Development of a pentavalent broadly protective nucleoside-modified mRNA vaccine against influenza B viruses

Abstract

Messenger RNA (mRNA) vaccines represent a new, effective vaccine platform with high capacity for rapid development. Generation of a universal influenza virus vaccine with the potential to elicit long-lasting, broadly cross-reactive immune responses is a necessity for reducing influenza-associated morbidity and mortality. Here we focus on the development of a universal influenza B virus vaccine based on the lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) platform. We evaluate vaccine candidates based on different target antigens that afford protection against challenge with ancestral and recent influenza B viruses from both antigenic lineages. A pentavalent vaccine combining all tested antigens protects mice from morbidity at a very low dose of 50 ng per antigen after a single vaccination. These findings support the further advancement of nucleoside-modified mRNA-LNPs expressing multiple conserved antigens as universal influenza virus vaccine candidates.

Fig. 1. Characterization of antigen-encoding mRNAs by flow cytometry or Western blot.

HEK 293 T cells were transfected with HA-, NA- or luciferase control-encoding mRNAs and protein production from each influenza virus antigen-encoding mRNA was assessed via flow cytometry. Positive binding of the antibodies specific for B/Phu HA (a), B/Col HA (b), and B/Col NA (c) relative to luciferase control. HEK 293 T cells were transfected with B/Col NP- or luciferase-encoding mRNAs and protein production from NP-encoding mRNA was assessed via Western blotting. All experiments were performed once. (d) NT non-transfected, L luciferase, GAPDH glyceraldehyde 3-phosphate dehydrogenase. Source data are provided as a source data file.

Fig. 2. Experimental plan: vaccines, immunizations, serology and challenge studies.

Mice received a single I.D. immunization with 5 µg monovalent mRNA-LNP vaccine or 5 µg (1 µg from each component) or 25 µg (5 µg from each component) of the pentavalent mRNA-LNP vaccine. Animals in the negative control group received 5 µg luciferase mRNA-LNP. Four weeks after vaccination, mice were intranasally (I.N.) infected with influenza virus containing 1x or 5x the mLD₅₀. Additionally, mice were bled for serological analysis (ELISA, MNT, NAI, and ADCC reporter assay) at this timepoint (a). For prime-boost vaccination studies mice were vaccinated twice as described with a 4-week interval between vaccinations. Four weeks after the boost, mice were I.N. challenged with 5mLD₅₀ of B/Malaysia/2506/2004 virus. Additionally, mice were bled for serological analysis (ELISA, MNT, NAI, and ADCC reporter assay) at this time point (b).

Fig. 3. Serum reactivity to different IBV antigens.

Mice were vaccinated with a prime only or prime-boost vaccination regimen (28 days apart) I.D. with 5 μg of monovalent mRNA-LNPs or with 5 μg/antigen or 1 μg/antigen of the pentavalent mRNA-LNP formulation. Control animals received 5 μg of luciferase mRNA-LNP. Sera were collected on day 28 post prime and post boost and binding of antibodies to influenza antigens was measured by ELISA. Binding of sera against B/Phuket/307/2013 HA (n = 25 prime-only samples, n = 10 prime-boost samples). (a) B/Colorado/06/2017 HA (n = 25 prime-only samples, n = 10 prime-boost samples). (b) B/Phuket/307/2013 NA (n = 10) (c) B/Colorado/06/2017 NA (n = 25 prime-only samples, n = 10 prime-boost samples) (d) B/Colorado/06/2017 NP (n = 10) (e) and B/Colorado/06/2017 M2 (n = 5) (f). Each symbol represents one animal. AUCs with a cutoff value of the average background plus three SDs are shown. Bars represent the mean of each group. The dotted lines indicate the limit of detection. Source data are provided as a source data file.

Fig. 4. Functional characterization of antibodies against B/Colorado/06/2017 (V) and B/Phuket/3073/2013 (Y).

Mice were vaccinated and sera were collected as described in Fig. 2. Functional characterization of antibodies in the prime-only and prime-boost sera of mice vaccinated with mRNA-LNPs was performed in microneutralization (MNT) (n = 5) (p-values left to right <0.0001, 0.0047, <0.0001, 0.0011, <0.0001, 0.0154) (a), NAI (n = 3) (b), or ADCC reporter (n = 5) (p < 0.0001 for all comparisons) (c) assay against B/Colorado/06/2017 (V) influenza virus. Functional characterization of antibodies in the prime-only and prime-boost sera of mice vaccinated with mRNA-LNPs was performed in MNT (n = 5) (p-values left to right <0.0001, <0.0001, <0.0001, <0.0001) (d), NAI (n = 3) (e), or ADCC reporter (n = 5) (p-values left to right 0.0059, 0.0006, <0.0001, <0.0001, 0.0017, 0.0002, <0.0001, 0.0001) (f) assay against B/Phuket/3073/2013 (Y) influenza virus. In b and e, solid lines show NAI values obtained from prime-only sera, dashed lines show NAI values obtained from prime-boost sera. Sera from 3 to 5 randomly selected animals from each vaccination group was assessed in each assay. For a, c, and f the dotted lines indicate the limit of detection. For a, c, d and f, significance was assessed using a one-way ANOVA and groups were compared to the luciferase control group at the prime-only or prime-boost time point. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For a, c, d and f, bars represent the group mean and error bars represent SD. Each symbol represents an individual animal. For b and e, data are presented as group mean at each sera dilution and error bars represent SD. Source data are provided as a source data file.

Fig. 5. Cellular immune responses induced by IBV mRNA-LNP vaccines.

