Development of an Influenza/COVID-19 Combination mRNA Vaccine Containing a Novel Multivalent Antigen Design That Enhances Immunogenicity of Influenza Virus B Hemagglutinins

Abstract

Background/objectives: Developing next-generation mRNA-based seasonal influenza vaccines remains challenging, primarily because of the relatively low immunogenicity of influenza B hemagglutinin (HA) antigens. We describe a systematic vaccine development strategy that combined vector and antigen design optimization.

Methods: Novel untranslated region (UTR) sequences and a hybrid poly(A) tail were used to increase plasmid stability and mRNA expression. Fusion proteins containing HA antigens linked by T4 foldon domains were engineered to enhance the immune responses against influenza B HA antigens and to permit the expression of multiple HA ectodomains from a single mRNA species. The vaccine performance was verified in a traditional encapsulated lipid nanoparticle (LNP) formulation that requires long-term storage at temperatures below -15 °C as well as in a proprietary thermo-stable LNP formulation developed for the long-term storage of the mRNA vaccine at 2-8 °C.

Results: In preclinical studies, our next-generation seasonal influenza vaccine tested alone or as a combination influenza/COVID-19 mRNA vaccine elicited hemagglutination inhibition (HAI) titers significantly higher than Fluzone HD, a commercial inactivated influenza vaccine, across all 2024/2025 seasonal influenza strains, including the B/Victoria lineage strain. At the same time, the combination mRNA vaccine demonstrated superior neutralizing antibody titers to 2023/2024 Spikevax, a commercial COVID-19 comparator mRNA vaccine.

Conclusions: Our data demonstrate that the multimerization of antigens expressed as complex fusion proteins is a powerful antigen design approach that may be broadly applied toward mRNA vaccine development.

Keywords: hemagglutinin; influenza/COVID-19 combination vaccine; multimeric antigens; seasonal influenza mRNA vaccine.

Figure 1

Vaccine development strategy.

Figure 2

Vector optimization. (A) Improved stability of plasmids containing hybrid poly(A)-T₂ sequence: E. coli Stbl3^TM cells were transformed with pLuc-A₁₂₀ or pLuc-A₃₀(T₂A₃₀)₃ plasmids containing homopolymeric poly(A) sequence (A₁₂₀) or hybrid poly(A)-T₂ sequence (A₃₀(T₂A₃₀)₃). Poly(A) tail length was analyzed for individual bacterial clones. (B) In vitro activity of Luc-A₁₂₀ and Luc-A₃₀(T₂A₃₀)₃ mRNA in transfected BHK-21 cells. Luciferase activity was determined in RLU (relative luminescence units). (C) In vivo imaging of Luc-A₁₂₀ and Luc-A₃₀(T₂A₃₀)₃ mRNA expression following i.m. delivery in mice. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) indicate statistically significant differences; not significant (ns).

Figure 3

Bivalent HA antigen design and immunogenicity. (A) Ribbon model of the native trimeric form of the ectodomain of the influenza HA protein; and a graphic showing the mRNA design for monovalent HA antigens, which contain the full HA protein, including transmembrane and cytoplasmic domains. (B) Ribbon model of the bivalent antigen comprising two HA ectodomains linked by T4 foldon and forming a homotrimer and a graphic showing the mRNA encoding the secreted bivalent “dumbbell” HA. Ribbon models are conceptual visual aids describing theoretical structures based on published HA (PDB 5W6R) and T4 foldon (PDB 1RFO) structures. (C–F) Day 35 serum HAI geometric mean titers (GMT) against seasonal influenza viruses. Graph bars represent HAI GMTs of individual mouse sera (N = 6 per group): LNP Control (200 μL); A_H1 + A_H3 + B_V + B_Y vaccine (3 µg each mRNA) expressing monovalent HA antigens from influenza A(H1N1), A(H3N2), B-Victoria and B-Yamagata strains; B_YA_H1+B_VA_H3 vaccine (6 µg each mRNA) expressing bivalent HA antigens with both HA dumbbells including one B- and one A- influenza strain-derived HA ectodomain (B/A design); B_YB_V + A_H3A_H1 vaccine (6 µg each mRNA) expressing bivalent HA antigens with the HA dumbbells including either two B- or two A-strain-derived HA ectodomains (B/B and A/A design). Striped graph bars in (E) indicate GMT elicited against matched 2022/23 (H1N1) seasonal influenza A-strain; the remaining data represent HAI titers against the 2023/24 seasonal strains. Summaries of HAI GMTs and seroconversion rates are included in Table S2A. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) indicate statistically significant differences; not significant (ns).

