Broad protection and respiratory immunity of dual mRNA vaccination against SARS-CoV-2 variants

Abstract

While first-generation, spike (S)-based COVID-19 vaccines were effective against early SARS-CoV-2 strains, the rapid evolution of novel Omicron subvariants have substantially reduced vaccine efficacy. As such, broadly protective vaccines against SARS-CoV-2 are needed to prevent future viral emergence. In addition, it remains less clear whether peripheral immunization, especially with mRNA vaccines, elicits effective respiratory immunity. Our group has developed a nucleoside-modified mRNA vaccine expressing the nucleocapsid (N) protein of the ancestral SARS-CoV-2 virus and has tested its use in combination with the S-based mRNA vaccine (mRNA-S). In this study, we examined efficacy of mRNA-N alone or in combination with mRNA-S (mRNA-S+N) against more immune evasive Omicron variants in hamsters. Our data show that mRNA-N alone induces a modest but significant protection against BA.5 and that dual mRNA-S+N vaccination confers complete protection against both BA.5 and BQ.1, preventing detection of virus in the hamster lungs. Analysis of respiratory immune response in mice shows that intramuscular mRNA-S+N immunization effectively induces respiratory S- and N-specific T cell responses in the lungs and in bronchoalveolar lavage (BAL), as well as antigen-specific binding IgG in BAL. Together, our data further support mRNA-S+N as a potential pan-COVID-19 vaccine for broad protection against current and emerging SARS-CoV-2 variants.

Fig. 1. mRNA-N vaccine is efficacious against Omicron variant BA.5 in hamsters.

A Experimental design and timeline (Created with BioRender.com). Two groups of hamsters (n = 10/group) were vaccinated intramuscularly with either empty LNP (mock) or mRNA-N vaccine (2 µg/dose) at weeks 0 and 3. At week 5, hamsters were intranasally challenged with Omicron BA.5 (2 × 10⁴ pfu). B Lungs were harvested at 2 and 4 DPI (days post infection; n = 5 at each time point) for quantification of viral RNA copies by RT-qPCR. C Hamster body weight was monitored from 0 to 4 DPI. In (B) symbols represent individual animals, midlines represent the median, error bars represent the interquartile range (IQR), and the dashed lines represent the lower limit of detection (LOD). The number of animals with viral loads above the LOD is noted. Log₁₀ normalized data was compared by Mann-Whitney test. In (C) symbols represent the mean, error bars represent the standard deviation, and the dashed line highlights 0% weight change. Weight change at individual time point between the two groups was compared by unpaired t-test. *p < 0.05, **p < 0.01.

Fig. 2. Respiratory T cell response induced by mRNA-N in mice.

A Mouse experimental design and timeline (Created with BioRender.com). Two groups of C57BL/6 mice (n = 5/group) were intramuscularly vaccinated with either empty LNP (mock) or mRNA-N vaccine (1 μg) at weeks 0 and 3. Two weeks after booster dose (week 5), immune responses were analyzed. B, C Analysis of tetramer positive, N-epitope specific T cells in the lungs by flow cytometry. B Representative flow cytometry plots for N-tetramer staining of CD8 T cells from LNP and mRNA-N vaccinated mice. C Frequencies of N epitope-specific CD8 T cells in the lungs of LNP and mRNA-N vaccine group. Analysis of total and N epitope-specific T cells in the BAL. Frequencies of activated (CD44⁺) CD4 and CD8 T cells (D), CXCR6⁺ CD4 and CD8 T cells (E), and N epitope-specific CD8 T cells (F) in BAL were examined. Data were presented as median and interquartile range, and were compared by Mann-Whiteney test between the two groups. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3. Dual mRNA-S+N vaccination protects hamsters from Omicron BA.5.

