June 1, 2021  |  

Next generation sequencing of full-length HIV-1 env during primary infection.

Background: The use of next generation sequencing (NGS) to examine circulating HIV env variants has been limited due to env’s length (2.6 kb), extensive indel polymorphism, GC deficiency, and long homopolymeric regions. We developed and standardized protocols for isolation, RT-PCR amplification, single molecule real-time (SMRT) sequencing, and haplotype analysis of circulating HIV-1 env variants to evaluate viral diversity in primary infection. Methodology: HIV RNA was extracted from 7 blood plasma samples (1 mL) collected from 5 subjects (one individual sampled and sequenced at 3 time points) in the San Diego Primary Infection Cohort between 3-33 months from their estimated date of infection (EDI). Median viral load per sample was 50,118 HIV RNA copies/mL (range: 22,387-446,683). Full-length (3.2 kb) env amplicons were constructed into SMRTbell templates without shearing, and sequenced on the PacBio RS II using P4/C2 chemistry and 180 minute movie collection without stage start. To examine viral diversity in each sample, we determined haplotypes by clustering circular consensus sequences (CCS), and reconstructing a cluster consensus sequence using a partial order alignment approach. We measured sample diversity both as the mean pairwise distance among reads, and the fraction of reads containing indel polymorphisms. Results: We collected a median of 8,775 CCS reads per SMRT Cell (range: 4243-12234). A median of 7 haplotypes per subject (range: 1-55) were inferred at baseline. For the one subject with longitudinal samples analyzed, we observed an increasing number of distinct haplotypes (8 to 55 haplotypes over the course of 30 months), and an increasing mean pairwise distance among reads (from 0.8% to 1.6%, Tamura-Nei 93). We also observed significant indel polymorphism, with 16% of reads from one sample later in infection (33 months post-EDI) exhibiting deletions of more than 10% of env with respect to the reference strain, HXB2. Conclusions: This study developed a standardized NGS procedure (PacBio SMRT) to deep sequence full-length HIV RNA env variants from the circulating viral population, achieving good coverage, confirming low env diversity during primary infection that increased over time, and revealing significant indel polymorphism that highlights structural variation as important to env evolution. The long, accurate reads greatly simplified downstream bioinformatics analyses, especially haplotype phasing, increasing our confidence in the results. The sequencing methodology and analysis tools developed here could be successfully applied to any area for which full-length HIV env analysis would be useful.

June 1, 2021  |  

Full-length HIV-1 env deep sequencing in a donor with broadly neutralizing V1/V2 antibodies.

Background: Understanding the co-evolution of HIV populations and broadly neutralizing antibodies (bNAbs) 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 HIV-1 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 loop region. We developed a PacBio 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 and viral escape motif evolution were interpreted in the context of the development V1/V2-targeting broadly neutralizing antibodies. Results: We collected a median of 6799 (range: 1770-14727) high quality full-length HIV env circular consensus sequences (CCS) per SMRT Cell, per time point. Using only CCS reads comprised of 6 or more passes over the HIV env insert (= 16 kb read length) ensured that our median per-base accuracy was 99.7%. A phylogeny inferred with PacBio and 100 cloned env sequences (10 time points) found the cloned sequences evenly distributed among PacBio sequences. Viral escape from the V1/V2 targeted bNAbs was evident at V2 positions 160, 166, 167, 169 and 181 (HxB2 numbering), exhibiting several distinct escape pathways by 40 months post-infection. Conclusions: Our PacBio full-length env sequencing method allowed unprecedented view and ability to characterize HIV-1 env dynamics throughout the first four years of infection. 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 will prove critical for increasing our understanding of how env evolution drives the development of antibody breadth.

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.