Mice were vaccinated I.D. with a single dose of 5 μg of NP, NA, HA or control luciferase mRNA-LNPs. Splenocytes collected from immunized animals 10 days after immunization were stimulated with NP or NA or HA overlapping peptide pools, and cytokine production by CD4⁺ and CD8⁺ T cells was assessed by flow cytometry (a). Percentages of B/Col NP- (p-values left to right 0.0023, <0.0001, 0.028, <0.0001, 0.0004, 0.0478) (b), B/Col NA- (p-values left to right <0.0001, <0.0001, 0.0014, <0.0001, 0.0001, 0.2702) (c), B/Col HA- (p-values left to right 0.1794, 0.0002, 0.0047, 0.0037, 0.0023, 0.1322) (d) and B/Phu HA- (p-values left to right 0.026, <0.0001, 0.0031, 0.0006, 0.0003, 0.5177) (e) specific CD4⁺ and CD8⁺ T cells producing IFN-γ, IL-2 and TNF-α are shown. Each symbol represents one animal and error is shown as SEM (n = 8 mice per group for B/Col NP and n = 7 for B/Col NA, B/Col HA, B/Phu HA and luciferase). Statistical analysis: two-tailed unpaired t-test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗p < 0.0001. Source data are provided as a source data file.

Fig. 6. Morbidity and survival graphs for mRNA-LNP-vaccinated mice following I.N. challenge with ancestral through recent IBV strains.

Morbidity of mice vaccinated with a single dose of mRNA-LNPs containing monovalent or pentavalent formulations following I.N. challenge with 5mLD₅₀ of B/Lee/1940 (n = 5) (a) B/Malaysia/2506/2004 (V) (n = 5) (b), B/New York City/PV01181/2018 (V) (n = 5 for all groups except M2 where one mouse was found dead on day 1) (c), B/Florida/04/2006 (Y) (n = 5) (d), or B/New York City/PV00094/2017 (Y) (n = 4) (e) influenza viruses 4 weeks after immunization. Data are shown as the group mean and error bars represent SD. Survival of mice vaccinated with a single dose of mRNA-LNPs containing monovalent or pentavalent formulations following I.N. challenge with 5mLD₅₀ of B/Lee/1940 (f), B/Malaysia/2506/2004 (V) (g), B/New York City/PV01181/2018 (V) (h), B/Florida/04/2006 (Y) (i), or B/New York City/PV00094/2017 (Y) (j) influenza viruses 4 weeks after immunization. Source data are provided as a source data file.

Fig. 7. A single immunization with IBV pentavalent mRNA-LNP vaccine prevents virus replication.

Mice were I.D. vaccinated once with 5 μg of monovalent mRNA-LNPs or with 5 μg/antigen or 1 μg/antigen of the pentavalent mRNA-LNP formulation. Control animals received 5 μg of luciferase mRNA-LNP. Virus titers in the lung homogenate of mRNA-LNP-vaccinated mice at 3 and 6 dpi following I.N. challenge with 1mLD₅₀ of B/New York City/PV01181/2018 (V) (n = 3 mice/group) (a). Virus titers in the lung homogenate of mRNA-LNP-vaccinated mice at 3 and 6 dpi following I.N. challenge with 1mLD₅₀ of B/New York City/PV00094/2017 (Y) (n = 3 mice/group) (b). Each symbol represents one animal and the average is presented as the group mean. The dotted lines indicate the limit of detection. Source data are provided as a source data file.

Fig. 8. A single immunization with the pentavalent IBV mRNA-LNP vaccine protects mice in the nanogram dose range.

Mice were I.D. vaccinated once with 5, 0.5, 0.05, 0.005, or 0.0005 µg of each antigen in the pentavalent mRNA-LNP formulation. Four weeks later sera were collected and antibody responses towards B/Colorado/06/2017 (V) influenza virus-infected cells were assessed (p-values from left to right <0.0001, <0.0001) (a). Each symbol represents one animal, and the bar represents the mean. Mice were then I.N. infected with 5mLD₅₀ of B/Malaysia/2506/04 (V) influenza virus and morbidity (b) and survival (c) were monitored for 14 dpi. Data are shown as individual data points and the group average presented as the group mean (n = 5 per group). Significance was assessed using a one-way ANOVA and groups were compared to the luciferase control group at each time point. The dotted line in b indicates the maximum body weight loss (25%) for the experiment. Source data are provided as a source data file.

Fig. 9. Humoral protection from IBV challenge is afforded by antibodies that target glycoproteins.

Mice were vaccinated twice (with a 4-week interval) I.D. with 5 µg of mRNA-LNPs. Sera were collected from vaccinated animals 4 weeks after the boost and then assessed for antibody reactivity towards B/Colorado/06/2017 (V) influenza virus-infected cells (n = 10 mice/group) (a). Sera from vaccinated mice were transferred into naïve mice. Two hours after the transfer, sera from naïve recipients of passive sera were assessed for antibody reactivity towards B/Colorado/06/2017 (V) influenza virus-infected cells (n = 5 mice/group) (b). For a and b bars represent the mean of each group and each point represents an individual animal. Error bars are representative of SD. One mouse was excluded from the NP group due to failed serum transfer. Naïve mice were then I.N. challenged with 5mLD₅₀ of B/New York City/PV01181/2018 (V) influenza virus and weight loss was monitored for 14 days. Data are shown as mean and error bars represent SD (n = 5 per group) (c). The dotted line in c indicates the maximum body weight loss (25%) for the experiment. Source data are provided as a source data file.