3.1.1 Physicochemical characteristics and basic biological characteristics of W541vaccine protein
The W541 vaccine protein sequence contains 704 amino acid residues with a molecular weight of 74.6 kDa and a theoretical isoelectric point of 6.29. It exhibits an average hydrophobicity of -0.294, an aliphatic index of 68.85, an instability index of 45.37, and possesses 57 phosphorylation sites and 7 glycosylation sites. The expressed vaccine protein lacks signal peptide and transmembrane domains. Upon expression in human cells, it localizes to lysosomes and the cytoplasm. It had a half-life of 1.1 hours in mammalian reticulocytes, 3 minutes in yeast, and 2 minutes in E. coli . These data indicate that the W541 vaccine protein is a relatively large soluble protein prone to degradation. The W541 vaccine protein also displays commendable antigenicity, non-allergenicity, non-toxicity, and no homology with human proteins (Table 2).
3.1.2 The spatial structures of the W541 vaccine protein and docking with TLR4
According to the analysis conducted by the SOPMA server, the secondary structure of the W541 protein exhibited a distribution of α-helices, β-sheet, β-turns, and random coils, accounting for 26.99%, 19.03%, 11.51%, and 42.47% of the total sequence, respectively. These four secondary structures were arranged alternately throughout the overall structure but had one distinct feature: the α-helices primarily exist in the central and posterior parts of the protein (figure 3A). Due to their inherently flexible nature, the random coils and β-turns tend to be situated on the protein surface, showcasing a prominent structure. This region is usually enriched with epitopes that are advantageous for B-cell recognition.
For the W541 vaccine protein, AlphaFold2 predicted five possible tertiary structures exhibiting high similarities. Both Ag85A and Ag85B sequences were folded separately, while the three C-terminal epitopes exist independently as α-helices and random coils. The Prosa server scored respectively the five predicted structures as -10.99, -10.96, -10.65, -10.60, and -10.97, which fall within the expected range of the server’s protein model database. This result indicates a high reliability of the predicted results (figure 3B).
The top-ranking W541 vaccine protein A structure was selected to study the interaction with TLR4 to evaluate the immune system activation effect of the vaccine. The central region of the W541 vaccine protein showed a remarkable docking affinity with the B chain of TLR4, with a binding energy of -10.7 kcal/mol and an interface area of 2209.7Ų (figure 3C). The lower the binding energy indicates the closer vaccine-TLR binding, the more likely it is to activate TLR4 and induce the production of cytokines and chemokines to enhance immune responses.
3.1.3 B-cell epitopes of W541 vaccine protein
B-cell epitopes can bind to B cell receptors (BCRs), activating downstream signaling cascades that regulate B-cell activation and antibody production. Activated B cells can also act as antigen-presenting cells. The prediction results from the ElliPro server showed that the W541 vaccine protein had 15 linear epitopes with prediction scores ranging from 0.511 to 0.755 and 11 discontinuous epitopes with predicted scores ranging from 0.543 to 0.987 (shown in Supplementary Table 1).
3.1.4 HTL and CTL epitopes of W541 vaccine protein
Based on the selection criteria, the W541 vaccine protein contained a total of 138 HTL epitopes (Supplementary Table 2) and 73 CTL epitopes (Supplementary Table 3), in which 101, 63, 3, and 19 HTL epitopes could induce IFN-γ, IL-4, IL-6, and IL-10, respectively. The W541 protein contained 10 HTL toxic epitopes and 6 CTL toxic epitopes. These toxic epitopes were concentrated within two discreet regions of the amino acid chain, totaling 21 residues (Supplementary Table 4).
3.1.5 Population coverage analysis and molecular docking of T lymphocyte epitopes with MHC for W541 vaccine protein
The population coverage analyses on the HTL and CTL epitopes of the W541 vaccine protein and their corresponding MHCs were performed using the Population Coverage server. The results revealed that the population coverage rates of HTL and CTL epitopes were 99.68% and 98.36%, respectively. 138 HTL epitopes that stimulate IFN-γ secretion (Supplementary Table 2) and 73 CTL epitopes (Supplementary Table 3) could recognize 24 MHC class II molecules and 26 MHC class I molecules, respectively. Some of these epitopes had a binding affinity for a single MHC molecule, while others could bind to multiple MHC molecules. From this epitope library, 18 HTL epitopes (antigenicity threshold > 0.8, inducing only IFN, IC50 < 500) and corresponding MHC molecules are displayed in Table 3A; 27 CTL epitopes (antigenicity threshold > 0.8, class I immunogenicity > 0.1, IC50 < 500) and corresponding MHC molecules are shown in Table 3B. Molecular docking results of two selected T-cell epitopes with their corresponding MHC molecules are shown in figure 4.
3.1. 6 Immune simulation in silico
3.1.6.1 Activation of innate immune cells by