To generate influenza B vaccines that protect against viruses from both the Victoria- and Yamagata-lineages, we first compared the HA1 amino acid sequences of representative Victoria- and Yamagata-lineage viruses. We selected B/Phuket/3073/2013 (Phuket/Yam, the Yamagata-lineage vaccine strain since the 2017–2018 season) and B/Washington/02/2019 (Wash/Vic, the Victoria-lineage vaccine strain in the 2020–2022 southern hemisphere influenza season). The Wash/Vic and Phuket/Yam HA1 proteins differ by 39 amino acids in HA1, including a two-amino acid deletion in Wash/Vic (Fig. 1). Using commercial gene synthesis services, we synthesized two gene libraries (based on the Wash/Vic and Phuket/Yam HA1 sequences) that encoded either parental amino acid at each of the 37 respective amino acid positions (Table 1). No mutations were introduced at the amino acid deletion of Wash/Vic HA compared to Phuket/Yam HA. Thus, the synthetic HA1 gene libraries theoretically encoded 237 mutant HA genes. The amino acid changes in the Wash/Vic or Phuket/Yam HA1 regions could create structural and/or functional incompatibilities with HA2 or neuraminidase (NA). To account for this possibility, we employed molecular cloning techniques to first join the mutant Wash/Vic or Phuket/Yam HA1 fragments with Wash/Vic or Phuket/Yam HA2 sequences. We then used our established reverse genetics system19 to create virus libraries composed of the six internal genes of high-yield B/Yamagata/173 virus20, the NA genes of Wash/Vic or Phuket/Yam, and the Wash/Vic or Phuket/Yam HA libraries, resulting in eight virus libraries (Table 2). Forty-eight hours after transfecting cells with plasmids for virus library generation, the virus libraries were collected from plasmid-transfected human embryonic kidney (293 T) cells and inoculated onto hCK cells for plaque assays. Mutations at up to 37 amino acid positions of HA may result in many non-functional mutants; however, at the mutated positions, we started with only two amino acids that are known to be functional in the context of Wash/Vic or Phuket/Yam, respectively. Moreover, the strategy used here––reverse genetics followed by viral plaque assays––eliminated any non-viable mutants, which would not form virus plaques. Thus, while many mutants are theoretically possible, only viruses with a functional HA would be isolated from plaque assays.

a Shown are the HA amino acid differences between B/Phuket/3073/2013 (Phuket/Yam) and B/Washington/02/2019 (Wash/Vic). Dots indicate the same amino acid at this position; hyphens represent deletions at this position. b Three-dimensional structure of influenza B virus HA (Protein Structure Database, PDB code: 6FYW). Different HA trimers are colored in light green, blue, and beige. The amino acid differences in the HA1 subunits of Phuket/Yam and Wash/Vic are shown in red.

We isolated 384 virus plaques from Phuket/Yam HA1 virus libraries and 163 virus plaques from Wash/Vic HA1 libraries and established their HA sequences by Sanger sequencing. In total, we identified 217 different genotypes, 188 with a Phuket/Yam-based HA1 and 29 with a Wash/Vic-based HA1 (Supplementary Data 1).

All mutant viruses were tested with ferret sera raised against Wash/Vic and Phuket/Yam. Hemagglutination inhibition (HI) assays demonstrated that wild-type Phuket/Yam reacts poorly with serum to Wash/Vic and vice versa (Supplementary Data 1). Forty-four mutant viruses were antigenically Phuket/Yam-like, defined by an HI titer ≥80 against anti-Phuket/Yam serum and an HI titer ≤40 against anti-Wash/Vic serum. Seventy-one mutants were Wash/Vic-like, defined by an HI titer ≥80 against anti-Wash/Vic serum and an HI titer ≤40 against anti-Phuket/Yam serum. Sixty-nine mutants displayed HI titers of ≤40 against both ferret sera. Importantly, we also identified 33 mutants with HI titers ≥80 to both sera (Supplementary Data 1). These mutants retained reactivity with serum raised against the homologous parent virus and gained reactivity with serum raised against the heterologous parent virus; thus, these mutants may be antigenically located between the two influenza B virus lineages.

