Abstract
The haemagglutinin (HA) glycan binding selectivity of H1N1 influenza viruses is an important determinant for the host range of the virus and egg-adaption during vaccine production. This study integrates glycan binding data with structure-recognition models to examine the impact of the K123N, D225G and Q226R mutations (as seen in the HA of vaccine strains of the pandemic 2009 H1N1 swine influenza A virus). The glycan-binding selectivity of three A/California/07/09 vaccine production strains, and purified recombinant A/California/07/09 HAs harboring these mutations was examined via a solid-phase ELISA assay. Wild-type A/California/07/09 recombinant HA bound specifically to α2,6-linked sialyl-glycans, with no affinity for the α2,3-linked sialyl-glycans in the array. In contrast, the vaccine virus strains and recombinant HA harboring the Q226R HA mutation displayed a comparable pattern of highly specific binding to α2,3-linked sialyl-glycans, with a negligible affinity for α2,6-linked sialyl-glycans. The D225G A/California/07/09 recombinant HA displayed an enhanced binding affinity for both α2,6- and α2,3-linked sialyl-glycans in the array. Notably its α2,6-glycan affinity was generally higher compared to its α2,3-glycan affinity, which may explain why the double mutant was not naturally selected during egg-adaption of the virus. The K123N mutation which introduces a glycosylation site proximal to the receptor binding site, did not impact the α2,3/α2,6 glycan selectivity, however, it lowered the overall glycan binding affinity of the HA; suggesting glycosylation may interfere with receptor binding. Docking models and ‘per residues’ scoring were employed to provide a structure-recognition rational for the experimental glycan binding data. Collectively, the glycan binding data inform future vaccine design strategies to introduce the D225G or Q226R amino acid substitutions into recombinant H1N1 viruses.
Highlights
The influenza viruses belong to the family of single-stranded negative sense RNA viruses or Orthomyxoviridae [1,2]
To understand how these important mutations impact on the receptor binding preference of these key H1N1 2009 vaccine strains, we have examined the glycan selectivity of each HA variant using a solid-phase ELISA glycan binding assay with both whole viruses and purified recombinant HAs [35,36]
We examined the receptor binding specificity for egg-adapted vaccine strains of A/California/07/09, and purified recombinant HA proteins with the aforementioned RBS mutations associated with high egg-growth
Summary
The influenza viruses belong to the family of single-stranded negative sense RNA viruses or Orthomyxoviridae [1,2]. Others identified mutation sites in the HA molecule that significantly facilitated viral rescue and amplification in eggs namely, K123N, D225G and Q226R [32,33,34] To understand how these important mutations impact on the receptor binding preference of these key H1N1 2009 vaccine strains, we have examined the glycan selectivity of each HA variant using a solid-phase ELISA glycan binding assay with both whole viruses and purified recombinant HAs [35,36]. This study provides the structure-recognition rational for the glycan binding mechanism of important egg-adaption mutations in the HA receptor binding pocket of vaccine production variants of the 2009 H1N1 pandemic influenza A virus The introduction of these HA mutations into recombinant viruses will enable the rapid generation of future.
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