High-throughput sequencing-based neutralization assay reveals how repeated vaccinations impact titers to recent human H1N1 influenza strains

Abstract

<p>The high genetic diversity of influenzaviruses means that traditional serological assays have too low throughput to measure serum antibody neutralization titers against all relevant strains. To overcome this challenge, we developed a sequencing-based neutralization assay that simultaneously measures titers against many viral strains using small serum volumes using a workflowsimilar to traditional neutralization assays. The key innovation is to incorporate unique nucleotide barcodes into the hemagglutinin (HA) genomic segment, and then pool viruses with numerous differentbarcoded HA variants and quantify the infectivity of all of them simultaneously using next-generation sequencing. With this approach, a single researcher performed the equivalent of 2,880 traditional neutralization assays (80 serum samples against 36 viral strains) in approximately 1 month. We applied the sequencing-based assay to quantify the impact of influenzavaccination on neutralization titers against recent human H1N1 strains for individuals who had or had not also received a vaccine in the previous year. We found that the viral strain specificitiesof the neutralizing antibodies elicited by vaccination vary among individuals and that vaccination induced a smaller increase in titers for individuals who had also received a vaccine the previous year-although the titers 6 months after vaccination were similar in individuals with and without the previous-year vaccination. We also identifieda subset of individuals with low titers to a subclade of recent H1N1 even after vaccination. We provide an experimental protocol (dx.doi.org/10.17504/protocols.io.kqdg3xdmpg25/v1) and computational pipeline (https://github.com/jbloomlab/seqneut-pipeline) for the sequencing-based neutralization assays to facilitate the use of this method by others.<br></p>