Molecular signatures of hemagglutinin stem-directed heterosubtypic human neutralizing antibodies against influenza A viruses

Recent studies have shown high usage of the IGHV1-69 germline immunoglobulin gene for influenza hemagglutinin stem-directed broadly-neutralizing antibodies (HV1-69-sBnAbs). Here we show that a major structural solution for these HV1-69-sBnAbs is achieved through a critical triad comprising two CDR-H2 loop anchor residues (a hydrophobic residue at position 53 (Ile or Met) and Phe54), and CDR-H3-Tyr at positions 98±1; together with distinctive V-segment CDR amino acid substitutions that occur in positions sparse in AID/polymerase-η recognition motifs. A semi-synthetic IGHV1-69 phage-display library screen designed to investigate AID/polη restrictions resulted in the isolation of HV1-69-sBnAbs that featured a distinctive Ile52Ser mutation in the CDR-H2 loop, a universal CDR-H3 Tyr at position 98 or 99, and required as little as two additional substitutions for heterosubtypic neutralizing activity. The functional importance of the Ile52Ser mutation was confirmed by mutagenesis and by BCR studies. Structural modeling suggests that substitution of a small amino acid at position 52 (or 52a) facilitates the insertion of CDR-H2 Phe54 and CDR-H3-Tyr into adjacent pockets on the stem. These results support the concept that activation and expansion of a defined subset of IGHV1-69-encoded B cells to produce potent HV1-69-sBnAbs does not necessarily require a heavily diversified V-segment acquired through recycling/reentry into the germinal center; rather, the incorporation of distinctive amino acid substitutions by Phase 2 long-patch error-prone repair of AID-induced mutations or by random non-AID SHM events may be sufficient. We propose that these routes of B cell maturation should be further investigated and exploited as a pathway for HV1-69-sBnAb elicitation by vaccination.
Author Summary The quest for universal influenza vaccine has gained wide interest with the discovery of human neutralizing antibodies that are able to variably cross neutralize and protect against different influenza strains, subtypes, groups and lineages. These antibodies, which bind to a highly conserved epitope in the hemagglutinin stem, are often encoded by rearranged IGHV1-69 germline genes that alone make contact with HA and prevent virus entry and emergence of escape mutants. Our study was undertaken to gain an understanding of what structural requirements enable a rearranged IGHV1-69 Ab to become a potent cross-neutralizing antibody. We found that in addition to a critical amino acid triad consisting of a pair of anchor residues in CDR-H2 and a properly positioned CDR-H3 Tyr, distinctive V-segment substitutions that arise in positions that are distinct from phase I AID somatic hypermutation (SHM) hotspot motifs are often required. As few as two V-segment SHM can fulfill this role which appears to facilitate the optimal binding of CDR-H2 Phe54 and CHR-H3-Tyr into adjacent hydrophobic pockets in the HA stem. These studies provide new information on the SHM requirements for IGHV1-69-encoded B cells to produce HV1-69-sBnAbs and suggest that there may exist alternative routes to their elicitation by vaccination.