Assessment of a quadrivalent nucleoside-modified mRNA vaccine that protects against group 2 influenza viruses

Abstract

Combined vaccine formulations targeting not only hemagglutinin but also other influenza virus antigens could form the basis for a universal influenza virus vaccine that has the potential to elicit long-lasting, broadly cross-reactive immune responses. Lipid nanoparticle (LNP)-encapsulated messenger RNA (mRNA) vaccines can be utilized to efficiently target multiple antigens with a single vaccine. Here, we assessed the immunogenicity and protective efficacy of nucleoside-modified mRNA-LNP vaccines that contain four influenza A group 2 virus antigens (hemagglutinin stalk, neuraminidase, matrix protein 2, and nucleoprotein) in mice. We found that all vaccine components induced antigen-specific cellular and humoral immune responses after administration of a single dose. While the monovalent formulations were not exclusively protective, the combined quadrivalent formulation protected mice from all challenge viruses, including a relevant H1N1 influenza virus group 1 strain, with minimal weight loss. Importantly, the combined vaccine protected from morbidity at a dose of 125 ng per antigen after a single vaccination in mice. With these findings, we confidently conclude that the nucleoside-modified mRNA-LNP platform can be used to elicit protection against a large panel of influenza viruses.

Keywords: T cells; influenza virus; lipid nanoparticle; mRNA vaccine; nucleoside modification.

Fig. 1.

Assessment of binding to vaccine antigens in the prime-only and prime-boost sera using ELISA. Mice were vaccinated once or twice (4 wk apart) ID with 5 μg of monovalent mRNA-LNP or with 5 μg/antigen or 1.25 μg/antigen of the quadrivalent mRNA-LNP formulation. Negative control animals received 5 μg of Luc mRNA-LNP. Sera were collected on day 28 post single- or double-shot vaccination and binding of antibodies to A/Singapore/INFIMH-16-0019/2016 (H3N2) HA (A) NA (B), NP (C) and M2 (D) was assessed using ELISA. Each symbol represents one animal, sera from 5 to 20 animals were assessed. AUCs with a cutoff value of the average background plus three SDs are shown. Bars represent the geometric mean for each group and error bars depict SD. Statistical significance was calculated using one-way ANOVA and groups were compared to the Luc control for their respective vaccination regimen (prime-only/prime-boost vaccination). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2.

Determination of functional antibody responses in the prime-only and prime-boost sera. Mice were vaccinated once or twice (4 wk apart) ID with 5 μg of monovalent mRNA-LNP or with 5 μg/antigen or 1.25 μg/antigen of the quadrivalent mRNA-LNP formulation. Negative control animals received 5 μg of Luc mRNA-LNP. Functional characterization of antibodies in the sera of vaccinated mice was assessed using MNT (A), NI (B) and ADCC reporter (C) assay against the A/Singapore/INFIMH-16-0019/2016 (H3N2, IVR-186) virus. MNT data are presented as endpoint titers of five randomly selected animals. ADCC data are presented as AUCs with a cutoff value of the average background plus five SDs. For MNT and ADCC data, bars represent the geometric mean for each group and error bars depict SD. Statistical significance was calculated using one-way ANOVA and groups were compared to the Luc control for their respective vaccination regimen (single/double vaccination). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NI data are presented as inhibition calculated against a virus-only control. Each data point represents inhibition of sera from five randomly selected animals and error bars are representative of SD. Complete lines represent data obtained from prime-only sera, whereas dashed lines represent data obtained from prime-boost sera.

Fig. 3.

