Fig 1.
The flow of vaccine design and evaluation.
Table 1.
The key properties of candidate vaccine proteins.
Fig 2.
Results of amino acid sequence similarity comparison between Brucella melitensis and other pathogenic Brucella species.
(A) Sequence comparison results for OMP31. (B) Sequence comparison results for LptE. (C) Sequence comparison results for VIRB2.
Fig 3.
Protein signal peptide prediction results (A-F).
(A) Predicted OMP31 signal peptide results using SignalP 6.0. (B) Predicted OMP31 signal peptide results using LiPOP 1.0. (C) Predicted LptE signal peptide results using Signal P 6.0. (D) Predicted LptE signal peptide results using LiPOP 1.0. (E) Predicted VirB2 signal peptide results using SignalP 6.0. (F) Predicted VirB2 signal peptide results using LiPOP 1.0.
Table 2.
Selected CTLs dominant epitopes of multi-epitope vaccines.
Table 3.
Selected HTLs dominant epitopes of multi-epitope vaccines.
Table 4.
Selected LBEs dominant epitopes of multi-epitope vaccines.
Table 5.
Selected CBEs dominant epitopes of multi-epitope vaccines.
Fig 4.
B-cell conformational epitopes (A-E).
(A-B) B-cell conformational epitope residues of OMP31. (C-D) B-cell conformational epitope residues of LptE. (E) B-cell conformational epitope residues of VirB2.
Fig 5.
The docked results of T cell epitopes with HLA (A-B).
(A) Molecular docking result of CTL epitopes to HLA-A*02:01. (B) Molecular docking result of HTL epitopes to HLA-DRB1*01:01.
Fig 6.
Multi-epitope vaccine construction diagram.
Table 6.
Properties of designed multi-epitope vaccines.
Fig 7.
Prediction of secondary and tertiary structures of multi-epitope vaccines(A-C).
(A-B) Prediction results of MEV secondary structure. (C) pymol visualization of Robetta prediction of MEV tertiary structure results.
Fig 8.
Prediction and refinement of tertiary structures of multi-epitope vaccines(A-C).
(A) Quality factor plot for the improved vaccine protein. (B) Ramachandran Plot of MEV tertiary structure after refinement. (C) Z-value plot for the improved vaccine protein.
Fig 9.
Disulfide bond engineering results for vaccines.
Vaccine construct after disulfide bond engineering. The thin yellow lines in the figure indicate constructed disulfide bonds.
Fig 10.
The docking results and interaction of TLR4-MEV (A-B).
(A) The docking of the TLR4 -MEV complex using Pymol to demonstrate. (B) Result of the interaction of TLR4-MEV complex and its 2D image using Ligplot + v.2.2.9. Hydrogen bonds are represented by dotted green lines, and red semicircles indicate residues involved in hydrophobic interactions.
Fig 11.
Basic analysis result of the interaction and stability between TLR4 and MEV.
(A) B-factor and NMA graph. (B) The plot variance associated to the each modes. (C) Eigenvalues plot. (D) The complex residue deformability graph. (E) The elastic network models plot. (F) Covariance matrix graph.
Fig 12.
Molecular dynamics simulation results.
(A) RMSD plots of TLR4 and the complex. (B) Rg plots of TLR4 and the complex. (C) RMSF plots of the four chains of the TLR4 protein bound to MEV. (D) SASA plots of TLR4 and the complex. (E) Hydrogen bond plots of the complex.
Fig 13.
The result of Optimization of MEV codons and in silico cloning (A-D).
(A) The GC content after codon optimization is 53.73%. (B) MEV DNA sequence of after PCR. (C) The red region is the amplified target gene sequence of MEV inserted into Escherichia coli-BCG shuttle plasmid pMV261. (D) Simulated agarose gel electrophoresis results. “1” represents MEV after PCR, “2” represents shuttle plasmid pMV261, “3” represents pMV261-MEV-DNA recombinant plasmid.
Fig 14.
Immunostimulation simulation results(A-F).
(A) Number of immunoglobulins produced after 3 injections. (B) The B cell population after 3 injections. (C) The B cell population per state after 3 injections. (D) The number of helper T cells after 3 injections. (E) The cytotoxic T cells population per state after 3 injections. (F) Number of cytokines and interleukins after 3 injections.