Nucleoside-modified mRNA immunization elicits influenza virus hemagglutinin stalk-specific antibodies

Abstract

Currently available influenza virus vaccines have inadequate effectiveness and are reformulated annually due to viral antigenic drift. Thus, development of a vaccine that confers long-term protective immunity against antigenically distant influenza virus strains is urgently needed. The highly conserved influenza virus hemagglutinin (HA) stalk represents one of the potential targets of broadly protective/universal influenza virus vaccines. Here, we evaluate a potent broadly protective influenza virus vaccine candidate that uses nucleoside-modified and purified mRNA encoding full-length influenza virus HA formulated in lipid nanoparticles (LNPs). We demonstrate that immunization with HA mRNA-LNPs induces antibody responses against the HA stalk domain of influenza virus in mice, rabbits, and ferrets. The HA stalk-specific antibody response is associated with protection from homologous, heterologous, and heterosubtypic influenza virus infection in mice.

Fig. 1

Nucleoside-modified HA mRNA-LNP immunization elicits potent neutralizing antibody responses in mice. Mice received two i.d. (3, 10, or 30 µg) or i.m. (10, 30, or 90 µg) immunizations of A/California/07/2009 HA mRNA-LNPs or 30 (i.d.) or 90 (i.m.) µg of poly(C) RNA-LNPs at week 0 (prime) and 4 (boost). HA inhibition (HAI) titers against A/California/07/2009 (a, b) and A/Puerto Rico/8/1934 (c, d) viruses were determined at week 4 and 8. n = 3–10 mice and each symbol represents one animal. Two independent experiments were performed. Horizontal lines show the mean; dotted line indicates the limit of detection. Statistical analysis: a, b two-way ANOVA with Bonferroni correction on log-transformed data, p < 0.05; all comparisons across doses and time points were statistically significant except for (a) 10 µg HA prime vs. 30 µg HA prime and 10 µg HA boost vs. 30 µg HA boost and for (b) 10 µg HA boost vs. 90 µg HA boost, 30 µg HA prime vs. 90 µg HA prime and 30 µg HA boost vs. 90 µg HA boost

Fig. 2

Nucleoside-modified HA mRNA-LNP immunization elicits HA stalk-specific antibody responses in mice. Mice received two i.d. (3, 10, or 30 µg) (a, b) or i.m. (10, 30, or 90 µg) (c, d) immunizations of A/California/07/2009 HA mRNA-LNPs or 30 (i.d.) or 90 (i.m.) µg of poly(C) RNA-LNPs at weeks 0 and 4. IgG binding to full-length H1 HA (a, c) and cH6/1 HA (b, d) proteins was determined at week 4 and week 8. n = 3–10 mice. Two independent experiments were performed. Error bars are SEM. Statistical analysis: a, b two-way ANOVA with Bonferroni correction, *p < 0.05 comparing A/California/07/2009 HA mRNA-LNP dose groups at each serum dilution, black star: significant difference in titers between the 3 and 30 µg groups, purple star: significant difference in titers between the 3 and 10 µg groups, green star: significant difference in titers between the 10 and 30 µg groups; c, d two-way ANOVA with Bonferroni correction, *p < 0.05 comparing A/California/07/2009 HA mRNA-LNP dose groups at each serum dilution, blue star: significant difference in titers between the 10 and 90 µg groups, green star: significant difference in titers between the 10 and 30 µg groups, gray star: significant difference in titers between the 30 and 90 µg groups

Fig. 3

Nucleoside-modified HA mRNA-LNP immunization elicits durable HA stalk-specific antibody responses in mice. Mice received a single i.d. dose of 30 µg of A/Puerto Rico/8/1934 HA mRNA-LNP vaccine and a HAI titers against A/Puerto Rico/8/1934 and IgG binding to full-length H1 HA (b) and cH5/1 HA (c) proteins in mouse serum obtained 28 and 238 days post single immunization were determined. n = 8 mice. a Each symbol represents one animal, horizontal lines show the mean, dotted line indicates the limit of detection. b, c Error bars are SEM. Statistical analysis: a two-way ANOVA with Bonferroni correction on log-transformed data, p < 0.05; all comparisons between the Luc and the day 28 and day 238 Puerto Rico/8/1934 HA groups were statistically significant. b, c Two-way ANOVA with Bonferroni correction comparing Puerto Rico/8/1934 HA day 28 and day 238 time points for different dilutions. *p < 0.05

