Background and Aim: African swine fever (ASF) is a viral disease of pigs caused by ASF virus (ASFV). High mortality and the lack of available treatments have severely impacted the swine industry resulting in huge global economic losses. In response to the dire necessity for vaccines, this study aims to identify highly conserved cytotoxic T-cell epitopes in ASFV structural proteins pp220, pp62, p72, p30, and CD2v through immunoinformatics approach.

Materials and Methods: The amino acid sequences of the structural proteins were retrieved from the National Center for Biotechnology Information protein database. The sequences were evaluated in CD-HIT Suite wherein resulting representative sequences were aligned in Clustal Omega. Highly conserved sequences were identified in the Protein Variability Server which were used as reference sequences for the cytotoxic T-cell epitope mapping. Epitopes were predicted using the tools in Immune Epitope Database. Peptides which bind to the swine major histocompatibility complex with IC₅₀ binding scores >500 nM were filtered out. Epitopes which are classified to be potentially toxic and cross-reactive with the swine proteome sequences were all excluded from the study. The epitopes were docked with the swine leukocyte antigen-1*0401 (SLA-1*0401) wherein the binding affinity, the binding energy, and the root-mean-square deviation (RMSD) per residue of epitope-SLA complexes formed were determined and compared with the influenza epitope as positive control.

Results: A total of 112 highly conserved fragments with Shannon variability index ≤0.1 were identified. These include 66, 12, 26, 6, and 2 highly conserved fragments from ASFV proteins pp220, pp62, p72, p30, and CD2v, respectively. From these reference sequences, 35 nonameric peptides were selected for the list of candidate cytotoxic T-cell epitopes. These include 26 epitopes for pp220, 7 for pp62, 6 for p72, and one each for p30 and CD2v. Bioinformatics analysis classified the peptides as non-toxic. Further evaluations of epitopes showed that these are less likely to cross-react with the domestic swine proteome sequences. This study identified candidate epitopes from pp220 (IADAINQEF, FLNKSTQAY, QIYKTLLEY, and SLYPTQFDY), and pp62 (GTDLYQSAM, FINSTDFLY, and STDFLYTAI) which can bind to at least two widely distributed SLAs in pig populations. The immunogenicity of candidate peptides RSNPGSFYW, DFDPLVTFY, AIPSVSIPF, and VVFHAGSLY was validated by the acceptable binding affinities, binding energies, and RMSD of the peptide-SLA complexes formed. Results were also comparable with the crystal structure of an SLA-epitope complex in the database.

Conclusion: This is the first study to identify highly conserved cytotoxic T-cell epitopes in the structural proteins of ASFV. Overall, the results of in silico evaluations showed that the identified highly conserved cytotoxic T-cell epitopes may be used as part of future vaccine formulations against ASFV infection in domesticated pigs. Nonetheless, these findings require in vitro and in vivo validation before application.

Keywords: African swine fever virus, cytotoxic T-cell epitopes, immunoinformatics, in silico.