In vitro evolution of an influenza broadly neutralizing antibody is modulated by hemagglutinin receptor specificity

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.

Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability

“…Since the CDRH3 is composed of V, D and J gene segments, it is the most variable region of an antibody in terms of both amino acid composition and length. The average length of CDRH3 in the naive human repertoire is 15 amino acids (34), but for a subset of influenza virus and HIV-1 broadly neutralizing antibodies, long CDRH3 regions of 20-35 amino acids are crucial for high affinity antigen-antibody interactions (35,36). Even though the mean CDRH3 length of isolated SARS-CoV-2 S protein-specific B cells did not differ substantially from that of a naive population (34), we observed a significant difference in the distribution of CDRH3 length (two sample Kolmogorov-Smirnov test, p = 0.006) ( Fig.…”

An influenza A hemagglutinin small-molecule fusion inhibitor identified by a new high-throughput fluorescence polarization screen

“…The most stringent in vitro analysis to assess the potential antiviral activity of chemical and/or biological interventions is with cell-based infection assays. MDCK-SIAT1 cells die after 72 hrs of incubation with influenza virus, as previously described (22,31,32). Increasing concentrations (976 nM to 500 µM) of F0045(S) and F0045(R) were co-incubated with MDCK-SIAT1 cells, respectively.…”

Structural Basis of Pan-Ebolavirus Neutralization by an Antibody Targeting the Glycoprotein Fusion Loop

“…Our structures, however, provide a valuable tool for using structure-based design in silico to generate new starting models, such as those with increased affinity, that can be subsequently integrated into an in vitro evolution pipeline (Adolf-Bryfogle et al, 2018). Such efforts have shown promise for influenza mAbs, although it seems that increases in specificity result in diminished cross-reactivity (Wu et al, 2017; Wu and Wilson, 2017, 2018). Future work should be aimed at optimizing potent antibodies such as ADI-15878 for clinical development.…”