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. 2022 Oct 18;119(42):e2211616119.
doi: 10.1073/pnas.2211616119. Epub 2022 Oct 10.

Substitutions near the HA receptor binding site explain the origin and major antigenic change of the B/Victoria and B/Yamagata lineages

Miruna E Rosu  1 , Pascal Lexmond  1 , Theo M Bestebroer  1 , Blake M Hauser  2 , Derek J Smith  2 , Sander Herfst  1 , Ron A M Fouchier  1
Affiliations

Substitutions near the HA receptor binding site explain the origin and major antigenic change of the B/Victoria and B/Yamagata lineages

Miruna E Rosu et al. Proc Natl Acad Sci U S A. .

Abstract

Influenza B virus primarily infects humans, causing seasonal epidemics globally. Two antigenic variants-Victoria-like and Yamagata-like-were detected in the 1980s, of which the molecular basis of emergence is still incompletely understood. Here, the antigenic properties of a unique collection of historical virus isolates, sampled from 1962 to 2000 and passaged exclusively in mammalian cells to preserve antigenic properties, were determined with the hemagglutination inhibition assay and an antigenic map was built to quantify and visualize the divergence of the lineages. The antigenic map revealed only three distinct antigenic clusters-Early, Victoria, and Yamagata-with relatively little antigenic diversity in each cluster until 2000. Viruses with Victoria-like antigenic properties emerged around 1972 and diversified subsequently into two genetic lineages. Viruses with Yamagata-like antigenic properties evolved from one lineage and became clearly antigenically distinct from the Victoria-like viruses around 1988. Recombinant mutant viruses were tested to show that insertions and deletions (indels), as observed frequently in influenza B virus hemagglutinin, had little effect on antigenic properties. In contrast, amino-acid substitutions at positions 148, 149, 150, and 203, adjacent to the hemagglutinin receptor binding site, determined the main antigenic differences between the Early, Victoria-like, and Yamagata-like viruses. Surprisingly, substitutions at two of the four positions reverted in recent viruses of the Victoria lineage, resulting in antigenic properties similar to viruses circulating ∼50 y earlier. These data shed light on the antigenic diversification of influenza viruses and suggest there may be limits to the antigenic evolution of influenza B virus.

Keywords: antigenic evolution; hemagglutinin; influenza B virus; vaccines.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Maximum likelihood phylogenetic tree inferred from 617 HA1 nucleotide sequences of viruses that span the period between 1940 and 2000. The phylogenetic tree was constructed under the GTR+G+I model. Dutch virus isolates described in this study are highlighted in red and vaccine and reference strains in blue. Bootstrap values are indicated for the main nodes in the tree. Vertical bars are shown for the B/Victoria and B/Yamagata lineages, starting from their respective reference viruses. Scale bar represents nucleotide substitutions per site.
Fig. 2.
Fig. 2.
Comparison of the antigenic and genetic evolution of historical influenza B viruses from 1958 to 2000. (A) 2D antigenic map constructed from HI titers of Dutch virus isolates and global vaccine and reference antigens and their corresponding antisera. Viruses are represented by colored circles and antisera by uncolored squares. The vertical and horizontal axis both represent antigenic distance. The spacing between the grid lines denotes 1 antigenic unit (AU) of distance, corresponding to a twofold dilution of antisera in the HI assay. Viruses are colored based on the antigenic cluster to which they belong: green for Early, blue for Yamagata, and red for Victoria. Shades of red are used to denote the different genetic lineages to which Victoria-like viruses belong. Clusters were identified by proximity of the antigens and named after the influenza B lineages they represent. (B) Maximum likelihood phylogenetic tree of HA1 nucleotide sequences inferred under the GTR+G+I model, color-coded based on antigenic clusters. Viruses labeled with a triangle were used as prototypes to determine the genetic basis of major antigenic change. Years of isolation are shown for several viruses for reference. Scale bar represents nucleotide substitutions per site.
Fig. 3.
Fig. 3.
Fixation of amino acid substitutions across antigenic cluster boundaries. Antigenic map is the same as shown in Fig. 2 but color coded based on amino acid residues at positions 71, 88, 148, 149, 150, 162, 202, 203, and 209. Numbering is according to B/Brisbane/60/2008 HA.
Fig. 4.
Fig. 4.
Antigenic effect of amino acid substitutions in HA on cluster transitions. Antigenic maps show the effect of individual or combined amino acid substitutions on recombinant prototype viruses. Wild-type viruses are shown as colored triangles, mutant viruses as colored circles, and antisera as uncolored squares. Arrows indicate the antigenic distance and direction caused by amino acid substitutions. Viruses are colored based on antigenic clusters, and cluster boundaries are schematically represented by gray-shaded ovals. The vertical and horizontal axis both represent antigenic distance. The spacing between the grid lines denotes 1 AU of distance, corresponding to a twofold dilution of antisera in the HI assay. (A and B) Analysis of the Early to Victoria cluster transition, with substitutions introduced (A) in the Early prototype virus NL69 and (B) in the Victoria prototype virus NL74. (C and D) Analysis of the Victoria to Yamagata cluster transition, with substitutions introduced (C) in the Victoria prototype virus NL84 and (D) in the Yamagata prototype virus Yam88. (E) Overview of amino acid substitutions explaining the cluster transitions. (F) Crystal structure of B/Yamanashi/166/1998 HA (PDB 4M40) with key positions explaining cluster transitions shown in red and position 148 in orange to mark its more-modest effect and the RBS in yellow. Numbering is according to B/Brisbane/60/2008 HA.
Fig. 5.
Fig. 5.
Effect of indels on the antigenic properties of historical influenza B viruses. (A) The maximum likelihood phylogenetic tree of Fig. 2 was colored based on the presence of zero (red), one (purple), two (blue), or three (gray) deletions in the 160 loop of the HA protein. (B) The antigenic map of Fig. 2 was color coded based on the presence of deletions in the 160 loop of the HA protein, matching A. (C) Antigenic map displaying the effects of aa substitutions 150KN and 203TK alone and in combination in NL69 (red triangle) and NL71 (gray triangle) viruses containing zero and three deletions in the 160 loop, respectively.
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