Multivalent next generation influenza virus vaccines protect against seasonal and pre-pandemic viruses

Abstract

Each year, new influenza virus vaccine formulations are generated to keep up with continuously circulating and mutating viral variants. A next-generation influenza virus vaccine would provide long-lasting, broadly-reactive immune protection against current and future influenza virus strains for both seasonal and pre-pandemic viruses. Next generation immunogens were designed using computationally optimized broadly reactive antigen (COBRA) methodology to protect against a broad range of strains over numerous seasons. Novel HA and NA amino acid sequences were derived from multilayered consensus sequence alignment for multiple subtypes of influenza. This multivalent formulation was hypothesized to elicit broadly protective immune responses against both seasonal and pre-pandemic influenza viruses. Mice were vaccinated with multivalent mixtures of HA and NA (H1, H2, H3, H5, H7, N1, N2) proteins. Multivalent COBRA vaccinations elicited antibodies that recognized a broad panel of strains and vaccinated mice were protected against viruses representing multiple subtypes. This is a promising candidate for a universal influenza vaccine that elicits protective immune responses against seasonal and pre-pandemic strains over multiple seasons.

Figure 1

Serum HAI antibody titers for seasonal influenza viruses after vaccination in mice. Immunologically naïve DBA/2J mice were vaccinated intramuscularly (i.m.) three times at four-week intervals with quadrivalent (left) or heptavalent (right) formulations of COBRA recombinant HA and NA proteins, with AddaVax™ as adjuvant. Sera collected two weeks after final vaccination were used for HAI assay against a panel of (a, b) H1N1 and (c, d) H3N2 viruses. The virus strains are listed along the x-axis. The y-axis indicates the log₂ HAI titers for each vaccinated group and presents them as absolute mean values ± SEM. The dotted lines indicate HAI titers ranging from 1:40 (lower line) and 1:80 (upper line). Statistical differences between quadrivalent and heptavalent vaccine groups were analyzed using multiple unpaired t-tests by GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p value of less than 0.05 was defined as statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Figure 2

Serum HAI antibody titers for pre-pandemic influenza viruses after vaccination in mice. Immunologically naïve DBA/2J mice were vaccinated intramuscularly (i.m.) three times at four-week intervals with heptavalent formulations of COBRA recombinant HA and NA proteins, with AddaVax™ as adjuvant. Sera collected two weeks after final vaccination were used for HAI assay against a panel of (a) H2Nx, (b) H5Nx, and (c) H7Nx. The virus strains are listed along the x-axis. The y-axis indicates the log₂ HAI titers for each vaccinated group and presents them as absolute mean values ± SEM. The dotted lines indicate HAI titers ranging from 1:40 (lower line) and 1:80 (upper line).

Figure 3

Serum NAI antibody titers after vaccinations in mice. Immunologically naïve DBA/2J mice were vaccinated intramuscularly (i.m.) three times at four-week intervals with quadrivalent (green) or heptavalent (blue) formulations of COBRA recombinant HA and NA proteins, with AddaVax™ as adjuvant. Sera collected two weeks after final vaccination were used for NAI assay against (a, b) N1 and (c, d) N2. Sera were diluted two-fold from starting dilution of 1:20 to quantify NAI titer (a, c). Non-linear regression was conducted from these results to obtain reciprocal titer that inhibited 50% of the NA activity (b, d). Statistical differences between NAI₅₀ titers were analyzed by unpaired t-tests by GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p value of less than 0.05 was defined as statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Figure 4

Neutralizing antibody titers for seasonal influenza viruses after vaccination in mice. Immunologically naïve DBA/2J mice were vaccinated intramuscularly (i.m.) three times at four-week intervals with quadrivalent (green line) or heptavalent (blue line) formulations of COBRA recombinant HA and NA proteins, with AddaVax™ as adjuvant. Sera collected two weeks after final vaccination were pooled for FRA against modern strains of (a, b) H1N1 and (c, d) H3N2 viruses. Top dashed line represents 50% neutralization, bottom dashed line represents 80% neutralization.

Figure 5

Protective efficacy against seasonal virus infection in vaccinated mice. Naïve DBA/2J mice (n = 11 per group) were vaccinated i.m. three times at four-week intervals with quadrivalent (green) or heptavalent (blue) formulations of COBRA recombinant HA and NA proteins, with AddaVax™ as adjuvant. Four weeks after final vaccination, mice were intranasally infected with (a, b) lethal dose of A/Brisbane/02/2018 (3 × 10⁶ PFU), (c, d) A/Kansas/14/2017 (6 × 10⁶ PFU), or (e, f) mouse-adapted A/Switzerland/2013 (1 × 10⁵ PFU). The animals were observed for clinical signs and their body weight was recorded daily post infection. Statistical differences after infection with H3N2 was analyzed by multiple unpaired t-tests by GraphPad Prism 9 software (GraphPad, San Diego, CA, USA). A p value of less than 0.05 was defined as statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Figure 6

Lung viral titers after seasonal challenge. Lungs were taken on days 2 or 3 (n = 3) and day 6 (n = 3) post infection with Bris/18 (a), KS/17 (b), or MA_Switz/13 (c). Viral titers in lung tissues are presented as PFU/g on the y-axis.

Figure 7

Protective efficacy against pre-pandemic virus infection in vaccinated mice. Immunologically naïve DBA/2J mice (n = 11 per group) were vaccinated i.m. three times at four-week intervals heptavalent (blue line) formulation of COBRA recombinant HA and NA proteins, with AddaVax™ as adjuvant. Four weeks after final vaccination, mice were intranasally infected with (a, b) lethal dose of A/Sichuan/26221/2014 (10⁶ PFU) or (c, d) lethal dose of A/Anhui/01/2013 (10² PFU), or (e, f) A/mallard/Minnesota/AI08-3437/2008 (10⁴ PFU) in 50 µL volume. The animals were observed for clinical signs and their body weight was recorded daily post infection.

Figure 8

Lung viral titers after pre-pandemic challenge. Lungs were taken on day 3 (n = 3) and day 6 (n = 3) post infection with Sich/14 (a), Anhui/17 (b), or mal/MN/08 (c). Viral titers in lung tissues are presented as PFU/g shown on the y-axis.