Multiplexed Antibody Sequencing and Profiling of the Human Hemagglutinin-specific Memory B Cell Response following Influenza Vaccination

Abstract

Influenza virus is a highly contagious respiratory pathogen causing between 9.4 and 41 million infections per year in the United States in the last decade. Annual vaccination is recommended by the World Health Organization, with the goal to reduce influenza severity and transmission. Ag-specific single B cell sequencing methodologies have opened up new avenues into the dissection of the Ab response to influenza virus. The improvement of these methodologies is pivotal to reduce the associated costs and optimize the operational workflow and throughput, especially in the context of multiple samples. In this study, PBMCs and serum samples were collected longitudinally from eight influenza vaccinees either vaccinated yearly for four consecutive influenza seasons or once for one season. Following the serological and B cell profiling of their polyclonal Ab response to a panel of historical, recent, and next-generation influenza vaccine hemagglutinin (HA) and virus strains, a single multiplexed Ag-specific single B cell sequencing run allowed to capture HA-specific memory B cells that were analyzed for preferential Ig H chain/L chain pairing, isotype/subclass usage, and the presence of public BCR clonotypes across participants. Binding and functional profiles of representative private and public clonotypes confirmed their HA specificity, and their overall binding and functional activity were consistent with those observed at the polyclonal level. Collectively, this high-resolution and multiplexed Ab repertoire analysis demonstrated the validity of this optimized methodology in capturing Ag-specific BCR clonotypes, even in the context of a rare B cell population, such as in the case of the peripheral Ag-specific memory B cells.

Figure 1.. Schematic representation of the study.

Following consecutive (repeaters) or non-consecutive (non-repeaters) administration of seasonal inactivated influenza vaccine (IIV), serum and PBMCs were collected from human subjects at day 0 (D0) and day 21 (D21). PBMCs were also collected at day 7 (D7). ELISA and HAI assays were performed with collected sera using a panel of historical and recent recombinant HA protein strains and influenza viruses, respectively, to assess the breadth of recognition and HAI of participants at the polyclonal level. D0 and D21 PBMC samples were in vitro stimulated (D0_st and D21_st) and used to assess the magnitude and frequency of IgA, IgG1 and IgG3 ASCs. D21 samples were then processed through FACS to sort HA-specific B_mem cells and single cell (sc) sequence their antibody repertoire by NGS. Following BCR repertoire analysis, antibodies of interest were cloned, recombinantly expresses and characterized for their binding and functional profile through in vitro and in vivo studies.

Figure 2.. Heatmaps of binding and functional activity of polyclonal sera collected from study participants (repeaters and non-repeaters) at baseline (D0) and 21 days (D21) following administration of IIV.

(A) Breadth of binding was determined by ELISA against a panel of rHA representing historical and recent influenza virus vaccine strains as well as the chimeric cH6/1 and cH7/3 and the H1N1 and H3N2 COBRA HAs. Influenza vaccine strains contained in the administered IIV are underlined (the H1N1 component for all the considered vaccine seasons was A/California/07/2009 [CA/09] while the H3N2 component for the 2013–2014, 2014–2015, 2015–2016, 2016–2017 was A/Victoria/361/2011 [Vic/11], A/Texas/50/2012 (TX/12), A/Switzerland/9715293/2013 [Switz/13] and A/Hong Kong/4801/2014 [HK/14], respectively). Binding is expressed as the heatmap of the AUC of the serum dilutions for each participant ID. (B) Breadth of functional activity was determined by HAI assay against a panel of influenza viruses representing historical and recent influenza vaccine strains. HAI is expressed as the heatmap of the Log2 of the reciprocal dilution of the last serum dilution point that inhibited the hemagglutination. Vertical line in the HAI color legend and corresponding to the 5.32 Log₂ value corresponds to the 1:40 seroprotective threshold value. ELISA and HAI were performed in duplicate for two independent experiments. Results represent one experiment performed in duplicate.

Figure 3.. Differences in IG class/subclass, IGHV and IGKV/IGLV gene usage in participants.

(A) Frequency of H1-, H3 and H1-H3-specific IGH class/subclass of participants. (B) Frequency of H1-, H3 and H1-H3-specific IGL isotypes of participants. (C) Heatmap showing IGHV usage of H1-, H3- and H1-H3-specific clonotypes. Usage of a given IGHV gene is expressed as a frequency of IGHV gene usage by each participant (ID). *p<0.05. (D) Heatmap showing IGKV/IGLV usage of H1-, H3- and H1-H3-specific clonotypes. Usage of a given IGKV/IGLV gene is expressed as a frequency of IGKV/IGLV gene usage by each participant (ID).

Figure 4.. Heatmaps of binding and functional activity of unique and public clonotype mAbs.

(A-B) Breadth of binding was determined by ELISA against a panel of recombinant HA representing historical and recent influenza vaccine strains as well as the chimeric cH6/1 and cH7/3, the H1N1 and H3N2 COBRA HAs, the HA monomers and HA1 region and HAs carrying the Y98F mutation (HAΔSA). Binding activity of mAbs is expressed as AUC obtained from the corresponding binding curves using different mAb dilutions. (C-D) Breadth of functional activity was determined by HAI assay against a panel of influenza viruses representing historical and recent influenza vaccine strains. HAI is expressed as the minimal tested concentration (endpoint concentration) that inhibited the hemagglutination. ELISA and HAI were performed in duplicate for two independent experiments. Results represent one experiment performed in duplicate.

Figure 5.. Neutralization activity of H1N1- and H3N2-specific mAbs.

Neutralizing activity was tested against representative H1N1 seasonal (Brisb/07), pandemic (CA/09) and pandemic-like (Brisb/18) viral strains (A-F) or H3N2 seasonal strains (TX/12 and HK/14) (G-N). Results represent two independent experiments performed in duplicate.

Figure 6.. Synopsis of competition for binding of human mAbs.

Competitive binding assays were performed using biolayer interferometry (BLI) and pairs of (A) H1N1- and (B) H3N2-specific human mAbs. Competition values are expressed as a percentage of the inhibition of binding of the probe mAb in presence of the competitor mAb. The equilibrium dissociation constant (K_D) between each antibody and the corresponding antigen is reported below the synopses. For the competition assays and the K_D measurement of the H1N1 and H3N2 HA-specific mAbs, the rHA CA/09 and TX/12 were used as antigens, respectively. Results represent two independent experiments performed in duplicate.

Figure 7.. In vivo prophylactic and therapeutic influenza virus challenge studies using passively administered mAbs.

Survival rate of H1N1 or H3N2 influenza virus challenged mice (n=7/group) prophylactically or therapeutically administered with mAbs (A-C). The D1.H1.H3.1 mAb was used for the studies involving the H1N1 challenge while the D1.H3.3.1, D1.H1.H3.2 and D4.H1.H3 mAbs were used for the studies involving the H3N2 challenge. For the H1N1 challenge studies, the pandemic CA/09 influenza virus strain was used while for the H3N2 challenge studies, the mouse-adapted Sw/13M influenza virus strain was used. Weight loss curves of the corresponding influenza virus challenged mice (n=7/group) prophylactically or therapeutically administered with mAbs (D-F). Clinical sign scores of influenza virus challenged mice (n=7/group) prophylactically or therapeutically administered with mAbs (G-I). Lung viral titers as determined by plaque assay of influenza challenged mice (n=3/group) prophylactically or therapeutically administered with mAbs (J-L). Results of lung viral titers represent one independent experiment performed in triplicate. *p<0.05.