Convergent antibody evolution and clonotype expansion following influenza virus vaccination

Abstract

Recent advances in high-throughput single cell sequencing have opened up new avenues into the investigation of B cell receptor (BCR) repertoires. In this study, PBMCs were collected from 17 human participants vaccinated with the split-inactivated influenza virus vaccine during the 2016-2017 influenza season. A combination of Immune Repertoire Capture (IRCTM) technology and IgG sequencing was performed on ~7,800 plasmablast (PB) cells and preferential IgG heavy-light chain pairings were investigated. In some participants, a single expanded clonotype accounted for ~22% of their PB BCR repertoire. Approximately 60% (10/17) of participants experienced convergent evolution, possessing public PBs that were elicited independently in multiple participants. Binding profiles of one private and three public PBs confirmed they were all subtype-specific, cross-reactive hemagglutinin (HA) head-directed antibodies. Collectively, this high-resolution antibody repertoire analysis demonstrated the impact evolution can have on BCRs in response to influenza virus vaccination, which can guide future universal influenza prophylactic approaches.

Fig 1. Serological landscape of D#103, a representative participant in this study.

(A) HAI titer against historical influenza virus strains. (B) Serological IgG antibodies and HAI titer against the 2016–2017 influenza vaccine strains. All samples were run in triplicate. (C) Cell sorting for influenza-specific PBs. (D) Number of antibody secreting cells against the four influenza vaccine strains. (E) Impact of influenza vaccination on the memory B cell compartment.

Fig 2. Chain usage and variable region pairing.

(A) Heavy and (B) light chain constant region subclass usage amongst the IgG PBs. (C) Variable segment usage of participants in this study. (D) Kappa:lambda light chain usage for each heavy chain variable segment. (E-F) Circos plots representing the pairing between heavy and light chain variable regions in the left and right hemisphere respectively. Different colors represent different heavy chain variable segments in (D), while blue represents the most underrepresented (IGHV1/IGLV1) and red represents the most overrepresented (IGHV1/IGLV4) segment pairing in (E).

Fig 3. Clonotype landscape and expanded clonotypes for each participant.

(A) Participants whose three most expanded clonotypes belonged to three different IGHV groups. (B) A phylogenetic tree was included for participants when at least two of the three most expanded clonotypes were of the same IGHV group. This tree only includes those of the top 3 most expanded clonotypes that belong to the same IGHV group. The largest such compartments are shown in orange, the second largest in blue, and the third largest (for participants whose three largest compartments were all the same IGHV group) are shown in brown. On occasion, the most expanded clonotype is not shaded in the pie chart, and is not included on the phylogenetic tree as it represents a different IGHV group than the second and third most expanded clonotypes (e.g. D#089). The number of individual PBs are shown next to the highlighted clades on the phylogenetic trees.

Fig 4. Pairwise distance heatmaps for all PBs in the four convergent groups.

For each panel, the two heat maps on the left show CDR3 peptide identity for the heavy and the light chain, and the two heat maps on the right show variable region nucleotide identity for the heavy and light chain. (A) Pairwise identity matrix for the pubCDR3-1 group. (B) Pairwise identity matrix for the pubCDR3-2 group. (C) Pairwise identity matrix for the pubCDR3-3 group. (D) Pairwise identity matrix for the pubCDR3-4 group.

Fig 5. Clonotype landscape for each participant displaying public BCRs.

Only the 10 participants who had at least one PB belonging to one of the four convergent pubCDR3 groups are shown. The number of individual PBs expressing a public clonotype is shown next to the highlighted clades on the phylogenetic trees.

Fig 6. Expression profile for the four selected mAbs.

(A) Synopsis of H1-, H3-, and IBV-specific human mAbs binding against H1N1, H3N2 and IBV rHA. Binding of mAbs was evaluated against a panel of H1, H3 and IBV rHAs from historical seasonal, pandemic (CA/09) and post-pandemic (Mich/15 and Brisb/18) strains. Binding activity was also tested against COBRA H1N1 P1, X3 and X6 and H3N2 T10 and T11 rHA, the chimeric cH6/1, cH5/3 and cH7/3 rHAs, a truncated HA1 rHA and an rHA monomer. For H1-, H3- and IBV-specific mAbs the HA1 and HA monomer from CA/09, WI/05 and MA/12 were used, respectively. All the mAbs were tested at different 3-fold dilutions, starting from 20 μg/mL. The area under the curve (AUC) was calculated from each dilution curve and reported on the heatmap table. (B) HAI activity of mAbs against the corresponding H1 (CA/09), H3 (HK/14) and IBV (Brisb/08) vaccine strains. All mAbs were tested across a 2-fold serial dilution series, starting from 10 μg/mL. For each mAb, the minimal concentration needed to inhibit hemagglutination is reported in the corresponding tables and expressed in microgram per milliliter. (C) Sequence analysis of heavy and light chain private and public mAbs.