2.1.4 The spatial structure analyses of W541 vaccine protein and molecular docking with Toll-like receptor (TLR)4 
Using the SOPMA serve predicted the secondary structure of the W541 vaccine protein (45). Utilize the bKunyun supercomputing platform to execute the AlphaFold2(46)to predict the tertiary structure of W541, which was then authenticated using the Prosa server(47).
The GRAMM server can systematically assess a series of docking postures between proteins and ligands and predict the most stable docking conformation(48). Firstly, the PDB files of TLR4 were downloaded from the PDB database(49) and uploaded to the GRAMM server along with the W541 PDB files predicted by AlphaFold to predict the docking status between the TLR4 and W541 vaccine. Finally, the PDBePISA server (50) was used to calculate the detailed docking data of the docking complexes, such as the interaction surface areas and binding energies.
2.1.5 Prediction of B-cell epitopes in the W541 vaccineprotein 
The linear and discontinuous B-cell epitopes of W541 vaccine proteins were predicted using the ElliPro server(51) with default parameters by uploading the AlphaFold-predicted W541 PDB file.
2.1.6 Prediction of HTL and CTL epitopes in the W541 vaccine protein
The helper T lymphocytes (HTL) epitopes were predicted by the MHC-II Binding Predictions server(52) using the “IEDB recommended method” specified for the Full HLA reference set with default parameters, which can predict all the 15 amino acid residues in the W541 vaccine protein. Later the epitopes(excluding epitopes containing linker amino acids) with IC50 values below 500 nM were further analyzed by the VaxiJen server with a threshold of 0.4, the IFN epitope server(53), IL4pred server (54), IL6pred server(55), and IL-10pred server (56) to predict their antigenicity, the abilities to stimulate IFN-γ, IL-4, IL-6, and IL-10 secretion with default parameters. 
The cytotoxic T lymphocytes (CTL) epitopes were predicted by the MHC-I Binding Predictions server(57) using the “recommended epitope predictor” specified for the default HLA allele reference set, which can predict all the 9 and 10 amino acid residues CTL epitopes of the vaccine. Further, the epitopes (excluding epitopes containing linker amino acids) with IC50 values below 500 nM were analyzed using the Class I Immunogenicity server with default parameters to predict their immunogenicity (58)and using the VaxiJen server to predict their antigenicity with default parameters.
At last, all the HTL and CTL epitopes of the W541 vaccine protein were submitted to AllerTOP v. 2.0 serve and ToxinPred serve to predict their allergenicity and toxicity with default parameters.
2.1.7 Analysis of population coverage and molecular docking of T-cell epitopes with MHC molecules for the W541 vaccine
The population coverage of the vaccine was predicted by the IEDB Population Coverage tool(59). The molecular docking between the vaccine epitopes and MHC molecules was performed using the GRAMM docking server. Firstly, the PDB files of the MHC molecules were downloaded from the RCSB PDB database and processed with Pymol software 2.0 (an open-source tool) to remove unnecessary ligands. Meanwhile, the structures of all docking epitopes were predicted using the PEP-FOLD 3.5 server (60). The PDB files of MHC molecules and corresponding epitope structures were then submitted to the GRAMM docking server using the ”free docking” mode. The docking results were analyzed using the PDBePISA server.
2.1.8 Immune simulation in silico
The immune responses to the W541 vaccine were simulated using the C-ImmSim server(61). In this study, we performed three rounds of in silico immunization with the W541 vaccine at 0, 14, and 28 days to simulate the immune effects of the W541 vaccine.