Fig 1.
Flowchart representation of the methodology process for the current study.
Table 1.
The antigenic scores of the six proteins- MSA-2c, AMA-1, TASP, SPAG-1, Vir B10, and OMP1 with their location.
Table 2.
Protparam analysis of the six outer membrane proteins- MSA-2c, AMA-1 (Babesia bovis) TASP, SPAG-1 (Theileria annulata) and Vir-B10, OMP-1 (Anaplasma marginale).
Table 3.
The lowest percentile ranks and highest scores achieved for selected MHC I epitopes.
Table 4.
The lowest percentile ranks and highest scores achieved for selected MHC II epitopes.
Table 5.
Assessment of the features of templates used to predict the tertiary model of bovine MHC II alleles.
Table 6.
Assessment of the protein-peptide interaction energy and average RMSD values for BoLA allele docking to the four MHC II epitopes.
Table 7.
Antigenicity prediction, screening of transmembrane topology, allergenicity, conservancy, along with toxicity assessment of the six best major histocompatibility complex class I epitopes.
Table 8.
Antigenicity prediction, screening of transmembrane topology, allergenicity, conservancy along with toxicity assessment of the six best major histocompatibility complex class II epitopes.
Fig 2.
Three algorithm methods for b-cell epitope prediction for the two outer membrane proteins of Babesia bovis- MSA-2c(A), AMA-1(B).
The X-axis of each graph displays position while the y-axis shows the score. Epitopes lying above the threshold value are situated in yellow color. Most favored position is represented by the longest peak found in yellow color. Bepipred linear Epitope Prediction Method (Left column), Emini Surface Accessibility Prediction Method (Centre), and Kolaskar and Tongaonkar Prediction Method (Right column).
Fig 3.
Three algorithm methods for b-cell epitope prediction for the two outer membrane proteins of Theileria annulata- SPAG-1(C), TASP(D). The X-axis of each graph displays position while the y-axis shows the score. Epitopes lying above the threshold value are situated in yellow color. Most favored position is represented by the longest peak found in yellow color. Bepipred linear Epitope Prediction Method (Left column), Emini Surface Accessibility Prediction Method (Centre), and Kolaskar and Tongaonkar Prediction Method (Right column).
Fig 4.
Three algorithm methods for b-cell epitope prediction for the two outer membrane proteins of Anaplasma marginale- Vir-B10(E) and OMP1(F). The X-axis of each graph displays position while the y-axis shows the score. Epitopes lying above the threshold value are situated in yellow color. Most favored position is represented by the longest peak found in yellow color. Bepipred linear Epitope Prediction Method (Left column), Emini Surface Accessibility Prediction Method (Centre), and Kolaskar and Tongaonkar Prediction Method (Right column).
Table 9.
Antigenicity prediction, screening of transmembrane topology, allergenicity, conservancy along with toxicity assessment of the best b-cell epitopes generated from each of the six proteins AMA-1, MSA-2c (Babesia bovis) TASP, SPAG-1 (Theileria annulata) and Vir-B10, OMP-1 (Anaplasma marginale).
Fig 5.
Graphical illustration of the newly designed chimeric vaccine construct.
Table 10.
The best epitopes used for the vaccine construction along with adjuvants and linkers.
Table 11.
Allergenicity assessment, antigenicity prediction and solubility analysis of the three constructed vaccines.
Table 12.
Physicochemical characteristics of the three chimeric vaccine constructs.
Fig 6.
Structure prediction of the chimeric vaccine, V3.
(A) Three-dimensional structure of the chimeric vaccine construct, V3 (ribbon model) predicted by the RaptorX server. This shows that the construct consists of three domains. The three types are: alpha-helix, beta sheet, and coils. (B) Secondary structure of the chimeric vaccine construct, V3 predicted by CFSSP server. This shows that the construct V3 consists of 61.0% of Helix, 15.3% of Beta-pleated sheet and 15.3% of Turns in their structure. There are a total of 358 helix residues, 90 Beta-sheet residues and 90 Turn residues. The letter ‘H’ is denoted for helix. The letter ‘E’ is denoted for Beta-sheet and the letter ‘T’ is denoted for turns.
Fig 7.
Ramachandran plot analysis of the chimeric vaccine construct, V3 (after refinement) predicted by the PROCHECK program of the SAVE v6.1 server.
Fig 8.
Disulphide engineering of the chimeric vaccine construct, V3 performed by the DbD2 v2.13 server.
The right picture shows the original structure of the chimeric vaccine construct while the left picture shows the mutant chimeric vaccine construct with one disulphide bond (Yellow cylinders) created between single pair of amino acids.