Antigenic cartography21 is widely used to assess the antigenic properties of influenza viruses. To generate an antigenic map for influenza B viruses, we downloaded all available influenza B virus HA sequences from GISAID (>52,000) and generated a phylogenetic tree of the >8000 unique amino acid sequences by using RaxML. Using this tree, we classified all known influenza B virus HA sequences as either ‘Ancestral’, ‘Victoria’ or ‘Yamagata’. Based on the phylogenetic tree, we selected 36 viruses (4 ancestral, 18 Yamagata-, and 14 Victoria-lineage viruses), spanning several decades and including major sublineages. For the selected viruses, ferret sera were already available in our group or were generated by immunizing ferrets with live virus, followed by a boost with inactivated virus administered with adjuvant. Next, we determined the HI titers of the ferret sera against wild-type viruses and against the 33 influenza B mutants that reacted with ferret sera raised against Wash/Vic and Phuket/Yam (Supplementary Data 2). Since the mutant viruses differ in their HA2 and NA sequences (see Table 2), we also included recombinant control viruses in which wild-type Wash/Vic or Phuket/Yam HA1 sequences were combined with wild-type Wash/Vic or Phuket/Yam HA2 and/or NA sequences (Supplementary Data 2). Based on these HI titers, an antigenic map for wild-type and mutant influenza B viruses was generated (Fig. 2).

Viruses are depicted by circles; sera are depicted by squares. Sera generated against mutant viruses are depicted by small squares. Red, ancestral viruses; green, Victoria-lineage viruses; blue, Yamagata-lineage viruses. Mutant viruses are shown in light green (Victoria-lineage mutants) or light blue (Yamagata-lineage mutants), respectively. The two mutants selected for vaccination and challenge studies are indicated by red circles. Anti-sera generated against the two mutants are indicated by orange squares. The parent Wash/Vic and Phuket/Yam viruses are labeled and indicated by large circles in dark green or purple, respectively.

The antigenic map (Fig. 2) shows the antigenic separation of influenza B viruses into ancestral viruses (red circles; circulating before the split into the Yamagata- and Victoria-lineages), Yamagata-lineage viruses (blue circles), and Victoria-lineage viruses (green circles). The antigenically most advanced viruses are the Wash/Vic and Phuket/Yam vaccine viruses (shown in large blue and green circles, respectively, in Fig. 2) that were used as parent viruses in our study. The Wash/Vic and Phuket/Yam control viruses that differ in their HA2 and/or NA sequences are located close to each other in the antigenic map, confirming that the antigenic properties are primarily determined by amino acids in HA1 (Fig. 2).

Most mutant Wash/Vic viruses (Fig. 2, small light green circles) are antigenically different from the parent Wash/Vic viruses (Fig. 2, large dark green circles); compared with the wild-type Washington-lineage viruses (large- and mid-sized green circles), they are located closer to the Yamagata-lineage viruses (Fig. 2, blue circles). Similarly, most mutant Phuket/Yam viruses (Fig. 2, small light blue circles) are antigenically different from the parent Phuket/Yam viruses (Fig. 2, large blue circles) and compared to the wild-type Yamagata-lineage viruses, they located closer to the Wash/Vic viruses. Most of the mutant viruses are located between the two influenza B virus lineages.

Based on the HI titers and the location in the antigenic map, we selected seven Wash/Vic and seven Phuket/Yam mutants to generate ferret sera, which were then tested against wild-type viruses and selected mutant viruses (Supplementary Data 2). These data were integrated into the antigenic map (Fig. 2, small squares). Several of the sera raised against the mutant influenza B viruses displayed robust HI titers of 40 or higher against influenza B viruses from the homologous lineages but, importantly, also against influenza B viruses from the heterologous lineage.

Based on the HI titers against wild-type and mutant viruses from both lineages (Supplementary Data 2) and their position in the antigenic map (Fig. 2), two mutants, namely Phuket/YamHA1-Phuket/YamHA2-Wash/VicNA-76 [Phuket/Yam-Mut, whose HA1, HA2, and NA are derived from Phuket/Yam (HA1 and HA2) and Wash/Vic (NA)] and Wash/VicHA1-Phuket/YamHA2-Phuket/YamNA-29 [Wash/Vic-Mut, whose HA1, HA2, and NA are derived from Wash/Vic (HA1) and Phuket/Yam (HA2 and NA)] (Fig. 2) were selected for immunization and challenge studies in ferrets. Ferrets (four per group) were immunized with recombinant HAs (used to avoid the contribution of other viral proteins to the immune responses) for Phuket/Yam-Mut, Wash/Vic-Mut, Phuket/Yam, or Wash/Vic (Fig. 3a). The ferrets were immunized with 15 µg of recombinant HA (with Alhydrogel® adjuvant 2%). Three weeks later, they received a booster with the same dose, followed by a second booster with the same dose eight weeks after the initial immunization. Additional animals were mock-vaccinated as controls. Sera collected five weeks after the second booster were tested for their reactivity against the four viruses tested here. Sera from ferrets immunized with recombinant wild-type Wash/Vic HA reacted with the parental viruses, but not with the HA of the other viruses (Fig. 3b). Immunization with recombinant wild-type Phuket/Yam HA elicited antibodies against the homologous virus, but also against the Phuket/Yam-Mut virus, indicating cross-reactivity. Likewise, sera from ferrets immunized with recombinant mutant Phuket/Yam-Mut HA reacted with Phuket/Yam virus. Moreover, immunization with recombinant mutant Phuket/Yam-Mut HA elicited low levels of cross-reactive antibodies against Wash/Vic-Mut virus, and vice versa (Fig. 3b).

