Ultrasensitive Single-Genome Sequencing to Study HIV Transmission and Evolution

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

BACKGROUND: In this project, we developed a new method for NGS library construction of viral genomes that reduces PCR bias and error, eliminates PCR recombinants from the final datasets, and generates thousands of single-genome HIV sequences of the same quality as standard sequencing but with 100-fold more variants (Boltz et al., Retrovirology 13:87, 2016). This new, ultrasensitive SGS (uSGS) assay has been used not only for detecting linkage of rare alleles, including drug-resistance mutations (Boltz, et al. JCI Insight 2021), but also for in-depth phylogenetic analyses of viral populations. Although it is generally believed that most HIV infections are initiated with a single virion, this conclusion rests on experiments in which the number of viral genomes analyzed was relatively small (about 10-100 HIV genomes). We will utilize our uSGS method to perform a deep-dive study of HIV genetics in acute infection. The underlying question is whether, in some of the individuals that appear to have been infected with a single virus, there were also minority variants that were missed due to shallow sampling. We were able to obtain plasma samples from 15 donors enrolled in the RV254/South East Asia Research Collaboration with Hawaii 010 Trial (101). The trial was conducted by the Thai Red Cross AIDS Research Centre and the Department of Retrovirology, U.S. Army Medical Component, Armed Forces Research Institute of Medical Sciences. The study was designed to identify acute HIV infection in high-risk populations in Thailand and offer immediate initiation of ART. Fifteen samples were collected from participants during acute HIV infection. Ten of the donors were previously reported to have a single founder virus in acute infection by standard SGS, and five donors were shown to have two to five founders using the same approach. In our study, we will investigate the presence of a single founder virus verses transmission of multiple founders in acute HIV infection. In total, we aim to obtain 10,000 HIV genomes from each donor sample, constituting the deepest look into the genetics of acute HIV infection. Results from this aim will inform studies using bNAbs in acute infection to target founder viruses and will contribute to our understanding of HIV transmission, evolution, and population dynamics. In addition to our goals of applying uSGS to studies of transmission and evolution, we will also develop the next generation of uSGS using Oxford Nanopore Technologies (ONT). We have begun these studies by demonstrating that ONT can be used to generate accurate near full-length HIV sequencing (Wright, et al. Cells 2021) and by developing a pipeline to annotate the sequences generated (Wright, et al. Retrovirology 2021). Moving forward, we will improve our current ONT assays and pipeline to capture the HIV integration sites as well as the full-length HIV sequences in samples collected from donors on ART. This advancement will contribute significantly to our understanding of HIV persistence. Dr. Freed recently reported that HIV can develop drug resistance in vitro to multiple classes of antiretroviral drugs by acquiring Env mutations. However, it is not known if these mutations confer multidrug resistance in vivo as they do in vitro. To investigate the possible contribution of Env mutations to drug resistance in vivo, we obtained plasma samples from five donors failing INSTI [raltegravir (RAL)]-containing regimens without mutations in the integrase gene by standard HIV genotyping and with plasma drug levels confirming adherence. Using samples obtained from the ACTG study A5273, we are currently performing uSGS on donor samples from pre-ART, early RAL failure, and during late RAL failure. Sequences obtained thus far were analyzed for known drug resistance mutations in IN and for newly emergent Env mutations. Several Env mutations emerged upon drug failure in the highly conserved gp120/gp41 interface, similar to those confirmed to confer drug resistance in vitro (Hikichi, et al. mBio 2021). These preliminary findings suggest that Env mutations might contribute to HIV drug resistance in vivo. If Env mutations are demonstrated to confer resistance to ART, we will initiate evolutionary studies to determine the origin and selection pressures on the newly identified drug resistance mutations (e.g., if they existed prior to ART and, if so, at what frequencies, if they are found in donors failing other ART regimens, and if they are selected in donors without ART). Understanding the role of Env in ART resistance will have significant clinical impact if mutations are found to confer drug resistance alone or to be a "stepping stone" toward virologic failure. ____ACCOMPLISHMENTS: ___Using our SGS assays, we demonstrated that pre-exposure prophylaxis can fail to prevent HIV transmission when drug-resistance mutations are present in the donor population of viral variants (Spinelli et al., Clin. Infect. Dis. 72:2025-2028, 2021). ___In collaboration with the lab of Dr. Eric Freed (HIV Dynamics and Replication Program), we investigated the role of off-target mutations in the failure of ART to control HIV replication in vivo. We identified several mutations in the gp120-gp41 interface of the HIV env gene that are capable of conferring low- to medium-level resistance to multiple ARVs (Hikichi et al., mBio12:e03134-20, 2021). ___We developed new near full-length HIV single-genome sequencing assay using Oxford Nanopore Technologies and demonstrated it's accuracy to be equivalent to other approaches at a reduce cost and higher throughput. This assay will be used to characterize HIV proviruses that persist on ART (Wright, et al. Cells. 10, 2021). ___We developed a new bioinformatic pipeline to annotate near full-length HIV sequences and determine if they are intact and likely capable of fueling rebound viremia if ART is interrupted (Wright, et al. Retrovirology. 18: 16, 2021.).