Abstract
The relatively recent discovery and characterization of human broadly neutralizing antibodies (bnAbs) against influenza virus provide valuable insights into antiviral and vaccine development. However, the factors that influence the evolution of high-affinity bnAbs remain elusive. We therefore explore the functional sequence space of bnAb C05, which targets the receptor-binding site (RBS) of influenza haemagglutinin (HA) via a long CDR H3. We combine saturation mutagenesis with yeast display to enrich for C05 variants of CDR H3 that bind to H1 and H3 HAs. The C05 variants evolve up to 20-fold higher affinity but increase specificity to each HA subtype used in the selection. Structural analysis reveals that the fine specificity is strongly influenced by a highly conserved substitution that regulates receptor binding in different subtypes. Overall, this study suggests that subtle natural variations in the HA RBS between subtypes and species may differentially influence the evolution of high-affinity bnAbs.
Highlights
The relatively recent discovery and characterization of human broadly neutralizing antibodies against influenza virus provide valuable insights into antiviral and vaccine development
Against A/Aichi/2/68, WT C05 (EC50 potent than VPGSGW (EC50 1⁄4 4100 mg ml À 1) is more potent In contrast, when tested 1⁄4 15.5 mg ml À 1) is more mg ml À 1). These results suggest that the amino-acid identity at residue 190 of the HA receptor-binding site (RBS), at least partially, modulates the binding affinity of the C05 CDR H3 loop and especially in the variant VPGSGW, illustrating how a highly conserved and functionally important substitution in the RBS shifts the amino-acid preference in the paratope of a broadly neutralizing antibody
We investigated the functional sequence space of C05, a prototypic HA receptor-binding site (HA RBS)-targeted broadly neutralizing antibodies (bnAb), by focusing on the six amino-acid residues on the apex of its long CDR H3 that inserts into the HA RBS and make the most intimate contacts with the RBS
Summary
The relatively recent discovery and characterization of human broadly neutralizing antibodies (bnAbs) against influenza virus provide valuable insights into antiviral and vaccine development. These stem-binding antibodies have guided the development of immunogens that confer heterosubtypic protection[18,19,20] Another class of influenza bnAbs that target the HA receptor-binding site (RBS) has been identified and characterized[21,22,23,24,25,26,27,28,29,30,31]. While many antibodies induced during the 2009 pandemic H1N1 influenza season exhibited broad neutralization activity against the HA stem[37], this response was not sustained in later years[36,38] These observations indicate that our understanding of the evolution and maintenance of influenza bnAbs is far from complete
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