Analysis of the Immunogenicity of African Swine Fever F317L Protein and Screening of T Cell Epitopes

Abstract

The African swine fever virus (ASFV) encodes numerous proteins characterized by complex immune escape mechanisms. At present, the structure and function of these proteins, including the F317L protein, have yet to be fully elucidated. In this study, we examined the immunogenicity of the F317L protein. Mice were subcutaneously immunized with the F317L protein using initial and subsequent booster doses, and, at the 28th day post-treatment, we assessed the humoral and cellular immune responses of mice. The F317L protein stimulated production of specific antibodies and activated humoral immune responses. In addition, F317L stimulated the production of large amounts of IFN-γ by splenic lymphocytes, thereby activating cellular immune responses. Using informatics technology, we predicted and synthesized 29 F317L protein T cell epitopes, which were screened using IFN-γ ELISpot. Among these, the F25 (²⁴⁶SRRSLVNPWT²⁵⁵) peptide was identified as having a stronger stimulatory effect than the full-length protein. Collectively, our findings revealed that the ASFV F317L protein can stimulate both strong humoral and cellular immunity in mice, and that the F25 (²⁴⁶SRRSLVNPWT²⁵⁵) peptide may be a potential active T cell epitope. These findings will provide a reference for further in-depth studies of the F317L protein and screening of antigenic epitopes.

Keywords: African swine fever virus; F317L protein; T cell epitope; cellular immunity; humoral immunity.

Figure 1

Experimental procedures of the immunization and sample collection. The first immunization was performed on Day 0, with a booster immunization at Day 21, and serums were collected on Days 7, 14, 21, and 28, while splenic lymphocytes were collected on Day 28. Red dots represent serum.

Figure 2

The IFN-γ secreted by splenic lymphocytes of immunized mice detected by IFN-γ ELISpot assays. (A) IFN-γ ELISpot spot plots. Splenic lymphocytes stimulated with 100 ng/well of F317L protein and p72 protein. (B) Histogram of spots counted in IFN-γ ELISpot assays. Data are expressed as mean ± SD. According to the number of spots counted in IFN-γ ELISpot assays, the data are representative of the mean ± SD calculated based on three mice and three parallel holes in each mouse. The difference between the F317L group compared to the negative control group (p < 0.001) and the p72 positive control group (p < 0.01) was statistically significant. Note: ** p < 0.01; *** p < 0.001.

Figure 3

Results of T cell subpopulation assays. (A) Flow cytometry results for each immunization group. (B) Histogram of CD4⁺/CD8⁺ ratio. Statistical analysis of the T cell subset ratios showed significant differences in the F317L protein group compared with the negative control group (p < 0.01) and the p72 positive control group (p < 0.05). Note: * p < 0.05; ** p < 0.01.

Figure 4

Specific antibody levels in humoral immunity in immunized mice. The levels of specific antibodies in the serum of 7 d, 14 d, 21 d, and 28 d in each group were detected, and the line graphs of specific antibodies were plotted.

Figure 5

The contents of cytokines IL2, IL4, IFN-γ, and TNF-α in the serum of mice on 21 and 28 days. Notably, 21 d represents the serum sample on the 21st day after the first immunization, while 28 d represents the serum sample on the 7th day after the second strengthened immunization. It is statistically significant. Note: *** p < 0.001.

Figure 6

Results of IFN-γ ELISpot experiments. (A) F317L 29 peptide, protein, negative and positive control IFN-γ ELISpot spot plots (only one mouse spot plot is shown). (B) Histogram of IFN-γ ELISpot experiment results. With positive control established, the results showed that peptide F25 (²⁴⁶SRRSLVNPWT²⁵⁵) had a statistically significantly higher number of spots per SFC/10⁵ cells than F317L protein (p < 0.01). Note: ** p < 0.01.