A Single Immunization with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in Mice

Abstract

SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality associated with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full-length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4⁺ and CD8⁺ T cell responses, as well as long-lived plasma and memory B cell responses. Additionally, we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro. Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19.

Keywords: COVID-19; SARS-CoV-2; mRNA vaccine; mRNA-LNP; nucleoside-modified mRNA.

Graphical abstract

Figure 1

In Vitro Characterization of SARS-CoV-2 Nucleoside-Modified mRNA Constructs (A) Supernatant from 293F cells transfected with RBD-encoding mRNA or mock was tested for binding reactivity to D001 and hACE2-Fc by ELISA. Data shown are area under curve of the log-transformed concentrations (log AUC). Symbols represent independent experiments. (B) 293F cells were transfected with mRNA encoding SARS-CoV-2 full-length WT and Δfurin S protein. Binding reactivity of full-length WT and Δfurin S proteins to D001, hACE2-Fc, and negative control CH65 (an anti-influenza neutralizing antibody) was measured by flow cytometry. Binding capacity was expressed in mean fluorescence intensity (MFI). Each dot represents an independent experiment. p value indicates a paired t test; ^∗p < 0.05. Data represent mean plus SEM.

Figure 2

SARS-CoV-2 mRNA Vaccines Induce S Protein-Specific Type 1 Cellular Responses BALB/c mice were vaccinated i.m. with a single dose of 30 μg of mRNA-LNP vaccines. (A–C) Spleen and lungs were harvested and stimulated with SARS-CoV-2 S protein peptide pools 10 days after immunization. (A) CD8⁺ and (B) CD4⁺ T cells were stained for type 1 intracellular cytokine expression and (C) CD8⁺ T cells for cytolytic markers granzyme B and CD107a as well. (D–G) Cells were stained directly ex vivo for activation markers, showing the proportion of i.v.-label negative (tissue-“infiltrating”) and i.v.-label positive (“vascular”) T cells that are (D and E) CD69⁺ and (F and G) CD44⁺ CD62L⁻ in lung. n = 8 mice per vaccine group and n = 5 naive mice, pooled from two independent experiments. Naive mice were age matched, non-immunized BALB/c mice. (C–G) Symbols represent individual animals. Data shown are mean plus SEM. Statistical analysis: (A–C) Kruskal-Wallis and post hoc Mann-Whitney U tests with Bonferroni correction and (D–G) two-way repeated-measures ANOVA test with multiple post hoc comparisons with Dunnett’s correction. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001. See also Figures S1 and S2.

Figure 3

Humoral Immune Responses after SARS-CoV-2 mRNA Vaccination BALB/c mice received a single i.m. immunization with 30 μg of SARS-CoV-2 or Luc mRNA-LNP vaccines. (A and B) S protein-specific IgG levels were determined by endpoint dilution ELISA (A) and neutralizing antibody (Nab) levels were measured by a VSV-based pseudovirus neutralization assay (B) before immunization and 4 and 9 weeks post immunization. (C) Nab levels were further confirmed by microneutralization assay using serum obtained 9 weeks post vaccination. n = 10 mice/group. Naive mice were age matched, non-immunized BALB/c mice. (A–C) Symbols represent individual animals. Horizontal lines represent the limit of detection. End-point dilution ELISA, FRNT₅₀, and IC₅₀ titers below the limit of detection are reported as half of the limit of detection. Data shown are mean plus SEM. (D) HEK293T cells transfected to express mFcγR1 were infected with SARS-CoV-2 pseudovirus or ZIKV virus-like particles preincubated with serially diluted anti-SARS-CoV-2 sera obtained 9 weeks post immunization or anti-ZIKV sera, respectively. Serum samples were pooled from 5 mice belonging to the same experimental group. Infection level was measured by luciferase assays. Mean ± SEM of three independent experiments is presented. Statistical analysis: (A and B) two-way ANOVA and (C) one-way ANOVA with Tukey’s multiple comparison on log-transformed data. (D) SARS-CoV-2: there are no significant differences when analyzed by two-way ANOVA with Tukey’s multiple comparisons test; ZIKV: two-way ANOVA with Sidak’s multiple comparisons test. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001. See also Figure S3.

Figure 4

SARS-CoV-2 mRNA Vaccines Elicit Antigen-Specific MBC and LLPC Responses BALB/c mice received a single i.m. immunization with 30 μg of SARS-CoV-2 or Luc mRNA-LNP vaccines and sacrificed 9 weeks post immunization. (A and B) Representative flow cytometry staining of full-length Δfurin and RBD-specific splenic (A) IgG1 and (B) IgG2a/2b memory B cells (MBC). (C) Quantification of total splenic RBD-specific MBC. (D and E) Quantification of splenic full-length Δfurin-specific (D) IgG1 and (E) IgG2a/2b MBC. (F and G) Quantification of RBD-specific splenic (F) IgG1 and (G) IgG2a/2b MBC. (H and I) Quantification of bone marrow (H) RBD and (I) full-length Δfurin-specific IgG antibody secreting cells (ASC). (J) Quantification of bone marrow RBD-specific IgG1, IgG2a, IgG2b, IgG3, IgM and IgA ASCs. n = 10 mice per group, pooled from two independent experiments. Naive mice were age-matched, non-immunized BALB/c mice. Symbols represent individual animals. Data shown are mean plus SEM. Statistical analysis: one-way ANOVA with Bonferroni correction, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001. See also Figure S4.