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