B cell imprinting in children impairs antibodies to the hemagglutinin stalk

Immune imprinting or Original Antigenic Sin (OAS) is a phenomenon where the immune system preferentially recalls its initial response to a related, often evolving pathogen upon subsequent exposure. Despite its important implications for vaccine development, the causes of imprinting remain unclear. To understand the basis and impact of imprinting by influenza A viruses, we characterized the B cell responses of young children following consecutive first infections with divergent H1N1 and H3N2 strains. Children had a primary but otherwise similar B cell response to that of adults. Adult B cells commonly cross-reacted with past strains using more stereotyped and mutated immunoglobulin genes, indicating significant homosubtypic imprinting. In children, following consecutive heterosubtypic primary infections, up to 6% of memory B cells are H1/H3 cross-reactive and bind the highly conserved central stalk epitope, a lead target for broadly protective vaccine candidates. Over 90% of these B cells had higher affinity for the imprinting H3N2 strain, resulting in reduced breadth and neutralization potency against H1N1 strains. Mechanistically, the imprinting H3 and affected H1 strains shared a single residue change in the stalk epitope (D46N) that was central to the nearly universal shift in reactivity, despite differing by only a single atomic group. In conclusion, imprinting by influenza viruses can cause a deleterious shift of nearly the entire memory recall response against key, conserved epitopes. Overall design: Blood samples were collected from 2- to 6-year-old children following sequential first-time exposures to H3N2 then H1N1 influenza viruses or the reverse order. Blood from children only infected by H1N1 viruses and H1N1-infected adults were included as controls. Acute (d1) and convalescent (d30) HA-specific B cells were bait-sorted with oligonucleotide-barcoded probes together with bulk-sorted total CD19+ cells for 10X Genomics single-cell RNA sequencing, following a lab-adapted protocol similar to LIBRA-seq. The 5' transcriptome, B cell receptor immunoglobulin VDJ gene (BCR) repertoire, and surface feature barcode profiles were obtained and analyzed.