Plasticity of amino acid residue 145 near the receptor binding site (RBS) of H3 influenza A viruses and its impact on receptor binding and antibody recognition

Authors: SANTOS, JEFFERSON - University Of Georgia; Abente, Eugenio; OBADAN, ADEBIMPE - University Of Georgia; THOMPSON, ANDREW - Scripps Institute; FERRERI, LUCAS - University Of Georgia; GEIGER, GINGER - University Of Georgia; GONZALEZ-REICHE, ANA - The Icahn School Of Medicine At Mount Sinai; LEWIS, NICOLA - University Of Cambridge; BURKE, DAVID - University Of Cambridge; RAJÃO, DANIELA - University Of Georgia; PAULSON, JAMES - Scripps Institute; Baker, Amy; PEREZ, DANIEL - University Of Georgia

Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/13/2018
Publication Date: 10/24/2018
Citation: Santos, J., Abente, E.J., Obadan, A.O., Thompson, A.J., Ferreri, L., Geiger, G., Gonzalez-Reiche, A.S., Lewis, N.S., Burke, D., Rajão, D.S., Paulson, J.C., Vincent, A.L., Perez, D.R. 2018. Plasticity of amino acid residue 145 near the receptor binding site of H3 swine influenza A viruses and its impact on receptor binding and antibody recognition. Journal of Virology. 93(2):e01413-18. https://doi.org/10.1128/JVI.01413-18.
DOI: https://doi.org/10.1128/JVI.01413-18

Interpretive Summary: Swine influenza A virus (IAV) is an endemic and economically important pathogen in pigs with the potential to infect other host species including humans. The hemagglutinin (HA), a glycoprotein on the surface of IAV, binds to residues on host cells. The HA protein gradually accumulates amino acid (aa) substitutions that allow IAV to escape population immunity through a mechanism known as antigenic drift. The HA protein is the primary target of protective immune responses and the major component in swine IAV vaccines. We recently confirmed that a small set of positions in the HA protein are largely responsible for driving antigenic drift in swine-origin H3 IAV. All identified residues are located adjacent to the HA receptor binding site (RBS), suggesting that substitutions associated with antigenic drift may also influence receptor binding. We tested whether there are functional constraints to substitutions near the RBS and their impact on receptor binding and antigenic properties. Despite limited amino acid usage found in nature at one key position, most of the 20 substitutions were well tolerated and stably maintained without major impact on virus replication in vitro. All substitutions retained receptor binding specificity, but frequently led to decreased receptor binding or modulated binding profiles. Furthermore, antigenic characterization identified specific substitutions that altered antibody recognition. This work provides a better understanding of the impact of aa substitutions near the RBS and the interplay between receptor binding and antigenic drift and can help inform vaccine strain selection.

Technical Abstract: The hemagglutinin (HA), a glycoprotein on the surface of influenza A virus (IAV), initiates the virus life cycle by binding to terminal sialic acid (SA) residues on host cells. The HA protein gradually accumulates amino acid (aa) substitutions that allow IAV to escape population immunity through a mechanism known as antigenic drift. We recently confirmed that a small set of aa residues are largely responsible for driving antigenic drift in swine-origin H3 IAV. All identified residues are located adjacent to the HA receptor binding site (RBS), suggesting that substitutions associated with antigenic drift may also influence receptor binding. To determine whether there are functional constraints to substitutions near the RBS and their impact on receptor binding and antigenic properties, we carried out site-directed mutagenesis experiments at the single aa level using a swine-origin H3N2 virus backbone. We generated a panel of HA mutant viruses carrying substitutions at residue 145 (H3 numbering) representing all 20 amino acids. Despite limited amino acid usage in nature, most substitutions at residue 145 were well tolerated and stably maintained without major impact on virus replication in vitro. All substitutions retained receptor binding specificity, but frequently led to decreased receptor binding. Glycan microarray analysis showed that substitutions at residue 145 modulate binding to a broad range of glycans. Furthermore, antigenic characterization identified specific substitutions at residue 145 that altered antibody recognition. This work provides a better understanding of the functional effects of aa substitutions near the RBS and the interplay between receptor binding and antigenic drift.