Westfall, D. H. | Search bioRxiv

bioRxiv2024

Rare twin cysteine residues in the HIV-1 envelope variable region 1 link to neutralization escape and breadth development

The identification of HIV-1 Envelope glycoprotein (Env) traits associated with development of neutralization cross-reactivity in natural infection is critical for vaccine design. Here we describe the presence of additional Cysteine (Cys) residues in V1 that are enriched among people with elite neutralization breadth. Using >65,000 V1 sequences from the CATNAP database, the AMP trials and three large longitudinal HIV infection cohorts, the SHCS, ZPHI and CAPRISA studies, we show that Env variants with extra V1 Cys are present at low levels throughout infection and fluctuate in frequency over time within participants. We demonstrate an independent association of extra V1 Cys with elite plasma neutralization, and a strong preference for two versus one extra Cys, suggesting certain Envs introduce an additional disulfide bond for stabilization. We observed high levels of neutralization resistance among Envs from 34 bNAb donors, of which 17.6% had elongated V1 regions with extra Cys. We show that extra V1 Cys moderately increase neutralization resistance in an Env from a V2- Apex bNAb-inducer. Modulation of the accessibility of bNAb epitopes on this Env by extra V1 Cys enhanced epitope shielding of several regions, but increased V2 exposure. This suggests that escape from autologous neutralizing activity drove insertion of the extra V1 Cys, creating a modified antigen that may have favored V2 bNAb induction in this donor. Overall, we identify a rare motif of twin Cys in V1 that confers increased neutralization resistance and Env stabilization, is associated with bNAb induction, and may hold potential for incorporation into future HIV bNAb immunogens.

immunology↗

bioRxiv2023

Optimized SMRT-UMI protocol produces highly accurate sequence datasets from diverse populations - application to HIV-1 quasispecies

Pathogen diversity resulting in quasispecies can enable persistence and adaptation to host defenses and therapies. However, accurate quasispecies characterization can be impeded by errors introduced during sample handling and sequencing which can require extensive optimizations to overcome. We present complete laboratory and bioinformatics workflows to overcome many of these hurdles. The Pacific Biosciences single molecule real-time platform was used to sequence PCR amplicons derived from cDNA templates tagged with universal molecular identifiers (SMRT-UMI). Optimized laboratory protocols were developed through extensive testing of different sample preparation conditions to minimize between-template recombination during PCR and the use of UMI allowed accurate template quantitation as well as removal of point mutations introduced during PCR and sequencing to produce a highly accurate consensus sequence from each template. Handling of the large datasets produced from SMRT-UMI sequencing was facilitated by a novel bioinformatic pipeline, Probabilistic Offspring Resolver for Primer IDs (PORPIDpipeline), that automatically filters and parses reads by sample, identifies and discards reads with UMIs likely created from PCR and sequencing errors, generates consensus sequences, checks for contamination within the dataset, and removes any sequence with evidence of PCR recombination or early cycle PCR errors, resulting in highly accurate sequence datasets. The optimized SMRT-UMI sequencing method presented here represents a highly adaptable and established starting point for accurate sequencing of diverse pathogens. These methods are illustrated through characterization of human immunodeficiency virus (HIV) quasispecies. Author SummaryThere is a great need to understand the genetic diversity of pathogens in an accurate and timely manner, but many errors can be introduced during the sample handling and sequencing steps which may prevent accurate analyses. In some cases, the errors introduced during these steps can be indistinguishable from real genetic variation and prevent analyses from identifying true sequence variation present in the pathogen population. There are established methods which can help to prevent these types of errors, but can involve many different steps and variables, all of which must be optimized and tested together to ensure the desired effect. Here we show results from testing different methods on a set of HIV+ blood plasma samples and arrive at a streamlined laboratory protocol and bioinformatic pipeline which prevents or corrects for different types of errors that can arise in sequence datasets. These methods should be an accessible starting point for anyone wanting accurate sequencing without extensive optimizations.

molecular biology↗