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.