Viral receptor-binding site antibodies with diverse germline origins

Abstract

Vaccines for rapidly evolving pathogens will confer lasting immunity if they elicit antibodies recognizing conserved epitopes, such as a receptor-binding site (RBS). From characteristics of an influenza-virus RBS-directed antibody, we devised a signature motif to search for similar antibodies. We identified, from three vaccinees, over 100 candidates encoded by 11 different VH genes. Crystal structures show that antibodies in this class engage the hemagglutinin RBS and mimic binding of the receptor, sialic acid, by supplying a critical dipeptide on their projecting, heavy-chain third complementarity determining region. They share contacts with conserved, receptor-binding residues but contact different residues on the RBS periphery, limiting the likelihood of viral escape when several such antibodies are present. These data show that related modes of RBS recognition can arise from different germline origins and mature through diverse affinity maturation pathways. Immunogens focused on an RBS-directed response will thus have a broad range of B cell targets.

Figure 1. Properties of CH65-like antibodies

(A) Representative CDR H3s from antibodies identified from a lineage and CDR H3s of orphan antibodies. The donor and V_H gene usage are noted. The critical dipeptide that mimics sialic acid in the CDR H3 is boxed (or underlined) in red. Lineage 652 and antibody H2548 are separated from the rest of the list because of the length and positioning of the critical dipeptide of its CDR H3. (B) Lineage and (C) orphan antibody V_H gene summary. (D) Lineage and (E) orphan antibody V_L gene summary. (F) Critical residues present in CH65-like antibodies. The single amino acid codes and number of antibodies having the critical dipeptide are in parentheses. (G) Distribution of CDR H3 lengths from all antibodies. See also Figure S3 and Tables S2–4.

Figure 2. Comparison of receptor binding site antibodies

(A) 641 I-9 (B) H2526 (C) CH67 (PDB 4HKX) and (D) 5J8 (PDB 4M5Z) in complex with H1 Solomon Islands/03/2006 (for A-C) or H1 California/07/2009 HA (for D) (colored silver). The CDR H3 for each antibody is colored magenta; their V_H and V_L domains are colored blue and green, respectively. See also Table S3 for a list of antibody residues that contact HA.

Figure 3. Comparison of receptor binding site antibodies and receptor mimicry

(A) Schematic of LSTc (modified from PDB 3UBE) docked into the RBS of H1 Solomon Islands/03/2006, highlighting the interaction between the carboxylate and acetamido groups with HA. (B) 641 I-9 Fab (C) H2526 (D) CH67 and (E) 5J8 in complex with HA (colored silver). The CDR H3 for each antibody is colored magenta with the critical dipeptide and flanking residues colored yellow and in stick representation. Antibody and HA numbering follow their respective deposited PDB structures.

Figure 4. Contact residues of CH65-like antibodies and resistance to RBS-targeting antibodies

Antibody footprints of (A) CH65 (green), (B) H2526 (magenta), (C) CH67 (yellow) and (D) 641 I-9 (cyan) highlighting overlapping contacts with sialic acid and conserved residues (dark blue). (E) Conserved and sialic acid contacts (dark blue) with resistance mutation G189D (red) highlighted. (F) Neutralization and binding data of H1 Solomon Islands/03/2006 wildtype or a virus with resistance mutation G189D. Neutralization IC₅₀ values are expressed in µg/ml. See also Table S5 for antibody-antigen contacts.

Figure 5. Serum blocking from TIV donors

Serum pre-immunization (gray) and post-immunization (black) from TIV donors described in this study were assayed for competition with CH65 antibody from lineage 860; fold change from pre- to post-vaccination is noted in parentheses. Red asterisk denotes TIV donors for whom we identified CH65-like antibodies.