Molecular Signatures of Hemagglutinin Stem-Directed Heterosubtypic Human Neutralizing Antibodies against Influenza A Viruses

Abstract

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.

Restricted, canonical, stereotyped and convergent immunoglobulin responses

“…However, epitopes in the stalk portion of HA that attaches the protein to the viral envelope are highly conserved between most influenza strains. Interestingly, antibodies to the HA stalk are preferentially encoded by VH1-69 and the highly similar VH1-18 gene segments (figure 2c) [19,[24][25][26][27][28]. Such antibodies were preferentially induced in individuals infected or vaccinated with the 2009 pandemic H1N1 influenza strains such that, in some cases, half of all neutralizing antibodies induced were encoded by VH1-69 or VH1-18 [29][30][31].…”

“…It is not clear what is unique about B cells expressing VH1-69-encoded BCRs but its common use in stereotyped responses could relate to the unique molecular structure of VH1-69-encoded antibodies. VH1-69-encoded antibody proteins have a distinct hydrophobic region within the antigen-binding site that may make this antibody type highly unique and important in various contexts [24][25][26]. This unique molecular character could target a particular class of epitopes shared by different antigens.…”

Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity

“…The subunits of HA are described in SI Appendix, Table S2. AT10-005 contains the IGHV1-69 gene segment and harbors the hydrophobic signature commonly found in group 1-specific antibodies (SI Appendix, Table S3) (38,39). Antibody competition using AT10-005 and the stem-binding antibody CR6261 (40) AT10-002 is specific for HA proteins of group 2 viruses (SI Appendix, Table S1) and shows neutralizing activity against four group 2 viruses (two H3N2, one H7N1, and one H7N7 virus) ( Table 1).…”

Structural insights into the design of novel anti-influenza therapies

“…A conserved Tyr residue in CDR H3 provides the basis for the interaction with the HA stem. Together, these recognition elements, arising from CDR H2 (IF) and H3 (Y), form the so-called IFY motif 56, 57 . Two other stem-binding bnAbs, FI6v3 44 and 39.29 58 of the V H 3–30 germline family, which bind the HA at a very different angle and use both light and heavy chains for interaction, compared to V H 1–69, where the heavy chain makes the majority, if not all, of the stem interactions.…”