Structural and genetic basis for development of broadly neutralizing influenza antibodies

Abstract

Influenza viruses take a yearly toll on human life despite efforts to contain them with seasonal vaccines. These viruses evade human immunity through the evolution of variants that resist neutralization. The identification of antibodies that recognize invariant structures on the influenza haemagglutinin (HA) protein have invigorated efforts to develop universal influenza vaccines. Specifically, antibodies to the highly conserved stem region of HA neutralize diverse viral subtypes. These antibodies largely derive from a specific antibody gene, heavy-chain variable region IGHV1-69, after limited affinity maturation from their germline ancestors, but how HA stimulates naive B cells to mature and induce protective immunity is unknown. To address this question, we analysed the structural and genetic basis for their engagement and maturation into broadly neutralizing antibodies. Here we show that the germline-encoded precursors of these antibodies act as functional B-cell antigen receptors (BCRs) that initiate subsequent affinity maturation. Neither the germline precursor of a prototypic antibody, CR6261 (ref. 3), nor those of two other natural human IGHV1-69 antibodies, bound HA as soluble immunoglobulin-G (IgG). However, all three IGHV1-69 precursors engaged HA when the antibody was expressed as cell surface IgM. HA triggered BCR-associated tyrosine kinase signalling by germline transmembrane IgM. Recognition and virus neutralization was dependent solely on the heavy chain, and affinity maturation of CR6261 required only seven amino acids in the complementarity-determining region (CDR) H1 and framework region 3 (FR3) to restore full activity. These findings provide insight into the initial events that lead to the generation of broadly neutralizing antibodies to influenza, informing the rational design of vaccines to elicit such antibodies and providing a model relevant to other infectious diseases, including human immunodeficiency virus/AIDS. The data further suggest that selected immunoglobulin genes recognize specific protein structural 'patterns' that provide a substrate for further affinity maturation.

Characterization of pathogenic human monoclonal autoantibodies against GM-CSF

“…However, an antibody with a K D of 3 × 10 −4 M could still activate B cells when expressed as a BCR in mice (41). In another study, although somatic mutations were reverted in three affinity-matured antibodies and the unmutated mAbs do not bind to the antigen, when the unmutated mAbs were expressed in a B-cell line as a BCR, they signaled when antigen was added (42). Furthermore, the probability that antibodies that bind to one unrelated antigen should have fortuitous somatic mutations that bind to two or three nonoverlapping epitopes of a DSG3 is small.…”

Overlapping hotspots in CDRs are critical sites for V region diversification

“…In addition, 65% of the mutations at residue 104, which encodes the serine in the C-terminal end of CDR1, are transitions that would create an alanine, and the rest are transversions that would create a threonine or isoleucine; according to the Pymol program, these changes could also create instability at the bases of both CDR2 and CDR1. These mutations are reminiscent of the mutations at the bases of the CDRs in anti-HIV and anti-influenza antibodies that have no direct contact with the antigen but change the stability of the CDR loops in some cases, converting the antibodies from affinity-matured highly neutralizing antibodies with restricted specificity into broadly neutralizing and protective antibodies to those viruses (1,70,71). The glutamine and leucine encoded by the highly mutated AGCT at residues 9 and 10 ( Fig.…”

Structure of the apo anti-influenza CH65 Fab

“…However, the unmutated common ancestor (UCA) and the intermediate (I-2) precursor antibodies in the CH65-CH67 lineage have significantly weaker affinity for the H1 HAs (Schmidt et al, 2013). These studies have suggested that the mutations acquired through affinity maturation dictate the structural conformation of the antibody CDR loops, as for the V H 1-69 stem-targeted antibody CR6261 (Lingwood et al, 2012). Molecular-dynamics simulations and crystal structure comparisons of UCA, I-2, CH65-HA, apo CH67 and CH67-HA have suggested that the maturation pathway leads to a preconfiguration of the antibody HCDR3 conformation that is optimal for antigen recognition and which is observed in the Fab-HA complexes (Schmidt et al, 2013).…”

“…These bnAbs bind the influenza hemagglutinin glycoprotein (HA; Wilson et al, 1981) and target functionally conserved epitopes on the HA head and stem regions (Lee & Wilson, 2015). In conjunction with these advances in antibody discovery, the developmental pathways that the HA stem-targeted V H 1-69-encoded antibodies take to convert from germline to affinitymatured forms are an active area of investigation (Lingwood et al, 2012;Avnir et al, 2014;Pappas et al, 2014). These studies show that somatic mutations that arise during the affinitymaturation process can affect the conformations of the antibody complementarity-determining region (CDR) loops.…”