Bivalent circular RNA vaccines against porcine epidemic diarrhea virus and transmissible gastroenteritis virus

Abstract

Porcine Epidemic Diarrhea Virus (PEDV) and Transmissible Gastroenteritis Virus (TGEV) pose significant threats to neonatal piglets, leading to severe diarrhea and potentially lethal consequences. Beyond enforcing stringent biosecurity protocols, effective and safe vaccinations are crucial in mitigating the impact of these diseases. In this study, the PEDV S1 (PS1) and TGEV S1 (TS1) antigens were initially chosen as candidates for the development of circRNA vaccines. Recognizing the comparatively lower immunogenicity of the PS1 antigen in contrast to the TS1 antigen, we strategically conjugated the PS1 with the pig fragment crystallizable (Fc) region to form PS1F. Despite these efforts, the bivalent circRNA vaccine prepared using an equal amount of the circRNA^PS1F and circRNA^TS1 mixture still led to a reduction in the antibody levels against PS1. Subsequent dosage optimization of these two circRNA vaccines resulted in the induction of comparable levels of antigen specific antibodies and T cell immunity. Furthermore, sequential vaccination regimen with bivalent circRNA vaccine and commercial inactivated vaccines (IAV) could elicit a predominantly Th1-driven antibody responses and effectively neutralize both PEDV and TGEV. Our findings not only provide a potential strategy for the development of bivalent or multivalent circRNA/mRNA-based vaccines but also highlight the promising application of sequential vaccination strategies within the swine industry.

Keywords: bivalent; circRNA vaccine; porcine epidemic diarrhea virus; sequential vaccination; transmissible gastroenteritis virus.

Figure 1

Production and characterization of circRNA^PS1 and circRNA^TS1 vaccines. (A) Schematic representation of the spike (S) protein from PEDV or TGEV. PS: S protein of PEDV; TS: S protein of TGEV. (B) The genes encoding the PS1 and TS1 domains were optimized, synthesized and inserted into the vector with PIE system for circRNA production in vitro. SP: Signal peptide. (C) The RT-PCR products of circRNA^TS1 and circRNA^PS1 were analyzed by Sanger sequencing to validate the splicing sites of the circRNAs. (D) The expression levels of PS1 and TS1 in the supernatants were determined by western blot analysis at 48 hours post-transfection of 293F cells with circRNA^TS1 and circRNA^PS1. (E) Schematic depiction of the circRNA-LNP complex. (F) Size distributions of the circRNA-LNP complex. (G) Zeta potential of the circRNA-LNP complex. (H) TEM image of the circRNA-LNP complex. Scale bar, 50 nm.

Figure 2

Immunogenicity assessment of the circRNA^PS1 and circRNA^TS1 vaccines. (A) Schematic illustration of the immunogenicity assessment protocol for circRNA^PS1 and circRNA^TS1 vaccinations. (B, C) Quantitative analysis of PS1-specific (B) and TS1-specific (C) IgG antibody titers induced by the circRNA^PS1 and circRNA^TS1 vaccines, respectively, at the indicated dosages. These values are expressed as mean ± S.D, n=5. (ns, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3

The Fc fusion significantly amplifies the immunogenicity of PS1 in the form of circRNA. (A) Schematic illustration of the preparation of circRNA ^PS1F and circRNA ^TS1F . (B) The RT-PCR products of circRNA^PS1F and circRNA^TS1F were analyzed by Sanger sequencing to validate the splicing sites of the circRNAs. (C) The expression levels of PS1F and TS1F in the supernatants were determined by western blot analysis at 48 hours post-transfection of 293F cells with circRNA^PS1F and circRNA^TS1F. (D) Schematic depiction of the circRNA^PS1F/TS1F-LNP complex. (E) Schematic representation of the vaccination regimen with circRNA^PS1F and circRNA^TS1F. An equivalent dosage of circRNA^PS1/TS1-LNP or LNP control was administered following the same protocol. (F, G) Quantitative analysis of PS1-specific (F) and TS1-specific (G) IgG antibody titers post-vaccination. These values are expressed as mean ± S.D, n=5. (ns, *P < 0.05, ***P < 0.001).

Figure 4

Equal dosing of the circRNA^PS1F and circRNA^TS1 vaccines leads to i a significant reduction in antibody levels against PS1. (A) Schematic depiction of the vaccination regimen. A mixture of circRNA^PS1F (10 μg) and circRNA^TS1 (10 μg) was administered in accordance with the outlined protocol. Additionally, CircRNA^PS1F (10 μg), CircRNA^TS1 (10 μg), or LNP (as a control) was delivered following the identical vaccination regimen. (B-C) Quantitative analysis of PS1-specific (B) and TS1-specific (C) IgG antibody titers. (D) Expression analysis of PSIF was performed 48 hours after transfection of 293F cells with the specified circRNAs. These values are expressed as mean ± S.D, n=5. (ns, *P < 0.05, ***P < 0.001).

Figure 5

Optimize the dose ratio of bivalent vaccines to induce higher levels of humoral and cellular immune responses against both PEDV and TGEV. (A) Schematic depiction of the immunization regimen for the bivalent circRNA vaccine. (B) Quantitative analysis of PS1-specific IgG antibody titers induced by circRNA^PS1F vaccine (10 μg or 20 μg). (C, D) Assessment of PS1 or TS1-specific IgG antibody titers induced by bivalent vaccines comprising circRNA^PS1F-LNP (20 μg) and circRNA^TS1-LNP (0, 2.5, 5 or 10 μg). (E) Detection of PS1 and TS1 antigen-specific T cell responses, as indicated by IFN-γ⁺ CD8⁺ T cells, in the splenocytes from mice immunized with bivalent vaccine comprising circRNA^PS1F (20 μg) and circRNA^TS1 (5 μg). These values are expressed as mean ± S.D, n=5. (ns, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 6

Sequential vaccination elicits robust immunity against both PEDV and TGEV. (A, B) Quantitative analysis of PS1-specific (A) and TS1-specific (B) IgG antibody titers in serum samples from mice with various immunization strategies. (C, D) Assessment of PS1-specific (C) and TS1-specific (D) IgG1 and IgG2a antibody titers. (E, F) Calculation of the IgG2a/IgG1 ratios for PS1-specific (E) and TS1-specific (F) antibodies, as presented in panels (C) and (D). (G, H) Neutralization assays of authentic PEDV and TGEV were carried out using the serum from mice immunized with corresponding strategies. IAV: Commercially available inactivated bivalent vaccine against PEDV and TGEV; CircRNA: The optimized bivalent circRNA vaccine comprising 20 μg of circRNA^PS1F and 5 μg of circRNA^TS1. These values are expressed as mean ± S.D, n=5. (ns, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).