Figure 4

Optimization of bivalent HA antigen design. (A–D) Day 35 serum HAI GMTs (N = 6 per group) against 2023/24 seasonal influenza viruses were determined for LNP Control (200 μL); Fluad (50 μL) and B_VA_H3 + B_yA_H1 vaccines (5 μg each mRNA) comprising B/A dumbbell HA antigens expressed either as fusion proteins connected through a linker or an extended linker (L or EL), and/or in a secreted or membrane-bound (S or M) antigen format. Summaries of HAI GMTs and seroconversion rates are included in Table S2B. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) indicate statistically significant differences; not significant (ns).

Figure 5

Bi- and trivalent dumbbell antigen designs. (A) Graphic showing the mRNA design for trivalent HA antigens. (B–D) Day 35 serum HAI GMTs (N = 8 per group) against 2024/25 seasonal influenza viruses were determined for LNP Control (200 μL); Fluzone HD (50 μL); B_VA_H3 + A_H3A_H1 influenza vaccines expressing membrane (M) B/A and A/A HA bivalent dumbbells (5 μg each); B_VA_H3A_H1 mRNA vaccines (10 μg) expressing secreted (S) trivalent HA dumbbells. Summaries of HAI GMTs and seroconversion rates are included in p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) indicate statistically significant differences; not significant (ns).

Figure 6

Encapsulated LNP-formulated influenza/COVID-19 combination mRNA vaccine. (A–C) Day 35 serum HAI GMTs (N = 7 per group) against 2024/25 seasonal influenza viruses were determined for LNP Control (200 μL); Spikevax (50 μL); Fluzone HD (50 μL); B_VA_H3A_H1 tested as an influenza-only, or combination vaccine with COV_XBBCOV_XBB mRNA tested at low and high doses (3 μg or 12 μg total mRNA, with a 1:1 mRNA weight ratio of two mRNA species). (D) Average neutralizing antibody titers against SARS-CoV-2 Omicron XBB.1.5 pseudovirus were evaluated in mouse sera of the following vaccination groups: Spikevax (50 μL) and B_VA_H3A_H1 and COV_XBBCOV_XBB mRNA combination vaccine tested both at low and high doses (3 μg or 12 μg total mRNA). Summaries of HAI GMTs and seroconversion rates are included in Table S2D. (E) Average total RBD-specific IgG titers of Day 35 mouse sera were tested for the following vaccine groups: Spikevax (50 μL); B_VA_H3A_H1 with COV_XBBCOV_XBB in both a high and low dose (12 μg or 3 μg total, respectively). (F) Average total RBD-specific IgG1 and IgG2a titers of pooled D35 mouse serum. Numbers above IgG2a bars indicate IgG1/IgG2a ratio. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) indicate statistically significant differences; not significant (ns).

Figure 7

Tri- and tetravalent dumbbell antigen designs for combination influenza/COVID-19 mRNA vaccine. (A) Graphic showing the mRNA design for tetravalent chimeric HA and XBB antigen dumbbells. (B–D) Day 35 serum HAI GMTs (N = 7 per group) against 2024/25 seasonal influenza viruses were determined for LNP Control (200 μL); Spikevax (50 μL); COV_XBBCOV_XBB (6 μg mRNA); B_VA_H3A_H1 tested in combination vaccine with COV_XBBCOV_XBB (12 μg total mRNA, 6 μg each); COV_XBBB_VA_H3A_H1 mRNA vaccine (12 μg) and B_VA_H3A_H1COV_XBB mRNA vaccine (12 μg) expressing secreted tetravalent chimeric dumbbell antigens. Summaries of HAI GMTs and seroconversion rates are included in Table S2E. (E) Average total RBD-specific IgG titers of pooled D35 mouse sera were determined for the same experimental groups. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) indicate statistically significant differences; not significant (ns).