Cellular immune responses induced by influenza group 2 mRNA-LNP vaccines. Mice were vaccinated ID with a single dose of 5 μg of H3ss-TM (HA), NA, NP, M2, or control luciferase mRNA-LNPs. Splenocytes collected from immunized animals 10 d after immunization were stimulated with NP or M2-specific immunodominant MHC class I-restricted and MHC class II-restricted peptides, respectively, or NA or HA overlapping peptide pools, and cytokine production by CD4⁺ and CD8⁺ T cells was assessed by flow cytometry. (A) Schematic illustration of the experiment. (B) Percentages of cytokine producing HA-specific CD4⁺ T cells. (C) Percentages of cytokine producing NA-specific CD4⁺ (Upper panels) and CD8⁺ (Lower panels) T cells. (D) Percentages of cytokine producing NP-specific CD8⁺ T cells. (E) Percentages of cytokine producing M2-specific CD4⁺ T cells. Each symbol represents one animal and error is shown as SEM (n = 6 to 9 mice per group). Statistical analysis: two-tailed unpaired t test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4.

A prime-only vaccination with the quadrivalent mRNA-LNP formulation protects mice from challenge with influenza virus. Mice were ID vaccinated once with monovalent or quadrivalent mRNA-LNP as described in Fig. 1. Four weeks later, animals were IN challenged with 5mLD₅₀ of group 2 influenza viruses and morbidity and mortality were assessed. Morbidity and mortality in mice challenged with A/Switzerland/9715293/2013 (H3N2) (A), A/Philippines/2/1982 (H3N2, A/PR/8/1934 reassortant) (B), A/duck/Czechoslovakia/1956 (H4N6, A/PR/8/1934 reassortant) (C) or A/Shanghai/1/2013 (H7N9, A/PR/8/1934 reassortant) (D) influenza viruses. Survival is also represented in parentheses. Data are shown as mean and error bars represent SD (n = 5 per group).

Fig. 5.

Humoral protection from group 2 influenza virus challenge is afforded by antibodies that target the NA. Mice were vaccinated twice (4-wk intervals) ID with 5 µg of monovalent or quadrivalent mRNA-LNPs. Sera were collected from vaccinated animals 4 wk after the boost and the sera were assessed for antibody reactivity toward A/Singapore/INFIMH-16-0019/2016 (H3N2, IVR-186) influenza virus-infected cells by cell-based ELISA (A). Sera from vaccinated mice were then transferred into naïve mice. Two hours after the transfer, sera from passively transferred naïve mice were assessed for antibody reactivity toward A/Singapore/INFIMH-16-0019/2016 (H3N2, IVR-186) influenza virus-infected cells by ELISA (B). Each symbol represents one animal, sera from 5 to 10 animals were assessed. AUCs with a cutoff value of the average background plus three SDs are shown. Bars represent the geometric mean for each group and error bars depict SD. Naïve mice were then I.N. challenged with 5mLD₅₀ of A/Switzerland/9715293/2013 (H3N2) and weight loss (C) and survival (D) were monitored for 14 d. Survival is also represented in parentheses in panel D. Data are shown as mean and error bars represent SD (n = 4 or 5 per group). One mouse passively transferred NP sera was excluded due to a failed transfer.

Fig. 6.

A single immunization with the quadrivalent mRNA-LNP formulation protects mice in the nanogram range. Mice were ID vaccinated with 5, 0.5, 0.05, or 0.005 µg of the quadrivalent mRNA-LNP formulation. Twenty-eight days later sera were collected and antibody response toward A/Singapore/INFIMH-16-0019/2016 (H3N2, IVR-186) influenza virus-infected cells was assessed by cell-based ELISA (A). Each symbol represents one animal, sera from five animals were assessed. AUCs with a cutoff value of the average background plus three SDs are shown. Bars represent the geometric mean for each group and error bars depict SD. Statistical significance was calculated using a one-way ANOVA and groups were compared to the Luc control for their respective vaccination regimen (prime-only/prime-boost vaccination). **P < 0.01, ****P < 0.0001. Mice were then I.N. infected with 5mL₅₀ A/Switzerland/9715293/2013 (H3N2) influenza virus and morbidity (B) and mortality (C) were monitored for 14 d post challenge. Survival is also represented in parentheses in panel C. Data are shown as mean and error bars represent SD (n = 5 per group).