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

Natalie N Kinloch,R Brad Jones,Christopher Cannon,Szu-Han Huang ,Winnie Dong,Winiffer D Conce Alberto,Aniqa Shahid,Yanqin Ren,Talia M Mota,Pragya Khadka,Don Kirkby,Erika Benko,Harris Goldstein,Rebecca M Lynch,Andrew Wilson,William Hardy ,Mario Ostrowski ,Lynsay Maclaren,Avery Wimpelberg,Chanson J Brumme,Guinevere Q Lee,Marianne Harris,Colin Kovacs,Perla M Del Río Estrada ,Zabrina L Brumme

doi:10.1038/s41467-020-20442-3

Abstract

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.

Highlights

The Intact Proviral DNA Assay (IPDA) was developed to address the critical need for a scalable method for intact HIV-1 reservoir quantification
We describe an IPDA failure rate of 28% in our cohort, where we used Quantitative Viral Outgrowth Assay (QVOA) to confirm that intact proviruses were present in these cases, and where we present the underlying sequence polymorphisms for each case of failure
We present an independent assessment of the scope of the challenge of addressing HIV-1 diversity in the IPDA, and— through sequence characterization—contribute to understanding the catalogue of polymorphisms that will need to be addressed with secondary primers and probes, towards improved iterations of this valuable assay

Summary

Introduction

The Intact Proviral DNA Assay (IPDA) was developed to address the critical need for a precise and scalable method to quantify intact HIV-1 proviruses, which represent the main barrier to achieving HIV-1 remission or cure[1] This duplexed droplet digital PCR (ddPCR) assay simultaneously targets two HIV-1 regions, the Packaging Signal (Ψ) near the 5’ end of the viral genome and the Rev Responsive Element (RRE) within Envelope (env), to distinguish genomically intact proviruses against a large background of defective ones. While the IPDA is undeniably a major methodological advance, further study is needed to delineate the impact of HIV-1 sequence diversity This is relevant to clinical trials, which would either require an accurate anticipated rate of failure for power calculations, or strategies to address diversity directly, which might include preenrollment screening to determine IPDA-detectability of an individual’s virus and/or development of secondary primers and probes. We present an independent assessment of the scope of the challenge of addressing HIV-1 diversity in the IPDA, and— through sequence characterization—contribute to understanding the catalogue of polymorphisms that will need to be addressed with secondary primers and probes, towards improved iterations of this valuable assay

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