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. 2024 Jan 31;16(2):211.
doi: 10.3390/v16020211.

Safety, Immunogenicity, and Mechanism of a Rotavirus mRNA-LNP Vaccine in Mice

Chenxing Lu  1 , Yan Li  1 , Rong Chen  1 , Xiaoqing Hu  1 , Qingmei Leng  1 , Xiaopeng Song  1 , Xiaochen Lin  1 , Jun Ye  1 , Jinlan Wang  1 , Jinmei Li  1 , Lida Yao  1 , Xianqiong Tang  1 , Xiangjun Kuang  1 , Guangming Zhang  1 , Maosheng Sun  1 , Yan Zhou  1 , Hongjun Li  1
Affiliations

Safety, Immunogenicity, and Mechanism of a Rotavirus mRNA-LNP Vaccine in Mice

Chenxing Lu et al. Viruses. .

Abstract

Rotaviruses (RVs) are a major cause of diarrhea in young children worldwide. The currently available and licensed vaccines contain live attenuated RVs. Optimization of live attenuated RV vaccines or developing non-replicating RV (e.g., mRNA) vaccines is crucial for reducing the morbidity and mortality from RV infections. Herein, a nucleoside-modified mRNA vaccine encapsulated in lipid nanoparticles (LNP) and encoding the VP7 protein from the G1 type of RV was developed. The 5' untranslated region of an isolated human RV was utilized for the mRNA vaccine. After undergoing quality inspection, the VP7-mRNA vaccine was injected by subcutaneous or intramuscular routes into mice. Mice received three injections in 21 d intervals. IgG antibodies, neutralizing antibodies, cellular immunity, and gene expression from peripheral blood mononuclear cells were evaluated. Significant differences in levels of IgG antibodies were not observed in groups with adjuvant but were observed in groups without adjuvant. The vaccine without adjuvant induced the highest antibody titers after intramuscular injection. The vaccine elicited a potent antiviral immune response characterized by antiviral clusters of differentiation CD8+ T cells. VP7-mRNA induced interferon-γ secretion to mediate cellular immune responses. Chemokine-mediated signaling pathways and immune response were activated by VP7-mRNA vaccine injection. The mRNA LNP vaccine will require testing for protective efficacy, and it is an option for preventing rotavirus infection.

Keywords: lipid nanoparticles; mRNA vaccine; neutralizing antibody; rotavirus; structural protein VP7.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Vaccine characterization and immunization strategy. (A) Model of vaccine construction. (B) Detection of mRNA expression after transfection of Cap-mRNA to HEK293 cells by Western blotting; β-Actin was used as an internal reference. (C) Particle size detection with a Malvin laser granmeter. (D) Electric potential of the mRNA-LNP. (E,F) Shape of mRNA-LNP particles observed by transmission electron microscopy. The scale of (E,F) are 500 nm and 200 nm, respectively. (G) Percentage encapsulation of the encapsulated vaccine. Data are represented as mean ± SD. (H) PDI of the encapsulated vaccine. Data are represented as mean ± SD. (I) Schedule of immunization with the VP7-mRNA vaccine and blood collection. Mice were divided into three groups: IM injection with adjuvant; SC injection with adjuvant; and IM injection without adjuvant. Each group had a dosage subgroup of 2, 5 and 10 μg. The immunization procedure was three doses with an interval of 21 days, and the spleen was removed 14 days after the final immunization.
Figure 2
Figure 2
The vaccine triggered humoral immune responses in mice. (A) Levels of IgG antibodies that the vaccine triggers in 2, 5, and 10 μg groups after intramuscular injection with adjuvant. (B) Levels of IgG antibodies that the vaccine triggers in 2, 5, and 10 μg groups after subcutaneous injection with adjuvant. (C) Levels of IgG antibodies that the vaccine triggers in 2, 5, and 10 μg groups by intramuscular injection without adjuvant. (D) The effects of the three immunization routes were compared in the 10 μg dose group. Data are presented as geometric mean with geometric SD. Significant differences were determined by a two-way ANOVA (* p < 0.05, ** p < 0.01 and **** p < 0.0001; ns. indicates not significant).
Figure 3
Figure 3
The vaccine triggered cellular immunity and neutralizing antibodies in mice. (A) In the group of 5 µg by intramuscular injection without adjuvant, the amount of interferon-γ produced by splenic lymphocytes in the control group and vaccine group was measured by ELISpot. (B) The number of spots in the control group and vaccine group was mapped and counted in ELISpot. Data are represented as mean ± SD. (C) In the group of 10 µg by intramuscular injection without adjuvant, the vaccine triggered neutralizing antibodies after the third immunization. Data are presented as geometric mean with geometric SD. Significant differences were determined by an unpaired t test. (*** p < 0.001). (D) The percentage of CD8+ cells in total spleen lymphocytes in the group of 10 µg by intramuscular injection with adjuvant. (E) The percentage of CD8+ cells in total spleen lymphocytes in the group of 10 µg by subcutaneous injection with adjuvant and (F) the group of 10µg by intramuscular injection without adjuvant. Data are presented as mean ± SD. Significant differences were determined by an unpaired t test (* p < 0.05).
Figure 4
Figure 4
Changes in gene expression of PBMCs. (A) The differences generated by the comparison are reflected in the volcano map. Gray shows genes with a non-significant difference in expression. Red and blue are genes with a significant difference in expression. The horizontal axis is log2 fold change. The vertical axis is −log10 of the p-value. (B) Analyses of functional enrichment (using the GO database) of the top 30 genes (based on selection of GO items corresponding to PopHits ≥ 5 in the three categories and ranking 10 items from largest to smallest according to the corresponding −log10 p-value of each item).
Figure 5
Figure 5
KEGG pathway classification. The horizontal axis is the percentage of the total number of genes annotated to each pathway (differentially expressed genes (DEGs)) and all genes annotated to the KEGG database (DEGs). The vertical axis represents the pathway name, and the number on the right side of the column represents the number of DEGs annotated to this pathway.
Figure 6
Figure 6
Changes in the body weight of mice. Starting at week 0, weight was measured weekly until the end of week 7. The weight of mice in different dose groups was divided into a (A) 2 μg group, (B) 5 μg group, and (C) 10 μg group. The weight data are presented in the form of mean ± SD.
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