Background: The HIV-1 proviral reservoir is incredibly stable, even while undergoing antiretroviral therapy, and is seen as the major barrier to HIV-1 eradication. Identifying and comprehensively characterizing this reservoir will be critical to achieving an HIV cure. Historically, this has been a tedious and labor intensive process, requiring high-replicate single-genome amplification reactions, or overlapping amplicons that are then reconstructed into full-length genomes by algorithmic imputation. Here, we present a deep sequencing and analysis method able to determine the exact identity and relative abundances of near-full-length HIV genomes from samples containing mixtures of genomes without shearing or complex bioinformatic reconstruction. Methods: We generated clonal near-full-length (~9 kb) amplicons derived from single genome amplification (SGA) of primary proviral isolates or PCR of well-documented control strains. These clonal products were mixed at various abundances and sequenced as near-full-length (~9 kb) amplicons without shearing. Each mixture yielded many near-full-length HIV-1 reads. Mathematical analysis techniques resolved the complex mixture of reads into estimates of distinct near-full-length viral genomes with their relative abundances. Results: Single Molecule, Real-Time (SMRT) Sequencing data contained near-full-length (~9 kb) continuous reads for each sample including some runs with greater than 10,000 near-full-length-genome reads in a three-hour sequencing run. Our methods correctly recapitulated exactly the originating genomes at a single-base resolution and their relative abundances in both mixtures of clonal controls and SGAs, and these results were validated using independent sequencing methods. Correct resolution was achieved even when genomes differed only by a single base. Minor abundances of 5% were reliably detected. Conclusions: SMRT Sequencing yields long-read sequencing results from individual DNA molecules, a rapid time-to-result. The single-molecule, full-length nature of this sequencing method allows us to estimate variant subspecies and relative abundances with single-nucleotide resolution. This method allows for reference-agnostic and cost-effective full-genome sequencing of HIV-1, which could both further our understanding of latent infection and develop novel and improved tools for quantifying HIV provirus, which will be critical to cure HIV.
The replication-competent HIV-1 latent reservoir is primarily established near the time of therapy initiation.
Although antiretroviral therapy (ART) is highly effective at suppressing HIV-1 replication, the virus persists as a latent reservoir in resting CD4+ T cells during therapy. This reservoir forms even when ART is initiated early after infection, but the dynamics of its formation are largely unknown. The viral reservoirs of individuals who initiate ART during chronic infection are generally larger and genetically more diverse than those of individuals who initiate therapy during acute infection, consistent with the hypothesis that the reservoir is formed continuously throughout untreated infection. To determine when viruses enter the latent reservoir, we compared sequences of replication-competent viruses from resting peripheral CD4+ T cells from nine HIV-positive women on therapy to viral sequences circulating in blood collected longitudinally before therapy. We found that, on average, 71% of the unique viruses induced from the post-therapy latent reservoir were most genetically similar to viruses replicating just before ART initiation. This proportion is far greater than would be expected if the reservoir formed continuously and was always long lived. We conclude that ART alters the host environment in a way that allows the formation or stabilization of most of the long-lived latent HIV-1 reservoir, which points to new strategies targeted at limiting the formation of the reservoir around the time of therapy initiation.Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
The macaque simian or simian/human immunodeficiency virus (SIV/SHIV) challenge model has been widely used to inform and guide human vaccine trials. Substantial advances have been made recently in the application of repeated-low-dose challenge (RLD) approach to assess SIV/SHIV vaccine efficacies (VE). Some candidate HIV vaccines have shown protective effects in preclinical studies using the macaque SIV/SHIV model but the model’s true predictive value for screening potential HIV vaccine candidates needs to be evaluated further. Here, we review key parameters used in the RLD approach and discuss their relevance for evaluating VE to improve preclinical studies of candidate HIV vaccines.Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.