Protective Immune Responses Induced by an mRNA-LNP Vaccine Encoding prM-E Proteins against Japanese Encephalitis Virus Infection

Abstract

Japanese encephalitis virus (JEV) is an important zoonotic pathogen, which causes central nervous system symptoms in humans and reproductive disorders in swine. It has led to severe impacts on human health and the swine industry; however, there is no medicine available for treating yet. Therefore, vaccination is the best preventive measure for this disease. In the study, a modified mRNA vaccine expressing the prM and E proteins of the JEV P3 strain was manufactured, and a mouse model was used to assess its efficacy. The mRNA encoding prM and E proteins showed a high level of protein expression in vitro and were encapsulated into a lipid nanoparticle (LNP). Effective neutralizing antibodies and CD8+ T-lymphocytes-mediated immune responses were observed in vaccinated mice. Furthermore, the modified mRNA can protect mice from a lethal challenge with JEV and reduce neuroinflammation caused by JEV. This study provides a new option for the JE vaccine and lays a foundation for the subsequent development of a more efficient and safer JEV mRNA vaccine.

Keywords: Japanese encephalitis virus; immunogenicity; mRNA vaccine; prM-E protein.

Figure 1

Construction and expression of mRNA-prM-E. (A) Characterization of vectors for in vitro transcription. (B) mRNA-prM-E was run in the 1% formaldehyde denaturing gel (RNase-free). (C) HEK-293T cells were transfected with the mRNA-prM-E, and lysate was analyzed by Western blotting with a monoclonal antibody 1H10 against the JEV E protein. The pcDNA3.1 plasmid encoding prM-E and JEV were used as the positive controls. (D) The particle size of the LNP-mRNA was measured three times using a Zetasizer spectrometer indicating an average size of approximately 80 nm.

Figure 2

Immune responses in C57BL/6 induced by the mRNA-prM-E vaccine. (A) Mice were intramuscularly injected with 15 μg of mRNA-prM-E or SA14-14-2 live-attenuated vaccine. The sera of mice in SA14-14-2 group and DMEM group were collected on day 21 while the sera of mice immunized with prM-E-mRNA or LNP were collected on day 42. (B) Serial dilutions of serum were tested for neutralization activity by PRNT assay. Neutralization curves (left panel) and the PRNT50 (right panel) are shown. Each experiment was independently repeated three times and PRNT50 was calculated by the Reed–Muench method (n = 16, ** p < 0.01; **** p < 0.0001). (C) The percentages of CD8 ⁺ and CD4 ⁺ T cells in spleen were analyzed by flowcytometry. Data are represented as mean ± SEM of 3 spleens from 3 mice from each group. (** p < 0.01; **** p < 0.0001). (D) The IFN-γ level in sera was measured by ELISA. Data are represented as mean ± SEM. (n = 16, ** p < 0.01; **** p < 0.0001).

Figure 3

Protective efficacy against JEV in C57BL/6 after immunization. Body weight changes (A), behavioral score (B) and survival rate (C) were recorded after JEV P3 strain challenge. Survival rate is displayed as Kaplan–Meier survival curves and behavioral changes from normal to severe are represented on a scale of 0 to 5 (n = 10). (D) Viral loads in mice brain were detected by plaque assay and qRT-PCR at day 5 after JEV-infection. Data are represented as mean ± SEM. (n = 3, **** p < 0.0001).

Figure 4

The mRNA vaccine protects against a JEV-induced inflammatory response in mice brain. (A) The mRNA expression levels of inflammatory cytokines (IL-6, CCL-2, CCL-5, TNF-α) in brain tissue lysates were quantified by qRT-PCR. (B,C) The activation of the pathological changes and glial cells in brain tissue of mice were analyzed by H&E staining and IHC. Integrated option density analysis of three visual fields of slides from each group was performed to quantify the results of immunohistochemical staining (the lower panels). Scale bar = 100 μm. Data are represented as mean ± SEM. (n = 3, **** p < 0.0001).