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

AbstractThe 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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HIV Diversity Considerations in the Application of the Intact Proviral DNA Assay (IPDA)

10.1101/2020.05.26.115006 ◽

2020 ◽

Author(s):

Natalie N. Kinloch ◽

Yanqin Ren ◽

Winiffer D. Conce Alberto ◽

Winnie Dong ◽

Pragya Khadka ◽

...

Keyword(s):

Digital Pcr ◽

Hiv Reservoir ◽

Dna Assay ◽

Proviral Dna ◽

Subtype B ◽

Clinical Trial Results ◽

Responsive Element ◽

Opening Paragraph ◽

Methodological Advance ◽

Hiv Diversity

Opening Paragraph (serves as abstract for submission) and BodyThe Intact Proviral DNA Assay (IPDA) was developed to address the critical need for a precise and scalable method for intact HIV reservoir quantification1. This duplexed droplet digital PCR (ddPCR) assay simultaneously targets the HIV Packaging Signal (Ψ) and the Rev Responsive Element (RRE) within Envelope (env) to distinguish genomically intact proviruses against a large background of defective ones2. The IPDA requires less time, resources, and biological material than the gold standard for replication-competent HIV reservoir measurement, the Quantitative Viral Outgrowth Assay (QVOA)3, and is being adopted in research and clinical studies4–7. In our cohort of HIV-1 subtype B-infected individuals from North America however, the IPDA yielded a 28% failure rate due to HIV polymorphism. We further demonstrate that within-host HIV diversity can lead the IPDA to underestimate intact HIV reservoir size, which could negatively impact clinical trial results interpretation. While the IPDA represents an important methodological advance, HIV diversity should be addressed before its widespread adoption.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2006816117 ◽

2020 ◽

Vol 117 (31) ◽

pp. 18692-18700 ◽

Cited By ~ 4

Author(s):

Francesco R. Simonetti ◽

Jennifer A. White ◽

Camille Tumiotto ◽

Kristen D. Ritter ◽

Mian Cai ◽

...

Keyword(s):

T Cells ◽

False Negative ◽

Basic Research ◽

The United States ◽

Quantitative Evidence ◽

Dna Assay ◽

Proviral Dna ◽

Negative Results ◽

Hiv 1

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, 54/106CD4+T cells) than proviruses detected by the quantitative viral outgrowth assay, which requires induction and in vitro growth (∼1/106CD4+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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Nonstructured Treatment Interruptions Are Associated With Higher Human Immunodeficiency Virus Reservoir Size Measured by Intact Proviral DNA Assay in People Who Inject Drugs

The Journal of Infectious Diseases ◽

10.1093/infdis/jiaa634 ◽

2020 ◽

Author(s):

Gregory D Kirk ◽

Jacqueline Astemborski ◽

Shruti H Mehta ◽

Kristen D Ritter ◽

Gregory M Laird ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Latent Reservoir ◽

Persons Who Inject Drugs ◽

Major Barrier ◽

Dna Assay ◽

Proviral Dna ◽

Hiv Rna ◽

Treatment Interruptions ◽

Immunodeficiency Virus ◽

Hiv 1

Abstract The latent reservoir for human immunodeficiency virus type 1 (HIV-1) in CD4+ T cells is a major barrier to cure. HIV-1–infected persons who inject drugs (PWID) often struggle to maintain suppression of viremia and experience nonstructured treatment interruptions (NTIs). The effects of injecting drugs or NTIs on the reservoir are unclear. Using the intact proviral DNA assay, we found no apparent effect of heroin or cocaine use on reservoir size. However, we found significantly larger reservoirs in those with frequent NTIs or a shorter interval from last detectable HIV RNA measurement. These results have important implications for inclusion of PWID in HIV-1 cure studies.

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

Nature Communications ◽

10.1038/s41467-021-23515-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Natalie N. Kinloch ◽

Yanqin Ren ◽

Winiffer D. Conce Alberto ◽

Winnie Dong ◽

Pragya Khadka ◽

...

Keyword(s):

Dna Assay ◽

Proviral Dna ◽

Hiv 1

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Prevalence of M184V and K65R in proviral DNA from PBMCs in HIV-infected youths with lamivudine/emtricitabine exposure

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkab080 ◽

2021 ◽

Author(s):

Santiago Jiménez de Ory ◽

Carolina Beltrán-Pavez ◽

Miguel Gutiérrez-López ◽

María Del Mar Santos ◽

Luis Prieto ◽

...