Fig. 4

Nucleoside-modified HA mRNA-LNP immunization induces HA stalk-reactive antibodies in ferrets. Ferrets were immunized two times i.m. with 60 µg of A/California/07/2009 HA mRNA-LNPs or 60 µg of poly(C) RNA-LNPs at week 0 (prime) and 4 (boost). HAI titers against the A/California/07/2009 (a) and A/swine/Jiangsu/40/2011 (b) viruses were determined at week 4 (prime) and week 13 (boost). c IgG binding to full-length H1 HA (total) and cH6/1 HA (stalk) proteins from serum samples obtained 9 weeks after the second immunization was determined. n = 12 ferrets. a, b Each symbol represents one animal, horizontal lines show the mean, dotted line indicates the limit of detection. c error bars are SEM. Statistical analysis: a, b one-way ANOVA with Bonferroni correction on log-transformed data, *p < 0.05. c Two-way ANOVA with Bonferroni correction comparing A/California/07/2009 HA and poly(C) immunizations for different dilutions. *p < 0.05

Fig. 5

Two immunizations with nucleoside-modified A/California/07/2009 HA mRNA-LNP vaccine elicits protection from A/California/07/2009 and Puerto Rico/8/1934 viruses. Mice received two i.d. (3, 10, or 30 µg) or i.m. (10, 30, or 90 µg) immunizations of A/California/07/2009 HA mRNA-LNPs or 30 (i.d.) or 90 (i.m.) µg of poly(C) RNA-LNPs at week 0 (prime) and 4 (boost). Animals were challenged with lethal doses of homologous A/California/07/2009 (a, b) or heterologous A/Puerto Rico/8/1934 viruses (c, d) 5 weeks after the second immunization and weight loss and survival were followed. Two independent experiments were performed. n = 5 mice and each weight loss line represents one animal

Fig. 6

Nucleoside-modified A/California/07/2009 HA mRNA-LNP vaccine elicits protection from A/California/07/2009 and A/Puerto Rico/8/1934 viruses after a single immunization. Mice received a single i.m. dose of 30 µg A/California/07/2009 HA mRNA-LNPs. Control animals were vaccinated i.m. with a single dose of 3 µg of monovalent A/California/07/2009 virus vaccine or 30 µg of poly(C) RNA-LNPs. HAI titers against the A/California/07/2009 and A/Puerto Rico/8/1934 virus (a) were determined 28 days post single immunization. Animals were challenged with lethal doses of homologous A/California/07/2009 (b) or heterologous A/Puerto Rico/8/1934 (c) viruses 28 days after immunization and weight loss and survival was followed. n = 5 mice. a Horizontal lines show the mean; dotted line indicates the limit of detection. b, c Each weight loss line represents one animal. Statistical analysis: a one-way ANOVA with Bonferroni correction on log-transformed data, *p < 0.05; b two-way ANOVA with Bonferroni correction on weight loss graphs comparing A/California/07/2009 mRNA-immunized animals to inactivated virus-immunized animals. p < 0.05 on days 4–6

Fig. 7

Nucleoside-modified A/California/07/2009 HA mRNA-LNP vaccine elicits protection from the A/Vietnam/1203/04 (H5N1) virus after two immunizations. Mice received two i.d. doses of 30 µg of A/California/07/2009 HA mRNA-LNPs at weeks 0 and 4. Control animals were vaccinated with two i.d. doses of 30 µg of poly(C) RNA-LNPs or two i.m. doses of 1 µg of inactivated H5N1 virus. (a) Animals were challenged with a lethal dose of A/Vietnam/1203/04 virus 28 days after the second immunization and weight loss and survival were followed. n = 10 mice. HAI titers (b) and in vitro microneutralization activity (c) against the A/Vietnam/1203/04 virus were determined 28 days after the first and 28 days after the second immunization. Pooled serum samples were used for MN assays. a Each weight loss line represents one animal. b, c Horizontal lines show the mean; dotted line indicates the limit of detection. Statistical analysis: a two-way ANOVA with Bonferroni correction on weight loss graphs comparing A/California/07/2009 mRNA-immunized animals to inactivated virus-immunized animals, p < 0.05 on days 5–7. b Unpaired t-test comparing post prime and post boost samples, *p < 0.05. c Two-way ANOVA with Bonferroni correction, *p < 0.05