA and Outgrowth Virus Frequencies in a Cohort of Individuals Receiving Antiretroviral Therapy

Shane D Falcinelli et al. J Infect Dis. .

Abstract

Background: The replication-competent human immunodeficiency virus (HIV) reservoir is the major barrier to cure. The quantitative viral outgrowth assay (QVOA), the gold-standard method to quantify replication-competent HIV, is resource intensive, which limits its application in large clinical trials. The intact proviral DNA assay (IPDA) requires minimal cell input relative to QVOA and quantifies both defective and intact proviral HIV DNA, the latter potentially serving as a surrogate marker for replication-competent provirus. However, there are limited cross-sectional and longitudinal data on the relationship between IPDA and QVOA measurements.

Methods: QVOA and IPDA measurements were performed on 156 resting CD4 T-cell (rCD4) samples from 83 antiretroviral therapy-suppressed HIV-positive participants. Longitudinal QVOA and IPDA measurements were performed on rCD4 from 29 of these participants.

Results: Frequencies of intact, defective, and total proviruses were positively associated with frequencies of replication-competent HIV. Longitudinally, decreases in intact proviral frequencies were strikingly similar to that of replication-competent virus in most participants. In contrast, defective proviral DNA frequencies appeared relatively stable over time in most individuals.

Conclusions: Changes in frequencies of IPDA-derived intact proviral DNA and replication-competent HIV measured by QVOA are similar. IPDA is a promising high-throughput approach to estimate changes in the frequency of the replication-competent reservoir.

Keywords: HIV; IPDA; QVOA; cure; decay; longitudinal; reservoir.

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Figures

Figure 1.
Figure 1.
Comparison of IPDA and QVOA HIV reservoir measurements. A, Frequency per million rCD4 T cells for each assay for individuals treated (A) during CHI on long-term (median 4.4 years) suppressive ART; and (B) during acute or early HIV infection on short-term (median 0.8 years) suppressive ART. Left-censored values are indicated with open symbols for each assay parameter. A,B, Black bars represent median values. C, Relationship between IPDA parameters and QVOA-derived IUPM for 156 resting CD4 T-cell samples from 76 individuals. Black, closed circles represent left-censored values for an IPDA and/or QVOA parameter. Mixed effects models of log10(IUPM) indicated associations between QVOA and log10(intact DNA) (estimated slope, 0.74; 95% CI, .64–.84), log10(total DNA) (estimated slope, 0.97; 95% CI, .80–1.14), log10(3′ deleted/hypermutated DNA) (estimated slope, 0.42; 95% CI, .24–.60), and log10(5′-deleted DNA) (estimated slope, 0.34; 95% CI, .17–.52). Abbreviations: ART, antiretroviral therapy; CHI, chronic HIV infection; CI, confidence interval; HIV, human immunodeficiency virus; IPDA, intact proviral DNA assay; IUPM, infectious units per million; QVOA, quantitative viral outgrowth assay; rCD4, resting CD4.
Figure 2.
Figure 2.
Longitudinal comparison of IPDA and QVOA measurements in participants treated during acute/early HIV infection. A-1 to A-6 indicates individual participants that were treated during acute/early HIV infection. Changes in HIV reservoir frequency over time for IPDA and QVOA measurements in (A) 3 participants with sampling 0.5–2.5 years post-ART suppression; (B) 2 participants with sampling 0.5–6 years post-ART suppression; and (C) 1 participant with sampling over 11 years of ART suppression. Open symbols indicate left-censored values. Time periods when participant received a study intervention (VOR and/or an autologous dendritic cell HIV vaccine as specified in the graph) are indicated by dotted line. Errors bars for QVOA represent 95% CIs from the maximum likelihood method [26]. Errors bars for IPDA measurements represent 95% CIs for the proviral frequency estimates using the total error from the QuantaSoft analysis software, which takes into account technical and subsampling error associated with ddPCR [27]. D, Percent change per year for each HIV reservoir frequency for all acute/early treated individuals (n = 9). Black lines represent the median for each parameter with the IQR. Mean difference between each IPDA measure and QVOA was assessed using linear mixed effect models (see “Methods”). Estimate mean differences versus QVOA were −0.1 (95% CI, −11.3 to 11.2) for intact DNA, −31.3 (95% CI, −56.1 to −6.6) for 3′-deleted/hypermutated DNA, −32.1 (95% CI, −59.4 to −4.9) for 5′-deleted DNA, and −8.1 (95% CI, −18.7 to 2.4) for total DNA. Abbreviations: ART, antiretroviral therapy; CI, confidence interval; ddPCR, digital droplet polymerase chain reaction; HIV, human immunodeficiency virus; IPDA, intact proviral DNA assay; IQR, interquartile range; QVOA, quantitative viral outgrowth assay; rCD4, resting CD4; VOR, vorinostat.
Figure 3.
Figure 3.
Longitudinal comparison of IPDA and QVOA measurements in participants treated during chronic HIV Infection. C-1 to C-10 indicates individual participants that were treated during chronic HIV infection. Changes in HIV reservoir frequency over time for IPDA and QVOA measurements in (A) 4 participants with sampling during the first 5 years post-ART suppression; (B) 4 participants with sampling following 5–10 years post-ART suppression; and (C) participants where the direction of change for intact DNA and QVOA were not concordant. Time periods when participants received a study intervention (VOR, CTLs, and/or an autologous dendritic cell HIV vaccine as specified in the graphs) are indicated by dotted lines. Errors bars for QVOA represent the 95% CIs from the maximum likelihood method [26]. Errors bars for IPDA measurements represent 95% CIs for the proviral frequency estimates using the total error from the QuantaSoft analysis software, which takes into account technical and subsampling error associated with ddPCR [27]. D, Percent change per year for each HIV reservoir frequency for all individuals treated during CHI (n = 20). Black lines represent the median for each parameter with the IQR. Mean difference between each IPDA measure and QVOA was assessed using a linear mixed effects model (see “Methods”). Estimate mean differences versus QVOA were 1.6 (95% CI, −10.2 to 13.4) for intact DNA, −23.4 (95% CI, −38.2 to −8.5) for 3′-deleted/hypermutated DNA, −31.0 (95% CI, −62.1 to 0.2) for 5′-deleted DNA, and −9.3 (95% CI, −19.1 to 0.4) for total DNA. Abbreviations: CTLs, expanded cytotoxic lymphocytes; ART, antiretroviral therapy; CI, confidence interval; ddPCR, digital droplet polymerase chain reaction; HIV, human immunodeficiency virus; IPDA, intact proviral DNA assay; IQR, interquartile range; QVOA, quantitative viral outgrowth assay; rCD4, resting CD4; VOR, vorinostat.
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