Keyword(s):

Resistance Testing ◽

Study Cohort ◽

Plasma Samples ◽

Resistance Mutations ◽

Proviral Dna ◽

Hiv Resistance ◽

Perinatal Hiv ◽

Subtype B ◽

Hiv 1

Abstract Objectives We analysed the prevalence of M184V/I and/or K65R/E/N mutations archived in proviral DNA (pDNA) in youths with perinatal HIV, virological control and who previously carried these resistance mutations in historic plasma samples. Methods We included vertically HIV-infected youths/young adults aged ≥10 years in the Madrid Cohort of HIV-1 Infected Children and Adolescents, exposed to lamivudine and/or emtricitabine, with M184V/I and/or K65R/E/N in historic plasma samples, on antiretroviral therapy (ART), virologically suppressed (HIV-1 RNA <50 copies/mL), and with available PBMCs in the Spanish HIV BioBank. Genomic DNA was extracted from PBMCs and HIV-1 RT gene was amplified and sequenced for resistance testing by Stanford HIV Resistance tool. Results Among the 225 patients under follow-up in the study cohort, 13 (5.8%) met selection criteria, and RT sequences were recovered in 12 (92.3%) of them. All but one were Spaniards, carrying subtype B, with a median age at PBMCs sampling of 21.3 years (IQR: 15.6–23.1) with 4 years (IQR 2.1–6.5) of suppressed viral load (VL). Nine (75%) youths did not present M184V/I in pDNA after at least 1 year of viral suppression. In December 2019, the remaining three subjects carrying M184V/I in pDNA maintained suppressed viraemia, and two still used emtricitabine in ART. Conclusions The prevalence of resistance mutations to lamivudine and emtricitabine in pDNA in a cohort of youths perinatally infected with HIV who remain with undetectable VL, previously lamivudine and/or emtricitabine experienced, was infrequent. Our results indicate that ART including lamivudine or emtricitabine may also be safe and successful in youths with perinatal HIV with previous experience of and resistances to these drugs detected in plasma.

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Reduced and highly diverse peripheral HIV-1 reservoir in virally suppressed patients infected with non-B HIV-1 strains in Uganda

Retrovirology ◽

10.1186/s12977-022-00587-3 ◽

2022 ◽

Vol 19 (1) ◽

Author(s):

Samira Joussef-Piña ◽

Immaculate Nankya ◽

Sophie Nalukwago ◽

Joy Baseke ◽

Sandra Rwambuya ◽

...

Keyword(s):

Limited Information ◽

Functional Cure ◽

Middle Income ◽

Proviral Dna ◽

Middle Income Countries ◽

Subtype B ◽

Cell Counts ◽

Immunodeficiency Virus ◽

Hiv 1

Abstract Background Our understanding of the peripheral human immunodeficiency virus type 1 (HIV-1) reservoir is strongly biased towards subtype B HIV-1 strains, with only limited information available from patients infected with non-B HIV-1 subtypes, which are the predominant viruses seen in low- and middle-income countries (LMIC) in Africa and Asia. Results In this study, blood samples were obtained from well-suppressed ART-experienced HIV-1 patients monitored in Uganda (n = 62) or the U.S. (n = 50), with plasma HIV-1 loads < 50 copies/ml and CD4+ T-cell counts > 300 cells/ml. The peripheral HIV-1 reservoir, i.e., cell-associated HIV-1 RNA and proviral DNA, was characterized using our novel deep sequencing-based EDITS assay. Ugandan patients were slightly younger (median age 43 vs 49 years) and had slightly lower CD4+ counts (508 vs 772 cells/ml) than U.S. individuals. All Ugandan patients were infected with non-B HIV-1 subtypes (31% A1, 64% D, or 5% C), while all U.S. individuals were infected with subtype B viruses. Unexpectedly, we observed a significantly larger peripheral inducible HIV-1 reservoir in U.S. patients compared to Ugandan individuals (48 vs. 11 cell equivalents/million cells, p < 0.0001). This divergence in reservoir size was verified measuring proviral DNA (206 vs. 88 cell equivalents/million cells, p < 0.0001). However, the peripheral HIV-1 reservoir was more diverse in Ugandan than in U.S. individuals (8.6 vs. 4.7 p-distance, p < 0.0001). Conclusions The smaller, but more diverse, peripheral HIV-1 reservoir in Ugandan patients might be associated with viral (e.g., non-B subtype with higher cytopathicity) and/or host (e.g., higher incidence of co-infections or co-morbidities leading to less clonal expansion) factors. This highlights the need to understand reservoir dynamics in diverse populations as part of ongoing efforts to find a functional cure for HIV-1 infection in LMICs.

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