Elite suppressors have low frequencies of intact HIV-1 proviral DNA

Kwaa, Abena K.; Garliss, Caroline C.; Ritter, Kristen D.; Laird, Gregory M.; Blankson, Joel N.

doi:10.1097/qad.0000000000002474

Cited by 21 publications

(18 citation statements)

References 25 publications

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“…More recent studies of 61 and 50 participants reported failure rates of 9.8% and 12%, respectively 6 , 7 . The largest study undertaken to date using the IPDA, a multi-cohort study published by the assay’s developers, reports an overall failure rate of 6.3% of 400 participants 5 , although it is important to note that this study includes a number of published cohorts including four with 0% failure rates 4 , 8 – 10 . As such, the inferred failure rate for some of the unpublished datasets could be as high as 18%, a value that approaches the 28% rate reported in the present study.…”

Section: Discussionmentioning

confidence: 99%

“…While detection failures are expected for any molecular assay targeting a genetically variable pathogen, this rate has varied markedly across studies, ranging from 0% in the initial two reports (0/62 1 and 0/81 4 ) to 12% (6/50) in the most recent publication 7 , where the assay was performed by the same group. A recent multi-cohort study comprising 400 individuals reports an overall 6.3% failure rate 5 ; however, because this study includes the above 4 and other published cohorts with 0% failure rates 8 – 10 it can be inferred that the rates for the unpublished datasets are higher. Thus, while the IPDA is undeniably a major methodological advance, further study is needed to delineate the impact of HIV-1 sequence diversity.…”

Section: Introductionmentioning

confidence: 94%

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HIV-1 diversity considerations in the application of the Intact Proviral DNA Assay (IPDA)

et al. 2021

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The Intact Proviral DNA Assay (IPDA) was developed to address the critical need for a scalable method for intact HIV-1 reservoir quantification. This droplet digital PCR-based assay simultaneously targets two HIV-1 regions to distinguish genomically intact proviruses against a large background of defective ones, and its application has yielded insights into HIV-1 persistence. Reports of assay failures however, attributed to HIV-1 polymorphism, have recently emerged. Here, we describe a diverse North American cohort of people with HIV-1 subtype B, where the IPDA yielded a failure rate of 28% due to viral polymorphism. We further demonstrate that within-host HIV-1 diversity can lead the IPDA to underestimate intact reservoir size, and provide examples of how this phenomenon could lead to erroneous interpretation of clinical trial data. While the IPDA represents a major methodological advance, HIV-1 diversity should be addressed before its widespread adoption as a principal readout in HIV-1 remission trials.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 94%

HIV-1 diversity considerations in the application of the Intact Proviral DNA Assay (IPDA)

et al. 2021

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“…The IPDA has already led to novel insights. For example, the assay has been used to provide more accurate estimates of the decay rate of the latent reservoir (19), to demonstrate differential decay rates for cells carrying intact and defective proviruses (9,19,20), to document low reservoir size in elite suppressors (21), and to explore the frequency and inducibility of intact HIV-1 proviruses in different subsets of CD4 + T cells (22).…”

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confidence: 99%

Intact proviral DNA assay analysis of large cohorts of people with HIV provides a benchmark for the frequency and composition of persistent proviral DNA

Simonetti

White

Tumiotto

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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A scalable approach for quantifying intact HIV-1 proviruses is critical for basic research and clinical trials directed at HIV-1 cure. The intact proviral DNA assay (IPDA) is a novel approach to characterizing the HIV-1 reservoir, focusing on the genetic integrity of individual proviruses independent of transcriptional status. It uses multiplex digital droplet PCR to distinguish and separately quantify intact proviruses, defined by a lack of overt fatal defects such as large deletions and APOBEC3G-mediated hypermutation, from the majority of proviruses that have such defects. This distinction is important because only intact proviruses cause viral rebound on ART interruption. To evaluate IPDA performance and provide benchmark data to support its implementation, we analyzed peripheral blood samples from 400 HIV-1+ adults on ART from several diverse cohorts, representing a robust sample of treated HIV-1 infection in the United States. We provide direct quantitative evidence that defective proviruses greatly outnumber intact proviruses (by >12.5 fold). However, intact proviruses are present at substantially higher frequencies (median, 54/106 CD4+ T cells) than proviruses detected by the quantitative viral outgrowth assay, which requires induction and in vitro growth (∼1/106 CD4+ T cells). IPDA amplicon signal issues resulting from sequence polymorphisms were observed in only 6.3% of individuals and were readily apparent and easily distinguished from low proviral frequency, an advantage of the IPDA over standard PCR assays which generate false-negative results in such situations. The large IPDA dataset provided here gives the clearest quantitative picture to date of HIV-1 proviral persistence on ART.

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“…Recently Bruner et al developed a high throughput intact proviral DNA assay (IPDA) that approximates the frequency of intact proviruses as determined by full genome sequencing [25] . Kwaa et al used the IPDA to compare the size of the viral reservoir in patients on ART and ECs and found that the frequency of both total and intact DNA in EC was approximately 20-fold lower than the frequency seen in patients on ART [26] . Jiang and colleagues sequenced 1,385 and 2,388 full length proviral genomes from 64 ECs and 41 CPs respectively and also found significantly lower levels of intact and total DNA in ECs [27] .…”

Section: Evidence Of Replication-competent Virus In Ecsmentioning

confidence: 99%

“…Kwaa et al described a 200-fold variation in the level of intact proviral DNA that could be detected in the IPDA in ECs. One subject who has routinely had negative culture assays over greater than 10 years also had undetectable proviral DNA levels [26] . Jiang et al identified 2 EC who have extremely low levels of intact proviral DNA.…”

Section: Exceptional Elite Controllersmentioning

confidence: 99%

Viral reservoirs in elite controllers of HIV-1 infection: Implications for HIV cure strategies

2020

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Elite controllers are HIV-1 positive subjects who control viral replication without antiretroviral therapy. Many of these subjects have replication-competent virus and thus represent a model of a functional cure. Peripheral CD4+ T cells in these subjects have small reservoirs with a low frequency of intact proviruses. Furthermore, recent studies suggest that many of these intact proviruses are disproportionally integrated at sites that have limited transcriptional activity raising the possibility that replication-competent viruses do not replicate because they are in a “blocked and locked” state. However, this feature is probably a consequence rather than a cause of elite control. Additionally, evolution of plasma virus has been detected in many elites suggesting that there continues to be ongoing viral replication in other compartments. While exceptional elite controllers with very limited viral reservoirs have recently been described, more work is needed to determine whether these patients have achieved a sterilizing cure.

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Elite suppressors have low frequencies of intact HIV-1 proviral DNA

Cited by 21 publications

References 25 publications

HIV-1 diversity considerations in the application of the Intact Proviral DNA Assay (IPDA)

HIV-1 diversity considerations in the application of the Intact Proviral DNA Assay (IPDA)

Intact proviral DNA assay analysis of large cohorts of people with HIV provides a benchmark for the frequency and composition of persistent proviral DNA

Viral reservoirs in elite controllers of HIV-1 infection: Implications for HIV cure strategies

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