A single-dose mRNA vaccine encoding the classical swine fever virus E2-ECD induces durable protective immunity in rabbits

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  Figure 1. Construction and characterization of CSFV-E2-based mRNA vaccines. A Illustration of mRNA vaccine constructs, which include the extracellular domain of E2 (E2-ECD, aa1-337), the N-terminus of the extracellular domain of E2 (E2-ECD-N, aa1-172), and the C-terminus of the extracellular domain of E2 (E2-ECD-C, aa173-337). The constructs contain a Cap1 structure at the 5’ end for enhanced mRNA stability and translation, a 5’ untranslated region (5′ UTR), a signal peptide (SP) for secreted protein expression, an antigen coding sequence, a 3’ untranslated region (3′ UTR), and a polyhistidine (His) tag for purification and detection. B Antigen expression in HEK293T cells. HEK293T cells were transfected with GFP (0.1 μg), E2-ECD (0.2 μg), E2-ECD-N (0.2 μg), or E2-ECD-C (0.2 μg) mRNAs for 16 h. Protein expression of the indicated mRNAs in the cell lysate and culture supernatant was analysed by immunoblotting analysis with anti-His-HRP and anti-GFP antibodies and rabbit antisera against CSFV. The nonspecific band at ~ 35 kDa is attributed to the cross-reactivity of the primary antibody with a host cell protein. ns, nonspecific. C Immunoblotting analysis of the expressed antigens under…

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  Figure 2. Humoral immune responses in CSFV-E2 mRNA-LNP-vaccinated mice. A Schematic representation of the experiments. BALB/c mice (n = 5 per group) were immunized intramuscularly with 0.5 or 5 μg of E2-ECD or E2-ECD-N mRNA-LNPs via a primer-only or prime-boost regimen. The control group received two doses of placebo (PBS) on Day 0 and Day 14. B E2-specific antibody titres in the serum were determined by ELISA on day 28 after immunization. The data are presented as mean ± SEM of five mice per group. The data were analysed using the Mann‒Whitney test with GraphPad Prism 8 (*P < 0.05; **P < 0.01; ND: not detected).

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  Figure 3. Cellular immune responses in CSFV-E2 mRNA-LNP-vaccinated mice. A Schematic representation of the experiments. Mice (n = 5 per group) were immunized intramuscularly with 5 μg of E2-ECD or E2-ECD-N mRNA-LNPs via a prime-only regimen. The control group received a placebo (PBS). Spleens were harvested 10 days post-vaccination. B Frequencies of IFN-γ- and IL-2-producing splenic CD4+ and CD8+ T cells following E2-ECD protein restimulation were detected via ICS assays and analysed by flow cytometry. Splenocytes were isolated on day 10 and stimulated with the recombinant E2-ECD protein for 48 h. C Measurement of CSFV-E2-specific IFN-γ- and IL-2-producing splenocytes by ELISpot assay. Splenocytes (5 × 105) were stimulated with E2-ECD protein for 36 h, and the spots were then counted. D Measurement of CSFV-E2-specific IL-2 and IL-4 responses in splenocytes by ELISA. IL-2 and IL-4 secretion by splenocytes after stimulation with the E2-ECD protein for 72 h. The cell culture supernatant was collected for detection by ELISA. The data shown are mean ± SEM of five mice per group. The data were analysed using two-way ANOVA with GraphPad Prism 8 (*P < 0.05; **P < 0.01; ns, not significant; ND, not…

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  Figure 4. E2-ECD and E2-ECD-N mRNA-LNPs trigger long-term humoral immune responses. A Schematic representation of the experiments. Mice (n = 7 per group) were immunized intramuscularly with 0.5 μg or 5 μg of E2-ECD or E2-ECD-N mRNA-LNPs via a primer-only regimen. Serum samples were collected at the indicated times. B E2-specific antibody titres in E2-ECD- and E2-ECD-N mRNA-LNP-vaccinated mice were determined by ELISA. Serum samples were collected at 2- or 4-week intervals for 24 weeks post-immunization, and the E2-specific antibody titres in the serum samples were determined via ELISA. The data shown are the mean ± SEM of seven mice per group.

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  Figure 5. E2-ECD mRNA-LNPs induce complete protection from CSFV infection in rabbits. A Schematic representation of vaccination, antibody analysis and CSFV challenge in rabbits. Rabbits (n = 5 per group) were immunized subcutaneously with 100 μg of E2-ECD or E2-ECD-N mRNA-LNPs via a prime-only regimen. The control group received a placebo (PBS). The serum was harvested on days 14, 21 and 28 post-vaccination. The rabbits were challenged with the CSFV C-strain on day 31 and sacrificed at 4 days post-challenge. B The Erns-specific antibody titres in rabbit sera were determined by ELISA. Serum samples were collected on day 28 after primary immunization to detect CSFV Erns protein-binding IgG antibody titres. Serum samples from the CSFV-C challenge group, collected 14 days post-challenge, were included as positive controls for infection-induced antibodies. C E2-specific antibody titres in rabbit sera were determined by ELISA. Serum samples were collected on days 14, 21 and 28 after primary immunization to evaluate the time course of CSFV E2 protein-binding IgG antibody titre. D The neutralizing antibody titres in rabbit sera were determined via a virus neutralization assay. Neutralizing…

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  Figure 6. E2-ECD mRNA-LNPs induce long-term protective immunity against CSFV in rabbits. A Schematic representation of vaccination, antibody analysis and CSFV challenge in rabbits. Rabbits (n = 4 per group) were immunized subcutaneously with 100 μg of E2-ECD mRNA-LNP in a prime-only regimen. The control group received a placebo (PBS). Serum samples were collected at the indicated times. The rabbits were challenged with the CSFV C-strain at week 18 and sacrificed at 4 days post-challenge. B Measurement of CSFV-E2-specific IFN-γ-producing splenocytes by ELISpot assay. Peripheral blood mononuclear cells (5 × 105) isolated from vaccinated rabbits on days 14 and 120 were stimulated with E2-ECD protein for 36 h, and the spots were then counted. The data were analysed using the Mann‒Whitney test with GraphPad Prism 8 (*P < 0.05; ND, not detected). C E2-specific antibody titres in E2-ECD mRNA-LNP-vaccinated rabbits were determined by ELISA. Serum samples were collected at 2- or 4-week intervals for 18 weeks post-immunization, and the E2-specific antibody titres in the serum samples were determined via ELISA. The data shown are the mean ± SEM of four rabbits per group. D Rectal temperatures of the…