Neutralizing antibodies against West Nile virus identified directly from human B cells by single-cell analysis and next generation sequencing

Abstract

West Nile virus infection (WNV) is an emerging mosquito-borne disease that can lead to severe neurological illness and currently has no available treatment or vaccine. Using microengraving, an integrated single-cell analysis method, we analyzed a cohort of subjects infected with WNV - recently infected and post-convalescent subjects - and efficiently identified four novel WNV neutralizing antibodies. We also assessed the humoral response to WNV on a single-cell and repertoire level by integrating next generation sequencing (NGS) into our analysis. The results from single-cell analysis indicate persistence of WNV-specific memory B cells and antibody-secreting cells in post-convalescent subjects. These cells exhibited class-switched antibody isotypes. Furthermore, the results suggest that the antibody response itself does not predict the clinical severity of the disease (asymptomatic or symptomatic). Using the nucleotide coding sequences for WNV-specific antibodies derived from single cells, we revealed the ontogeny of expanded WNV-specific clones in the repertoires of recently infected subjects through NGS and bioinformatic analysis. This analysis also indicated that the humoral response to WNV did not depend on an anamnestic response, due to an unlikely previous exposure to the virus. The innovative and integrative approach presented here to analyze the evolution of neutralizing antibodies from natural infection on a single-cell and repertoire level can also be applied to vaccine studies, and could potentially aid the development of therapeutic antibodies and our basic understanding of other infectious diseases.

Hierarchical Clustering Can Identify B Cell Clones with High Confidence in Ig Repertoire Sequencing Data

“…Reconstruction of the B cell clonal lineages using methods such as maximum parsimony or likelihood (24) allows tracing somatic mutations through the Ig sequences and helps in understanding the evolution of neutralizing antibodies (17, 18). Lineage relationships have also been used to gain insight into the mechanisms underlying isotype switching (25, 26) and to show that B cell clones in the central nervous system in subjects with multiple sclerosis are first activated in the periphery (15).…”

“…However, these algorithms have run times that scale exponentially, which is computationally intractable for large sequencing datasets (29). In practice, most studies cluster sequences based on junction sequence similarity (13, 15, 18, 21, 30–32). …”

“…, identical V and J gene segments, and junction length), and then apply hierarchical clustering on the junction sequence of these smaller groups (8, 15, 18, 21, 30–34). Several distance metrics have been proposed, including Hamming distance, which is simply the absolute count of differences between two amino acid (31, 33) or nucleotide (21, 32) sequences, normalized edit distance (30), and a metric that incorporates hot/cold-spot biases in SHM targeting (15, 18). In addition to metrics defining distance between two sequences, linkage methods define how distance is calculated between groups of sequences.…”

A Model of Somatic Hypermutation Targeting in Mice Based on High-Throughput Ig Sequencing Data

“…250ng of RNA input was used for each sample. Immune sequencing library construction for human samples was performed as previously described using human VH and VL primer mix in each reaction (19–21), and for mouse samples was performed using mouse VL-λ and VL-κ primer mix (21). Briefly, RNA was reverse transcribed using biotin-dT oligonucleotide, and barcoded on the 5′ end in a template switch-like reaction using an oligonucleotide harboring a semi-degenerate 17 nucleotide long Unique Identifier barcode (UID) and a universal flanking sequence.…”

“…The cDNA was purified using streptavidin bead pull down and then subjected to a first round of 12 cycles of PCR. The human samples used a human constant region primer mix composed of IGHC, IGKC and IGLC flanked by another universal sequence (19–21), and the mouse samples used a mouse primer mixed composed of IGKC and IGLC (21). A second round of 12 cycles of PCR was then performed using universal overhang from both ends to add the required Illumina sequencing and clustering adapters.…”