Preclinical evaluation of a universal inactivated influenza B vaccine based on the mosaic hemagglutinin-approach

Abstract

We have developed a new universal influenza B vaccination strategy based on inactivated influenza B viruses displaying mosaic hemagglutinins (mHAs). Recombinant mHA viruses were constructed by replacing the four major antigenic sites of influenza B virus HAs, with those from exotic avian influenza A virus HAs. Sequential vaccination of naïve mice with mHA-based vaccines elicited higher immune responses towards the immuno-subdominant conserved epitopes of the HA than vaccination with wildtype viruses. Among the different preparations tested, mHA split vaccines were less immunogenic than their whole inactivated virus counterparts. This lower immunogenicity was overcome by the combination with adjuvants. mHA split vaccines adjuvanted with a Toll-like receptor-9 agonist (CpG 1018) increased Th1 immunity and in vivo cross-protection, whereas adjuvanting with an MF59-like oil-in-water nano-emulsion (AddaVax) enhanced and broadened humoral immune responses and antibody-mediated cross-protection. The mHA vaccines with or without adjuvant were subsequently evaluated in mice that were previously immunized to closely mimic human pre-existing immunity to influenza B viruses and the contribution of innate and cellular immunity was evaluated in this model. We believe these preclinical studies using the mHA strategy represent a major step toward the evaluation of a universal influenza B virus vaccine in clinical trials.

Fig. 1. Influenza B virus surveillance and universal influenza virus vaccine design.

A Surveillance of influenza B viruses in the US from 1997 to 2023 and (B) percentage of influenza B/Yamagata/16/1988-like and B/Victoria/2/1987-like lineage viruses identified from 2015 to 2023 (data obtained from FluNet, (who.int) as of 1st Aug 2023). C Phylogenetic tree of HA sequences of historical annual formulations for IBV vaccine strains from 1999 to 2023 (data obtained from the Global Influenza Programme (who.int) as of 1st Aug 2023 and reported in Supplementary Fig. 1). Influenza B virus HA sequences used in this study as prime vaccination (B/Yamagata/16/1988) and as universal influenza virus vaccines (B/Brisbane/60/2008 and B/Phuket/3073/2013) are highlighted in green and dark red, respectively. The phylogenetic tree was constructed using the maximum likelihood method and Tamura-Nei model and visualized with Mega11^,. D Mosaic HA (mHA) universal influenza vaccine approach. Sequential vaccination with mHA vaccines, where the major immunodominant epitopes are replaced in each vaccination to refocus the immune response to subdominant head and stalk epitopes of the HA glycoprotein (representation of an HA glycoprotein based on PDB accession no. 4M44). E mHA influenza B virus rescue scheme. B mHA and wildtype (WT) viruses were rescued following the reverse genetics method as previously described. Thereafter, viruses were propagated in embryonated chicken eggs and harvested in the allantoic fluid. The mosaic viruses were based on B/Yamagata/16/1988 (Yam), B/Brisbane/60/2008 (Bris) and B/Phuket/3073/2013 (Phu) resulting in the mH8/B_Yam, mH13/B_Bris and mH5/B_Phu viruses, respectively. F mHA influenza B virus vaccine preparation. Whole inactivated viruses (WIV) were generated by inactivating the harvested viruses with either formaldehyde (FA) or beta-propiolactone (BPL) and purified by sucrose cushion ultracentrifugation. To produce split versions of these vaccines, BPL inactivated and purified virus preparations were treated with Triton X-100 and the remaining detergent was removed using hydrophobic beads in batch mode chromatography.

Fig. 2. Antibody responses to the conserved HA stalk and immuno-subdominant head domains elicited by wildtype and mHA vaccines prepared by two different methods.

Whole inactivated virus (WIV) with beta-propiolactone (BPL) and inactivated split vaccine combining BPL inactivation and Triton X-100 splitting were compared for two different vaccination strategies with WT viruses or mHA viruses. Vaccines were tested without adjuvant or with the combination with CpG 1018 (30 µg per mouse). A, B Vaccination regimen and groups. BALB/c mice (n = 10) were vaccinated in a three-dose vaccination experiment with a dose of 1 µg of HA in a 3–4-week interval. mHA inactivated split vaccines adjuvanted with AddaVax (1:1 v:v) and an unvaccinated group (PBS) were included as controls. C, D Binding of serum antibodies towards the immuno-subdominant epitopes. A cH7/B_Yam protein with a group 2 avian H7 head and the B/Yamagata/16/1988 HA stalk was used to measure stalk-specific antibodies in the unadjuvanted and adjuvanted groups (C). A mH11/B_Yam protein displaying the H11 major antigenic sites in the B/Yamagata/16/1988 HA (mH11/B_Yam) was used to measure antibody binding to conserved epitopes in the HA head and stalk domains in the unadjuvanted and adjuvanted groups (D). The geometric mean endpoint titer was calculated as the readout. The statistics were calculated using an unpaired one-tailed t test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001).

