Sera Antibody Repertoire Analyses Reveal Mechanisms of Broad and Pandemic Strain Neutralizing Responses after Human Norovirus Vaccination

Figure 1. The Anti-NoV GII.4c IgG Serum Antibody Repertoire following Vaccination Is Polarized and Shaped by Previous HuNoV Infection (A) Donors (n = 3) were immunized with experimental HuNoV vaccine and serum and PBMCs were collected at the times indicated. (B) Relative abundance of GII.4c-specific IgG clonotypes pre- and post-vaccination. Each bar represents a CDR-H3 antibody clonotype observed at ≿0.5% of the GII.4c antigen-specific antibody repertoire. (C) Amount of anti-GII.4c serum antibodies (i.e., the product of anti-GII.4c IgG ELISA titer × relative antibody abundance as determined by LS-MS/MS; see STAR Methods) pre- and post-vaccination; ELISA titer shown over each bar. (D) Number of detected antibody clonotypes before and after vaccination. (E) Anti-GII.4c antibody V-gene somatic hypermutation rates for pre-existing and emergent antibodies; n.s. represents not statistically significant (two-tailed t test; p > 0.3). (F) V-gene family usage in the serological repertoire. Related to Table S1.

Figure 2. HuNoV Recombinant Serum Antibodies Recognize Conserved Binding, Ligand-Blocking, and GII.4 Neutralizing Epitopes Sigmoidal dose response curves were fit to the mean percent control binding for determination of (A) half-maximum binding (EC50) or (B) half-maximum blocking of HBGA binding (half-maximum inhibitory concentration, IC50) of pre-existing (P) and emergent (E) antibodies. Titers >2 μg/mL were scored as negative (white box). The HBGA blocking assay was used to characterize A1431 against a panel of pandemic norovirus GII.4 strains. Antibodies were tested in duplicate in a minimum of two independent experiments. (C) Sigmoidal dose response curves were fit to the mean percent viral genomic copies control for determination of IC50 in a neutralization assay. The neutralization assay was used to characterize A1431 and A1227 against the GII.P16-GII 4 Sydney strain. Error bars represent mean ± SEM from four independent experiments with 2 technical replicates for each dilution. Related to Figure S1.

Figure 3. Serum Antibodies that Block Historical GII.4 Strains Recognize Overlapping Epitopes within Epitope A (A) Homology model based on PDB: 3SLD (Shanker et al., 2011) of the GII.4 2006a P domain dimer of the HuNoV major capsid protein, VP1 with known P domain blockade antibody epitopes A (blue), D (dark gray), E (purple), and F (orange) highlighted. (B) Amino acid sequences of pandemic strains and select mutants of GII.4 1987. (C) Blockade potency binned by IC50. (D) Select mutants of GII.4 2006a.581 VP1. Highlighted areas indicate sequences important for antibody binding. (E) Antibody binding binned by EC50 values. Antibodies were tested in duplicate in a minimum of two independent experiments.

Figure 4. The Broadly Binding Non-Blockade Antibody A1227 Binds to a Highly Conserved Epitope within the P Domain (A) Crystal structure of A1227 Fab bound to GII.4.2002 P domain dimer shown in cartoon representation, with P domain monomers colored pink and orange and A1227 heavy and light chains in gray and blue, respectively. (B) The A1227 antibody binds near the P1 dimeric interface (P domain dimer in surface representation and A1227 Fab in cartoon). (C) A1227 Fab footprint is outlined in yellow on the P domain surface, mapped by GI-GII sequence conservation, with degree of conservation colored from variable (white) to conserved (purple). (D) Detailed view of A1227 Fab-P domain binding interface, with interacting residues depicted in stick and hydrogen bonds shown in yellow dotted lines. (E) Modelling of the GII.4.2002 P domain-A1227 Fab crystal structure onto the GI.1 capsid structure (PDB: 1IHM) reveals an antibody binding site that is occluded on the assembled VLP. (F) Close-up view from (E) showing the occlusion of A1227 Fab within the GI.1 capsid. (G) Quantification of volume overlap between A1227 Fab (or VH/VL) and GI.1 capsid proteins. Over 60% of the volume of A1227 Fab overlaps…

Figure 5. Broad GII.4-Blockade Antibody A1431 Recognizes an Accessible Semi-Conserved Site of Vulnerability (A) Overall structure of A1431 Fab bound to GII.4.2002 P domain dimer is shown in ribbon representation, with P domain monomers in pink and orange and A1431 heavy and light chains in magenta and cyan, respectively. (B) Mapped blockade epitopes are highlighted on a surface representation of GII.4.2002 P domain dimer (colored as in Figure 3A) with the A1431 Fab shown in cartoon. The crystal structure of GII.4 (Farmington Hills, 2004) P domain complexed to HBGA type B ligand (PDB: 4X05) was superimposed onto the GII.4.2002 P domain + A1431 complex to show location of HBGA binding site. (C) Details of A1431 Fab interactions with P domain residues are highlighted with interacting residues depicted in stick and hydrogen bonds shown in yellow dotted lines. (D) A1431 Fab footprint is outlined in yellow on the P domain dimer surface, mapped by GII.4 sequence conservation, with degree of conservation colored from variable (white) to conserved (purple). (E) The GII.4.2002 P domain-A1431 Fab crystal structure is modeled onto the GI.1 capsid structure (1IHM; Prasad et al., 1999), with a…