Immune Escape Adaptive Mutations in the H7N9 Avian Influenza Hemagglutinin Protein Increase Virus Replication Fitness and Decrease Pandemic Potential

Abstract

H7N9 avian influenza viruses (AIVs) continue to evolve and remain a huge threat to human health and the poultry industry. Previously, serially passaging the H7N9 A/Anhui/1/2013 virus in the presence of homologous ferret antiserum resulted in immune escape viruses containing amino acid substitutions alanine to threonine at residues 125 (A125T), 151 (A151T) and leucine to glutamine at residue 217 (L217Q) in the hemagglutinin (HA) protein. These HA mutations have also been found in the field isolates in 2019. To investigate the potential threat of the serum escape mutant viruses to humans and poultry, the impact of these HA substitutions, either individually or in combination, on receptor binding, pH of fusion, thermal stability and virus replication were investigated. Our results showed the serum escape mutant formed large plaques in Madin-Darby canine kidney (MDCK) cells and grew robustly in vitro and in ovo. They had a lower pH of fusion and increased thermal stability. Of note, the serum escape mutant completely lost the ability to bind to human-like receptor analogues. Further analysis revealed that N-linked glycosylation, as a result of A125T or A151T substitutions in HA, resulted in reduced receptor binding avidity toward both human and avian-like receptor analogues, and the A125T+A151T mutations completely abolished human-like receptor binding. The L217Q mutation enhanced the H7N9 acid and thermal stability while the A151T mutation dramatically decreased H7N9 HA thermal stability. To conclude, H7N9 AIVs that contain A125T+A151T+L217Q mutations in HA protein might pose a reduced pandemic risk but remain a heightened threat for poultry. IMPORTANCE Avian influenza H7N9 viruses have been causing disease outbreaks in poultry and humans. We previously determined that propagation of H7N9 virus in the virus-specific antiserum give rise to mutant viruses carrying mutations A125T+A151T+L217Q in their hemagglutinin protein, enabling the virus to overcome vaccine-induced immunity. As predicted, these immune escape mutations were also observed in the field viruses that likely emerged in the immunised or naturally exposed birds. This study demonstrates that the immune escape mutants also (i) gained greater replication ability in cultured cells and in chick embryo as well as (ii) increased acid and thermal stability, but (iii) lost preferences for binding to human-type receptor while maintaining binding for the avian-like receptor. Therefore, they potentially pose reduced pandemic risk. However, the emergent virus variants containing indicated mutations remain a significant risk to the poultry due to antigenic drift and improved fitness for poultry.

Co-infection of chickens with H9N2 and H7N9 avian influenza viruses leads to emergence of reassortant H9N9 virus with increased fitness for poultry and enhanced zoonotic potential

“…Viruses with preferable binding towards 3SLN(6-su) may have an increased propensity for circulation in terrestrial poultry (Gambaryan et al, 2008;Peacock et al, 2020), and this observation was reflected in the successful generation and transmission of these H9N9 genotypes in chickens in our in vivo experiment. The H9N2 and genotype 122 H9N9 viruses were found to have an optimal pH fusion of 5.4, which was slightly lower compared to that of Anhui/13 H7N9 having an optimal pH fusion 5.6 as seen previously (Chang et al, 2020). The results suggested that the reassortant H9N9 virus has a relatively more acid stable HA and stronger avidity for both human-like (6SLN) and avian-like (3SLN and 3SLN(6-su) receptors, thereby maintaining adaptation of such viruses for poultry while also enabling additional zoonotic potential (Peacock et al, 2020;Russier et al, 2016).…”

Hemagglutination Inhibition (HAI) antibody landscapes after vaccination with H7Nx virus like particles

“…There was no significant influence of the glycosylation site on host tropism, however, the potential change in antigenicity was not investigated [ 24 ]. The latest study published in 2020 also verified that the corresponding mutation A151T (A169T by our numbering) occurred in one of the escaping mutants and proved that the mutation results in glycosylation [ 25 ]. But both studies did not investigate its influence on cross-reactivity to other H7 strains.…”

Hemagglutination Inhibition (HAI) Antibody Landscapes after Vaccination with diverse H7 hemagglutinin (HA) proteins

“…There was no significant influence of the glycosylation site on host tropism, however, the potential change in antigenicity was not investigated (24). The latest study published in 2020 also verified that the corresponding mutation A151T (A169T by our numbering) occurred in one of the escaping mutants and proved that the mutation results in glycosylation (25). But both studies did not investigate its influence on cross-reactivity to other H7 strains.…”