Background: Understanding the co-evolution of HIV populations and broadly neutralizing antibody (bNAb) lineages may inform vaccine design. Novel long-read, next-generation sequencing methods allow, for the first time, full-length deep sequencing of HIV env populations.
Methods: We longitudinally examined env populations (12 time points) in a subtype A infected individual from the IAVI primary infection cohort (Protocol C) who developed bNAbs (62% ID50>50 on a diverse panel of 105 viruses) targeting the V1/V2 region. We developed a Pacific Biosciences single molecule, real-time sequencing protocol to deeply sequence full-length env from HIV RNA. Bioinformatics tools were developed to align env sequences, infer phylogenies, and interrogate escape dynamics of key residues and glycosylation sites. PacBio env sequences were compared to env sequences generated through amplification and cloning. Env dynamics were interpreted in the context of the development of a V1/V2-targeting bNAb lineage isolated from the donor.
Results: We collected a median of 6799 high quality full-length env sequences per timepoint (median per-base accuracy of 99.7%). A phylogeny inferred with PacBio and 100 cloned env sequences (10 time points) found cloned env sequences evenly distributed among PacBio sequences. Phylogenetic analyses also revealed a potential transient intra-clade superinfection visible as a minority variant (~5%) at 9 months post-infection (MPI), and peaking in prevalence at 12MPI (~64%), just preceding the development of heterologous neutralization. Viral escape from the bNAb lineage was evident at V2 positions 160, 166, 167, 169 and 181 (HxB2 numbering), exhibiting several distinct escape pathways by 40MPI.
Conclusions: Our PacBio full-length env sequencing method allowed unprecedented characterization of env dynamics and revealed an intra-clade superinfection that was not detected through conventional methods. The importance of superinfection in the development of this donor’s V1/V2-directed bNAb lineage is under investigation. Longitudinal full-length env deep sequencing allows accurate phylogenetic inference, provides a detailed picture of escape dynamics in epitope regions, and can identify minority variants, all of which may prove useful for understanding how env evolution can drive the development of antibody breadth.
Maximum likelihood phylogeny of the primary infection clade, inferred from longitudinal full-length deep sequenced HIV env. Terminal branches are colored by sample date (numbers in legend represent months post infection). Inset boxes show successively expanded phylogenetic detail, highlighting the sequencing depth. Very short terminal branches suggest low sequencing error rates.