A Hamster experimental design and timeline (Created with BioRender.com). Three groups of hamsters (n = 10/group) were vaccinated intramuscularly with empty LNP (mock), mRNA-S (2 μg), or mRNA-S+N (2 μg for each mRNA) at weeks 0 and 3, followed by intranasal challenge with SARS-CoV-2 Omicron BA.5 (2 × 10⁴ pfu) at week 5. Lungs were harvested at 2 (B) and 4 (C) DPI (n = 5 at each time) for quantification of viral RNA copies by RT-qPCR. (D) Hamster body weights were monitored from 0 to 4 DPI. In (B, C), symbols represent individual animals, midlines represent the median, error bars represent the interquartile range, and the dashed line represents the lower limit of detection (LOD). The number of animals with viral loads above the LOD is noted. Data were compared among the three groups by Kruskal-Wallis test. In (D), symbols represent the mean, error bars represent the standard deviation, and the dashed line highlights 0% weight change. Weight change was compared by 2-way ANOVA followed by Tukey’s multiple comparisons test (two factors; hamsters’ weight and time). *p < 0.05, **p < 0.01, ****p < 0.0001.

Fig. 4. Dual mRNA-S+N vaccination protects hamsters from Omicron BQ.1.

A Experimental design and timeline (Created with BioRender.com). Three groups of hamsters (n = 10/group) were vaccinated intramuscularly with empty LNP (mock), mRNA-S (2 μg), or mRNA-S+N (2 μg for each mRNA) at weeks 0 and 3, followed by intranasal challenge with SARS-CoV-2 Omicron BQ.1 (2 × 10⁴ pfu) at week 5. Lungs were harvested at 2 (B) and 4 (C) DPI (n = 5 at each time point) for quantification of viral RNA copies by RT-qPCR. D Hamster body weights were monitored from 0 to 4 DPI. In (B, C), symbols represent individual animals, midlines represent the median, error bars represent the interquartile range, and the dashed line represents the lower limit of detection (LOD). The number of animals with viral loads above the LOD is noted. Log₁₀ normalized data was compared among the three groups by Kruskal-Wallis test. In (D), symbols represent the mean, error bars represent the standard deviation, and the dashed line highlights 0% body weight change. Weight change was compared by 2-way ANOVA followed by Tukey’s multiple comparisons test (two factors; hamsters’ weight and time). *p < 0.01, **p < 0.01, ****p < 0.0001.

Fig. 5. Serum antibody response in hamsters following mRNA vaccination.

S-specific binding IgG (A), IgA (B), and IgM (C) endpoint titers (EPTs) in the hamster sera at week 5 after immunization (n = 4 for LNP; n = 10 for mRNA-S or mRNA-S+N). Log₁₀ normalized EPTs for each group is indicated. D Neutralization of the hamster sera (n = 10 for all three groups) against SARS-CoV-2 WA.1, BA.1, BA.5, and BQ.1 as measured by PRNT₅₀. Data are presented as median with interquartile range. Dotted lines in each plot indicates LOD for each assay. Number of animals in each group with neutralizing titers above the LOD is noted in (D). Data were compared among the three groups by Kruskal-Wallis test. *p < 0.05, **p < 0.01, ****p < 0.0001.

Fig. 6. Respiratory T cell response following different mRNA vaccination.

A Mouse experimental design and timeline (Created with BioRender.com). Three groups of C57BL/6 mice (n = 5/group) were intramuscularly vaccinated with either empty LNP (mock), mRNA-S vaccine (1 μg), or mRNA-S+N vaccine (1 μg for each mRNA) at week 0 and 3. Two weeks after final dose (week 5), mice were euthanized and immune analyses were performed. Analysis of activated T cells in the lungs (B), BAL (C), and spleens (D) of mice. Expression of CD44 on CD4 and CD8 T cells was examined by flow cytometry and shown as percent CD44⁺ of the parental population. Expression of CXCR6 on CD4 and CD8 T cells in the lungs (E), BAL (F), and spleens (G) was examined by flow cytometry and shown as percent CXCR6⁺ of the parental population. H Representative flow cytometry plots for S- and N-tetramer staining of CD8 T cells from LNP, mRNA-S, and mRNA-S+N vaccinated mice. Tetramer⁺ (S- and N-epitope specific CD8 T cells) in the lungs (I), BAL (J), and spleens (K) of mice. Data are presented as median and interquartile range. Kruskal-Wallis test was used for statistical comparison among the three groups. *p < 0.05, **p < 0.01.