Fig. 3. Cross-reactivity, HI-activity, and Fc effector function antibody responses elicited by wildtype and mHA vaccines prepared by two different methods.

Whole inactivated virus (WIV) with beta-propiolactone (BPL) and inactivated split vaccine combining BPL inactivation and Triton X-100 splitting were compared for two different vaccinations with WT viruses or mHA viruses. Vaccines were tested without adjuvant or with the addition of CpG 1018 (30 µg) as explained in Fig. 2. A Heatmap of IgG binding serum antibodies against a panel of recombinant influenza B HA proteins. B Heatmap of hemagglutination inhibition (HI) titer against a panel of influenza B viruses. C Heatmap of ADCC activity against three influenza B viruses. To perform the ADCC reporter assay, MDCK cells were infected with each virus at an MOI of 5 (single-cycle replication). One day after infection, mouse sera were added to the cells and incubated for 30 min and genetically modified Jurkat cells expressing the mouse FcγRIV with a luciferase reporter gene under transcriptional control of the nuclear factor-activated T (NFAT) cell promoter were then added and incubated for 6 h. Later luciferase activity was measured. The geometric mean AUC of fold induction of each group (pooled sera) was measured in technical duplicate.

Fig. 4. Cross-reactivity, Th1/Th2 immunogenicity, germinal center reaction and protection of mHA vaccines in a high pre-existing immunity model in mice.

A Vaccination regimen and experimental workflow. B Vaccination groups. BALB/c mice were vaccinated in a two-dose vaccination scheme with 1 µg of HA of the different WIV or split mHA vaccines in a 3-4 week interval after priming with 10⁵ PFU of mH8/B_Yam virus per mouse. WIV and split mHA vaccines were tested without adjuvant or with the addition of CpG 1018 (30 µg) or AddaVax (1:1 v:v). A QIV (Flulaval Quadrivalent) vaccinated group and an unvaccinated group (PBS) were included as controls. mH8/B_Yam virus infection was given intranasally in a total volume of 30 µL. C Heatmap of Th1/Th2 cytokine panel measured in sera taken 4 h after second boost vaccination (n = 4) using a 11-plex Luminex panel. Log2 fold change over PBS group and 2-way ANOVA test corrected using Dunnet’s test for multiple comparisons is shown (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). D, E Binding of serum antibody titers (n = 13–15) against subdominant epitopes analyzed as previously described in Fig. 3. Minimum weight loss (n = 5) of direct virus challenge with the ancestral B/Lee/40 virus (F), B/New York/PV01181/2018 virus (Victoria-lineage) (G) and B/New York/PV00094/2017 virus (Yamagata-lineage) (H). I Frequency of germinal center B cell formation. Inguinal lymph nodes were collected 4 weeks after second boost and frequency of germinal center B cells (live CD3^-B220⁺CD19⁺IgD^-GL7⁺CD38^low) was measured by FACS (n = 3–5). Serum passive transfer challenge with the B/Lee/40 virus (J–L) weight loss (J), survival of vaccination groups (K) and minimum weight loss (L) is shown. For direct challenge, BALB/c mice (n = 4–5) were challenged 8 weeks after second boost intranasally in a total volume of 30 µL. In the serum passive transfer experiment, BALB/c mice (n = 5) received 100 µL of pooled sera intraperitoneally and 2 hours later were challenged intranasally as previously described. In all cases a dose of 5× mLD50 was used. Weight loss and survival of mice were monitored for 2 weeks with a humane endpoint of ≥25% loss of the initial weight. Statistical significance was calculated using Kruskal-Wallis test corrected using Dunn’s test for multiple comparisons (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

Fig. 5. In vivo cross-protection of mice vaccinated with a low dose of mHA vaccines in a direct virus challenge and serum passive transfer/challenge experiment.

A, B Vaccination regimen and experimental workflow. BALB/c mice were vaccinated in a two-dose vaccination scheme with 1 or 0.1 µg of HA of the different vaccines in a 3–4-week interval after priming with 10⁵ PFU of mH8/B_Yam virus. WIV or split mHA vaccines were tested without adjuvant or with the addition of CpG 1018 (10 µg), CpG 1018 (10 µg) + Alum (50 µg) or AddaVax (1:1 v:v). A QIV (FluLaval Quadrivalent) vaccinated group and an unvaccinated group (PBS) were included as controls. mH8/B_Yam virus prime infection was given intranasally in a total volume of 30 µL 6 months prior to the two-dose immunization. C Four weeks after the second dose, mice (n = 5) were challenged with 50× mLD50 dose and viral titers were measured from harvested lung homogenate tissues 3 days post infection. Kruskal–Wallis test corrected using Dunn’s test for multiple comparisons against PBS is shown (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001).