a Immunization and challenge scheme. Ferrets were immunized three times with the indicated recombinant HA protein. Five weeks after the third immunization, ferrets were challenged with 106 pfu of wild-type Phuket/Yam or Wash/Vic virus. (Created in BioRender, Neumann, G (2024) https://BioRender.com/e49u244) (b) Ferret sera were collected 3 weeks after the third immunization (i.e., two weeks before challenge), and tested against Phuket/Yam, Wash/Vic, Phuket/Yam-Mut, and Wash/Vic-Mut viruses. c Virus titers in nasal swabs after challenge with wild-type Wash/Vic virus. d Virus titers in nasal swabs after challenge with wild-type Phuket/Yam virus. The values presented are the averages ±SD. P values were calculated by using a two-way ANOVA with multiple comparisons. Black asterisks indicate statistically significant differences between the PBS-treated control group and immunized animals. Blue asterisks (c) indicate statistically significant differences between the Phuket/Yam rHA-immunized group and any of the other immunized groups. Red asterisks (d) indicate statistically significant differences between the Wash/Vic rHA-immunized group and any of the other immunized groups. *P < 0.1; **P < 0.01; ***P < 0.001; ****P < 0.0001.

All immunized ferrets were challenged with 106 pfu of wild-type Wash/Vic or Phuket/Yam virus five weeks after the second boost (Fig. 3a). Nasal swab samples were collected each day for seven days post-challenge to assess virus replication (Fig. 3c, d). Additionally, the body temperature of infected ferrets was monitored daily for seven days; however, no significant differences were detected (Supplementary Fig. 1).

In mock-vaccinated animals, wild-type Wash/Vic and Phuket/Yam viruses replicated efficiently on Days 1 and 2 post-infection, whereas titers were lower on subsequent days and the infection was cleared by Day 7 post-infection (Fig. 3c, d). Virus titers after challenge were lowest in animals challenged with homologous wild-type virus, that is, in ferrets immunized with recombinant wild-type Wash/Vic HA and then challenged with wild-type Wash/Vic virus, or in ferrets immunized with recombinant wild-type Phuket/Yam HA and then challenged with wild-type Phuket/Yam virus. Immunization with recombinant wild-type Phuket/Yam HA followed by challenge with heterologous wild-type Wash/Vic virus reduced virus titers by 0.5–1.8 log units compared to mock-vaccinated animals, indicating low levels of cross-protection between the two influenza B virus lineages. Likewise, immunization with recombinant wild-type Wash/Vic HA followed by challenge with heterologous wild-type Phuket/Yam virus reduced virus titers by 0.9–1.5 log units compared to mock-vaccinated ferrets.

Importantly, immunization with recombinant mutant Phuket/Yam-Mut HA provided some protection against viruses from both lineages. Specifically, ferrets immunized with recombinant mutant Phuket/Yam-Mut HA were better protected against wild-type Wash/Vic challenge than ferrets immunized with recombinant wild-type Phuket/Yam HA (Fig. 3c, compare light and dark blue lines). Likewise, ferrets immunized with recombinant mutant Wash/Vic-HA were better protected against wild-type Phuket/Yam challenge than ferrets immunized with recombinant wild-type Wash/-Vic HA (Fig. 3d, compare light and dark red lines).

The Phuket/Yam and Wash/Vic mutants tested here differ by up to 39 positions in their HA1 amino acid sequences. We used a bespoke Bayesian model to identify HA positions with the greatest effect on HI titers (Fig. 4a, b). Briefly, effects were assigned to each individual HA position and for all pairs of amino acids. An HI titer between a virus and serum was then modeled as the sum of each position effect multiplied by the amino acid pair effect given the peptide sequences of the virus and virus used to generate the serum. Positive values in Fig. 4a, b indicate a positive effect on HI titers. The strongest effects were detected for amino acid positions 203, 133, and 149 (Fig. 4c).

a HA site effect sizes for all amino acid positions. Points show the posterior median effect size. Lines show the 95% highest density interval. b Top 10 effect sizes in (a). c Position effects colored on an HA monomer (PDB ID 4FQK3). Amino acid positions shown in gray were not variable in the dataset and therefore had no effect size estimated. The three positions with the highest effect sizes (133, 149, and 203) are labeled. The receptor-binding site is shown as a light red patch.