A Multi-Targeting, Nucleoside-Modified mRNA Influenza Virus Vaccine Provides Broad Protection in Mice

Abstract

Influenza viruses are respiratory pathogens of public health concern worldwide with up to 650,000 deaths occurring each year. Seasonal influenza virus vaccines are employed to prevent disease, but with limited effectiveness. Development of a universal influenza virus vaccine with the potential to elicit long-lasting, broadly cross-reactive immune responses is necessary for reducing influenza virus prevalence. In this study, we have utilized lipid nanoparticle-encapsulated, nucleoside-modified mRNA vaccines to intradermally deliver a combination of conserved influenza virus antigens (hemagglutinin stalk, neuraminidase, matrix-2 ion channel, and nucleoprotein) and induce strong immune responses with substantial breadth and potency in a murine model. The immunity conferred by nucleoside-modified mRNA-lipid nanoparticle vaccines provided protection from challenge with pandemic H1N1 virus at 500 times the median lethal dose after administration of a single immunization, and the combination vaccine protected from morbidity at a dose of 50 ng per antigen. The broad protective potential of a single dose of combination vaccine was confirmed by challenge with a panel of group 1 influenza A viruses. These findings support the advancement of nucleoside-modified mRNA-lipid nanoparticle vaccines expressing multiple conserved antigens as universal influenza virus vaccine candidates.

Keywords: influenza virus; lipid nanoparticle; mRNA; nucleoside-modification; universal vaccine; vaccine.

Graphical abstract

Figure 1

mRNA-Lipid Nanoparticle Vaccine Platform Utilized for the Delivery of a Combination of Conserved Influenza Virus Antigens (A) Schematic representation of the mRNA-lipid nanoparticle vaccine technology that incorporates a 1-methylpseudouridine-modified mRNA molecule into an 80 lipid nanometer vesicle for efficient delivery into host cells upon vaccination. (B) Diagrams illustrating the antigens used as immunogens in this study. Amino acid numbers are included under the mRNA coding for each antigen. Not drawn to scale.

Figure 2

Nucleoside-Modified mRNA-LNP Vaccines Encoding Conserved Influenza Virus Antigens Elicit Robust Immune Responses in Mice (A) Mice were vaccinated intradermally once with either monovalent or combined mRNA vaccines (20 μg per antigen). Sera were collected on day 28 post-vaccination, and binding of antibodies to corresponding antigen was measured by ELISA. (B–D) Mean optical density at 490 nm is plotted with SD for each dilution (n = 19–20 individual sera per group) against (B) Mini HA, (C) NA (Mich15), and (D) NP (Mich15). (E) Cell-based ELISAs were utilized to detect antibody binding to M2 (Mich15). Mean optical density at 490 nm is plotted with SD for each dilution displayed with SD (n = 4 repeats of pooled sera). (F) Endpoint titers of a multi-cycle microneutralization assay to determine the neutralization potential of antibodies elicited by vaccination. Sera from mice taken 4 weeks after vaccination with 1.5 μg of the 2018–2019 quadrivalent influenza virus vaccine (QIV) were included in this assay. Sera were pooled and run in duplicate against H1N1pdm virus. (G) ADCC activities of sera were measured using a reporter assay to determine engagement with the mouse FcγRIV. The positive control used was anti-influenza A group 1 monoclonal antibody KB2. Luminescence was measured and data from pooled sera run in triplicate are represented as fold change over background (average of negative wells plus 3 times the SD, indicated as a dashed line) with SD. Curves were fit using a nonlinear regression formula log(agonist) versus response minus variable slope (four parameters).

Figure 3

Vaccination with a Combination of Nucleoside-Modified mRNA-LNP-encoded Influenza Virus Antigens Protects Mice from a Highly Lethal Dose of Matched Challenge Virus (A) Sera collected 28 days after mRNA-LNP vaccination were measured against H1N1pdm virus. Individual data are represented as AUC with lines indicating mean and SD of responses (n = 19–20 per group). (B–D) Mice were challenged with (B) 5 × LD₅₀, (C) 50 × LD₅₀, or (D) 500 × LD₅₀ of H1N1pdm and weight loss was monitored for 14 days. Data are shown as mean and SEM (n = 5 per group). Mortality is reported as the percentage of surviving mice for each group.

