Expression and immunogenicity of S protein with two proline residue mutations of porcine deltacoronavirus

Abstract:

[Objective] Porcine deltacoronavirus (PDCoV) is a major porcine enteric coronavirus, causing huge economic losses to the pig breeding industry worldwide. However, there is no commercial vaccine available for this virus. The spike (S) protein is a key factor inducing host immune response. In this study, the two sites 855 and 856 in the loop between the heptapeptide repeat-1 (HR1) and the central helix of PDCoV S protein were mutated to proline (E855P and V856P). Then, the recombinant S protein and mutated S protein (S2P) were expressed and purified by the ExpiCHO-S eukaryotic expression system, and their immunogenicity and immunoprotecive performance were evaluated for developing a PDCoV subunit vaccine with good immune effect. [Methods] The serum level of the specific antibody IgG in immunized mice was measured by indirect enzyme-linked immunosorbent assay. The serum neutralization test was carried out to determine the titer of neutralizing antibodies in the immunized mice. The proliferation of T lymphocytes in immunized mice was detected by flow cytometry. The secretion levels of interferon (IFN)-γ, IFN-α, interleukin (IL)-2, and IL-4 were determined. RT-qPCR was employed to measure the PDCoV load in the intestinal tissue of mice after challenge. Tissue sections were prepared to observe the intestinal lesions of mice. The distribution of PDCoV antigen in the intestinal tissue of mice was detected by immunohistochemistry. [Results] High levels of anti-PDCoV specific IgG antibodies were produced in mice after intramuscular injection of S and S2P subunit vaccines, and the serum of mice 42 days after immunization had a neutralizing effect on PDCoV. The 50% neutralizing protective titer of LLC-PK cells in the S2P group was significantly higher than that in the S group. In addition, the immunization with S and S2P significantly induced the proliferation of CD₄⁺ T lymphocytes in mice, which was higher in the S2P group than in the S group. The immunization with S2P induced the proliferation of CD₈⁺ T lymphocytes in mice, and the level of CD₈⁺ T lymphocytes showed no difference between the S group and the PBS group. The levels of IFN-γ, IFN-α, IL-2, and IL-4 in the S and S2P groups were significantly higher than those in the PBS group and had no difference between the S and S2P groups. The challenge assay results showed that the PBS group presented PDCoV, pathological damage, and a large number of PDCoV antigens in the intestinal tissue, while neither PDCoV nor intestinal tissue damage was detected in the S and S2P groups, which showed no significant difference between the two groups. [Conclusion] S2P induces higher level of the humoral immune response against PDCoV in mice than S. The vaccines prepared with both S2P and S have protective effects on mice. The findings lays a foundation for the follow-up study of PDCoV subunit vaccines.

Key words:porcine deltacoronavirus (PDCoV);S protein;bisproline mutation;immunogenicity