Fig 9.
The discontinuous b-cell epitopes in the three-dimensional structure of the chimeric vaccine, V3 model.
The gray stick models represent the 3D models of the chimeric vaccine, and the yellow spheres represent the discontinuous b-cell epitopes (A) 38 residues with a score of 0.948; (B) 5 residues with a score of 0.874; (C) 66 residues with a score of 0.838; (D) 30 residues with a score of 0.837; (E) 3 residues with a score of 0.78; (F) 55 residues with a score of 0.674; (G) 53 residues with a score of 0.552; (H) 35 residues with a score of 0.542; (I) 35 residues with a score of 0.53.
Table 13.
List of discontinuous epitopes and their predicted scores.
Fig 10.
Molecular docking of the chimeric vaccine construct, V3 with Toll-like receptor TLR9 and with RP-105 (30% resemblance in sequence to TLR4) predicted by HDOC server.
(A) The red colored model represents the chimeric vaccine construct, V3 while the white colored model represents the Toll-like receptor 9 (sphere model). (B) The red colored model represents the vaccine construct, V3 while the blue colored model represents Toll-like receptor 9 (ribbon model). (C) The red colored model represents the vaccine construct, V3 while the white colored model represents RP-105 (sphere model). (D) The red colored model represents the vaccine construct, V3 while the blue colored model represents RP-105 (ribbon model).
Fig 11.
Amino acid residues of RP-105 involved in docking with the chimeric vaccine construct, V3.
The red colored ribbon model represents the chimeric vaccine, V3 and the blue colored stick model represents the RP-105 receptor. The amino acids Ser322, Glu149, Glu197, Gln198, Ala199, Ile220, Tyr135, Asp49, Asp334, Gln335, Gln338, Asp360, Ser385, Lys435, Ala436, Tyr187, Ser189, Lys191, Asp192, Glu215, Pro216, Asn234, Phe236, Ile237, Tyr262, Thr264, Ala266, Thr267, Ser287, Glu367, Lys393, Asp413, and Glu418 of RP-105 have docked with chimeric vaccine, V3.
Fig 12.
Amino acid residues of TLR9 involved in docking with the chimeric vaccine construct, V3.
The red colored ribbon model represents the chimeric vaccine, V3 and the blue colored stick model represents the TLR-9 receptor. The amino acids Lys50, His54, Ala57, His76, His78, Lys300, Arg304, Asp331, Arg388, Pro390, Arg495, Leu471, Arg522, Glu546, Arg576, Arg599, Arg654, Arg676, Gly698, Arg700, and Glu774 of TLR-9 have docked with chimeric vaccine, V3.
Table 14.
Global binding Energy, Hydrogen Bond Energy, Atomic contact energy (ACE) and docking scores of the docked complexes V3-TLR9 and V3-TLR4.
Fig 13.
The molecular docking of the peptides with the toll-like receptor TLR-9.
The peptides are represented in yellow colored surface model which is bonded to the rainbow-colored 3D surface models of TLR9.
Table 15.
Assessment of the docking energy scores obtained from the molecular docking of MHC I, MHC II and b-cell epitopes with TLR9 and RP-105.
Fig 14.
Molecular dynamic simulations of the chimeric vaccine construct, V3 with TLR-9 by the iMODS server.
(A) The docking of chimeric V3 to TLR9 (B) Main chain deformability (C) B-factor values (D) The eigenvalue (E) Variance (F) Co-variance (G) Elastic network of the model.
Fig 15.
The immune simulation of chimeric vaccine, V3 by C-ImmSimm server.
(A) Production of immunoglobulins in response to the chimeric vaccine, V3 (B) Population of B-cells in response to V3, γ2 represents the scale of memory B-cells (C) Population of Helper T-cells in response to V3, γ2 represents the scale of memory T-cells (D) Concentration of cytokines and interleukins produced.
Fig 16.
The insertion of the DNA sequence of the chimeric vaccine, V3 in to the plasmid vector, pET-28 (+).
(A) The map of plasmid pET-28 (+). We can see that the vector, pET-28(+) represented here is intact and was 5369 bp before insertion of the chimeric vaccine, V3. (B) The adapted DNA sequence of the chimeric vaccine V3, to which the restriction enzymes BglI and BglII are added at each end. The adapted DNA sequence is 1778 bp long. We can observe all kinds of restriction sites present in the adapted DNA sequence. (C) Insertion of chimeric vaccine V3 DNA into the vector, pET-28 (+). The vector, pET-28(+) is 3590 bp in length and the cloned product (DNA) is 5349 bp in length.