Figure 4

Nucleoside-Modified mRNA-LNP-Vaccine-Induced Protection from Influenza Virus Challenge Is Mediated Primarily by the Humoral Arm of the Immune System (A) Mice were vaccinated twice (4-week intervals) intradermally with 10 μg of mRNA-LNPs. Animals were euthanized on day 56 after initial vaccination and sera were collected and transferred into naive mice. Two hours after transfer, recipient mice were infected with 5 × LD₅₀ of H1N1pdm (IVR-180) and weight loss was monitored for 14 days. (B) ELISAs were performed to measure the ELISA reactivity of sera from hyper-immune mice to H1N1pdm before transfer (n = 9–10 per group). Lines indicate mean and SD. (C) Sera were pooled, transferred into naive mice, and reactivity to H1N1pdm was measured by ELISA from sera taken 2 h after transfer (n = 5 per group). Lines indicate mean and SD. (D) Weight loss curves of mice that received hyper-immune sera. Average weight loss with SEM is plotted (n = 5 per group). Mortality is reported as the percentage of surviving mice for each group.

Figure 5

Nucleoside-Modified Neuraminidase and Nucleoprotein mRNA-LNP Vaccines Elicit Robust Antigen-Specific T Cell Responses in Mice (A) Mice were vaccinated intradermally with a single dose of 20 μg of NA or NP mRNA-LNPs. Splenocytes were stimulated with NA or NP peptides 12 days after immunization, and cytokine production by CD4⁺ and CD8⁺ T cells was assessed by flow cytometry. Percentages of NA-specific (B) CD4⁺ and (C) CD8⁺ T cells producing IFN-γ, TNF-α, and IL-2 and frequencies of combinations of cytokines produced by (D) CD4⁺ and (E) CD8⁺ T cells are shown. Percentages of NP-specific (F) CD4⁺ and (G) CD8⁺ T cells producing IFN-γ, TNF-α, and IL-2 and frequencies of combinations of cytokines produced by (H) CD4⁺ and (I) CD8⁺ T cells are shown. Values from NA- and NP-immunized mice are compared to values from Luc-immunized animals for each cytokine combination (D, E, H, and I). Each symbol represents one animal and error is shown as SEM (n = 10 mice per group). Data from two independent experiments are shown. Statistical analysis: Mann-Whitney test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 6

The Combination of Nucleoside-Modified mRNA-LNP-Encoded Influenza Virus Antigens Enhances Porteection of NA-Mediated Immunity in the Nanogram Range Serum from mice vaccinated with a single intradermal dose of 5, 0.5, 0.05, or 0.005 μg of nucleoside-modified mRNA-LNPs of either (A) NA alone or (B) supplemented with Mini HA, M2, and NP constructs additively (combination) were tested against H1N1pdm in ELISA assays. Luciferase mRNA-LNP was used as a negative control at a dose of 5 μg, and quadrivalent inactivated influenza virus vaccine (QIV) was used as a standard of care control at a dose of 1.5 μg. Data are represented as AUC with the mean and SD plotted. (C and D) Mice were infected with 5 × LD₅₀ of H1N1pdm virus and body weight was monitored for 14 days. Weight loss curves after infection for mice vaccinated with NA alone or a combination of antigens. Luciferase and QIV groups are shown in both graphs. Mean plus SEM is plotted for each group (n = 5 per group). Mortality is reported as the percentage of surviving mice for each group.

Figure 7

A Single Immunization with a Combination of Nucleoside-Modified mRNA-Encoded Influenza Virus Antigens Protects Mice from Heterologous Challenge Twenty-eight days after a single intradermal vaccination with 20 μg of mRNA-LNPs, mice were bled and challenged with 5 × LD₅₀ of influenza virus. Weight loss was monitored for 14 days for challenge viruses: (A) A/New Caledonia/20/1999 H1N1 virus (n = 5 per group), (B) A/Puerto Rico/8/1934 H1N1 virus (n = 4–5 per group), (C) H5N8 virus (n = 5 per group), and (D) cH6/1N5 virus (n = 5 per group). Means and SEM are shown for weight loss curves. Mortality is reported as the percentage of surviving mice for each group. (E) Summarized maximum weight loss of all challenge experiments at 5 × LD₅₀ for each respective virus is represented. Mean plus SEM is plotted for each group. Statistical analysis: two-way ANOVA with Dunnett’s correction for multiple comparisons, ∗p < 0.0332, ∗∗p < 0.0021, ∗∗∗p < 0.0002, ∗∗∗∗p < 0.0001.