Whole Genome Deep Sequencing of HIV-1 Reveals the Impact of Early Minor Variants Upon Immune Recognition During Acute Infection

Abstract

Deep sequencing technologies have the potential to transform the study of highly variable viral pathogens by providing a rapid and cost-effective approach to sensitively characterize rapidly evolving viral quasispecies. Here, we report on a high-throughput whole HIV-1 genome deep sequencing platform that combines 454 pyrosequencing with novel assembly and variant detection algorithms. In one subject we combined these genetic data with detailed immunological analyses to comprehensively evaluate viral evolution and immune escape during the acute phase of HIV-1 infection. The majority of early, low frequency mutations represented viral adaptation to host CD8+ T cell responses, evidence of strong immune selection pressure occurring during the early decline from peak viremia. CD8+ T cell responses capable of recognizing these low frequency escape variants coincided with the selection and evolution of more effective secondary HLA-anchor escape mutations. Frequent, and in some cases rapid, reversion of transmitted mutations was also observed across the viral genome. When located within restricted CD8 epitopes these low frequency reverting mutations were sufficient to prime de novo responses to these epitopes, again illustrating the capacity of the immune response to recognize and respond to low frequency variants. More importantly, rapid viral escape from the most immunodominant CD8+ T cell responses coincided with plateauing of the initial viral load decline in this subject, suggestive of a potential link between maintenance of effective, dominant CD8 responses and the degree of early viremia reduction. We conclude that the early control of HIV-1 replication by immunodominant CD8+ T cell responses may be substantially influenced by rapid, low frequency viral adaptations not detected by conventional sequencing approaches, which warrants further investigation. These data support the critical need for vaccine-induced CD8+ T cell responses to target more highly constrained regions of the virus in order to ensure the maintenance of immunodominant CD8 responses and the sustained decline of early viremia.

Next-generation sequencing shows West Nile virus quasispecies diversification after a single passage in a carrion crow (Corvus corone) in vivo infection model

“…Subsampled reads were realigned against the consensus genome with gsMapper and SNVs were then called from the BAM alignment file with LoFreq version 0.5 (Wilm et al, 2012) with the recommended settings for RNA viruses (holmbonf strand bias filter and nonincorporation of mapping qualities). LoFreq was found to perform better in distinguishing errors from true viral variants on the plasmid control samples when compared with the variants called by gsMapper and those called by RC454 (Henn et al, 2012) and V-Phaser 2 (Yang et al, 2013) (Information S1). The cut-off used to exclude errors caused by PCR amplification was set to 1.81 % as this percentage corresponded to the most abundant variant obtained with the PCRamplified plasmid.…”

Dearth of full-length HIV-1 sequences obscures the true HIV-1 genetic subtypes distribution in sub-Saharan Africa

“…Analysis of full-length sequences could also help in the accurate identification of low frequency viral variants (Henn et al, 2012) and the use of multiple genes rather than single gene to identify HIV-1 subtypes can reduce the chances of false identification (Neogi et al, 2012). The fact that the full-length subtypes E and I isolates were never found, and have now been re-designated as circulating recombinant forms, CRF01_AE and CRF04_cpx respectively (Carr et al, 1996;Gao et al, 1998;Paraskevis et al, 2001), and the suggestion that the subtype G, was actually a recombinant, whose parental subtype included the CRF02_AG (Abecasis et al, 2007), justify the calls for subtype classification to be based only on analysis of full-length or near full-length genomes.…”

Computational Inference Methods for Selective Sweeps Arising in Acute HIV Infection

“…The onset of CTL response is temporally correlated with the end of the expansion period, suggesting a role for CTLs in controlling viral load. Numerous studies have shown that during acute infection, specific HIV mutations on CTL targeted epitopes sweep to fixation, providing more direct evidence that CTL response shapes the infecting HIV population; see Goulder and Watkins (2004) for a review.In recent years, full-genome sequencing studies have provided an increasingly detailed description of CTL response during acute infection, e.g., Fernandez et al Recent deep-sequencing data sets have provided a picture of HIV escape at the CTL targeted epitopes, e.g., Fisher et al (2010), Henn et al (2012), Bimber et al (2009). HIV escape mutations at the first CTL targeted epitope rise to significant proportions 1-3 weeks after peak viral load.…”

“…In recent years, full-genome sequencing studies have provided an increasingly detailed description of CTL response during acute infection, e.g., Fernandez et al Recent deep-sequencing data sets have provided a picture of HIV escape at the CTL targeted epitopes, e.g., Fisher et al (2010), Henn et al (2012), Bimber et al (2009). HIV escape mutations at the first CTL targeted epitope rise to significant proportions 1-3 weeks after peak viral load.…”

“…In recent years, full-genome sequencing studies have provided an increasingly detailed description of CTL response during acute infection, e.g., Fernandez et al (2005), Goonetilleke et al (2009), Liu et al (2006), Henn et al (2012), Bimber et al (2009), and see Boutwell et al (2010) for a review. A general picture in which initial CTL response targeting a single epitope occurs several days before peak viral load has emerged.…”