Immune memory shapes human polyclonal antibody responses to H2N2 vaccination

Summary: Influenza A virus subtype H2N2, which caused the 1957 influenza pandemic, remains a global threat. A recent phase I clinical trial investigating a ferritin nanoparticle displaying H2 hemagglutinin in H2-naïve and H2-exposed adults. Therefore, we could perform comprehensive structural and biochemical characterization of immune memory on the breadth and diversity of the polyclonal serum antibody response elicited after H2 vaccination. We temporally map the epitopes targeted by serum antibodies after first and second vaccinations and show previous H2 exposure results in higher responses to the variable head domain of hemagglutinin while initial responses in H2-naïve participants are dominated by antibodies targeting conserved epitopes. We use cryo-EM and monoclonal B cell isolation to describe the molecular details of cross-reactive antibodies targeting conserved epitopes on the hemagglutinin head including the receptor binding site and a new site of vulnerability deemed the medial junction. Our findings accentuate the impact of pre-existing influenza exposure on serum antibody responses.

Table S1: nsEMPEM map and deposition details.Related to Figures 2 and 4.
Table S3: nsEM map and deposition details for monoclonal immune complexes.Related to Figures 4 and  5.

Figure S1 :
Figure S1: Group 1 and 2 sample 2D classes.Related to Figure 2. Sample 2D classes that make up 3D models shown in Figure 2 for Group 1 (A) and Group 2 (B).Datasets with >7.3k particles are divided into 20 classes while those with <7.3k particles are divided into 9.All 2D classification datasets are shown in order of descending particle count.

Figure S2 :
Figure S2: Group 3 and 4 sample 2D classes.Related to Figure 2. Sample 2D classes that make up 3D models shown in Figure 2 for Group 3 (A) and Group 4 (B).Datasets with >7.3k particles are divided into 20 classes while those with <7.3k particles are divided into 9.All 2D classification datasets are shown in order of descending particle count.

Figure
Figure S3: B-cell sorting strategy and polyclonal stem responses observed by cryo-EM.Related to Figure 4 and 5. Flow cytometry gates used to detect and sort H2 HA head-specific B cells from the CD19+ CD3/14/56 (dump)-CD27 hi CD38 hi CD20 lo CD21 lo plasmablast (A) or CD19+ CD3/14/56 (dump)-CD20+ IgG+ memory B cell compartment (B).Each plot shows the cell population gated immediately to the left as indicated above each plot.H2 HA head-specific B cells were detected as H2 HA ectodomain+ H2 stem-.(C) 2D classes of HA bound to polyclonal antibodies.Classes featuring pFabs with stem specificities outlined in red.(D) 3D reconstruction of stem-specific pFab.

Figure S4 :
Figure S4: Local resolution plots of EM maps.Related to FigureS 5, 6, and 7. Local resolution was calculated according to a 0.143 FSC threshold in cryoSPARC 3.2 and visualized in ChimeraX.