Characterization of antigenically dominant regions in the hemagglutinin protein of B/victoria-lineage influenza B virus using monoclonal antibody escape mutants

Abstract

As of 2024, B/Victoria-lineage strains have emerged as the predominant influenza B viruses globally. To elucidate the antigenic regions responsible for variation within this lineage, three monoclonal antibodies (MAbs) targeting the hemagglutinin (HA) protein were employed to generate escape mutants of the B/Victoria strain B/Aichi/20/99, which was isolated approximately 10 years after the B/Victoria and B/Yamagata lineages began cocirculating. A total of 45 escape mutants were obtained. Sequencing of their HA genes identified six amino acid substitutions at four sites within two key antigenic regions-the 160-loop and 190-helix-specifically, N165Y, N165S, K167R, and an asparagine insertion between residues 164 and 165 in the 160-loop; and K203R and K203N in the 190-helix (numbering is based on the B/Brisbane/60/2008 HA sequence). Hemagglutination inhibition (HI) assays revealed that two MAbs affected binding of both mutants with mutations in the 160-loop and those with a mutation at residue 203. Mutations in the 160-loop did not affect reactivity with antiserum against the parental B/Aichi/20/99 strain, whereas K203N substitution reduced antiserum reactivity, indicating the antigenic importance of this residue. Further HI analyses using eight B/Victoria lineage strains isolated between 1997 and 2021 showed that all three MAbs lost reactivity with strains isolated after 2016, while the antiserum demonstrated reduced reactivity. Notably, the current vaccine strain, B/Austria/1359417/2021, which harbors substitutions at positions 150 and 203, also exhibited diminished reactivity. These findings suggest that both the 150-loop and 190-helix constitute antigenically dominant sites that contribute to immune escape and the emergence of drift variants within the B/Victoria-lineage.

Keywords: Antigenic structure; Escape mutant; Influenza B virus; Victoria-lineage.

Fig. 1

Replication kinetics of the escape mutants. The growth kinetics of the parent B/Aichi/20/99 virus and the escape mutants with the indicated substitutions were compared. MDCK cells were infected with each virus at a multiplicity of infection (MOI) of 0.001. Cell supernatants were collected at 8, 24, 48, and 72 h post-infection and used for virus titration in MDCK cells via plaque assay. Viral titers are presented as the mean ± standard deviation (SD) from three independent experiments.

Fig. 2

Phylogenetic tree of the HA1 genes of viruses used in the antigenic analysis. The tree shows the evolutionary relationships between the viruses analyzed. The numbers indicate the predominant amino acid changes inherited by subsequent B/Victoria-lineage strains.

Fig. 3

Locations of amino acid changes in escape mutants and natural isolates used in antigenic analysis, mapped onto the B/Brisbane/60/2008 HA structure (PDB: 4FQM). The residues within each antigenic region are color-coded as follows: cyan for the 120-loop, green for the 150-loop, blue for the 160-loop, and red for the 190-helix. Numbering is based on the B/Brisbane/60/2008 HA sequence.