3.2 Predication for B-cell and T-cell epitope
IEDB server help for B-cell and T-cell epitopes prediction. Non-allergic, antigenic, and non-toxic were identified. Most efficient B-cell epitopes recognized that can evoke B-lymphocytes. B-lymphocytes are critical for initiating pathogen-specific antibody-mediated responses. Therefore, an ideally designed vaccine construct should possess B-cell epitopes. From 20 to 100 amino acids are utilized to speed up the immune response, as result shown in Table 1. A total number of B cell epitopes with a threshold value greater than 5.0 were selected which have Non-Allergic and Non-Toxic. Then the epitopes were incorporated into the vaccine construct. Evaluation of antigenicity was performed where 0.5 was the average value, 0.5481 was the minimum value and 1.6237 was the maximum value. A value more than 0.5 is recognized as antigenic. To get the most effective T-cell epitopes, cytotoxic T-cell epitopes FIAENVRTNV, DSFVVGDANK, VVADFYRIR, ARPRQIAMSL, EQNSNLFTV, and DSFVVGDANK which show good antigenic score were selected Table 2. Here default 0.5 was set as a threshold value, where the epitope was selected on basis of the threshold value. A total number of 6 epitopes were selected with an average of 0.69 value, and 0.5053 was the minimum and 1.3369 maximum one respectively. The IEDB MHC-II tool was used to evaluate 7 HTL epitopes against seven human allele reference sets. The epitope sequences with a good antigenicity score that showed high binding affinities were finally selected for incorporation into the vaccine construct. Table 3 shows the selected epitopes and their properties.
Table 1
Final selected CTL epitopes for incorporation into the vaccine construct with their antigenicity and Toxicity
Epitopes | Position | Antigenicity Score | Allergenicity | Toxicity |
FIAGKEGT | 278–285 | 0.5481 | Non-Allergic | None-Toxic |
PNEIEGLEERLVSRFG | 311–326 | 0.7178 | Non-Allergic | None-Toxic |
FNHKDPTIE | 388–396 | 0.8271 | Non-Allergic | None-Toxic |
IKIQEKKVKIDNIQ | 406–419 | 1.2267 | Non-Allergic | None-Toxic |
RIRVKDLTSNQRSRN | 427–441 | 1.3676 | Non-Allergic | None-Toxic |
NAFGRD | 466–472 | 1.6237 | Non-Allergic | None-Toxic |
TKLRQSNTSISDDYE | 484–498 | 0,7693 | Non-Allergic | None-Toxic |
Table 2
Helper T lymphocyte epitopes along with their percentile rank for the final vaccine construct
Epitopes | Position | Antigenicity Score | IC50 Value (nM) | Percentile rank |
FIAENVRTNV | 13–22 | 1.3369 | 46.56 | 0.42 |
DSFVVGDANK | 38–47 | 0.5053 | 22.84 | 0.2 |
VVADFYRIR | 57–65 | 0.688 | 39.16 | 0.34 |
ARPRQIAMSL | 23–32 | 0.588 | 8.74 | 0.03 |
EQNSNLFTV | 45–53 | 0.578 | 29.81 | 0.32 |
DSFVVGDANK | 38–47 | 0.5053 | 22.84 | 0.2 |
Table 3
Linear B-cell epitopes ranging in antigenicity from 0.5481 to 1.6237
Epitopes | Position | Residues number | Antigenicity | Allergenicity |
FFIMTTWDK | 11–19 | 0.5481 | 0.9357 | Non-Allergic |
FFSTTSVEI | 34–43 | 0.7178 | 0.5819 | Non-Allergic |
IKIQEKKVK | 51–60 | 0.8271 | 1.6229 | Non-Allergic |
KTWIKPIHV | 30–38 | 1.2267 | 0.5995 | Non-Allergic |
RPRQIAMSL | 22–31 | 1.3676 | 0.5156 | Non-Allergic |
YGLTVSVDM | 41–50 | 1.6237 | 1.726 | Non-Allergic |
YRSADILLI | 52–59 | 0.7693 | 0.5476 | Non-Allergic |
3.3 MHC-1 and MHC-2 predication
SMM process was used for estimation of MHC HLA alleles on basis of IC50 value. The lower IC50 value represents the highest binding affinity which may bind with MHC-1 molecules. Lower the value shows maximum interaction. A value less than 0.4 considered toxic and allergic, was removed. Epitopes that have antigenic, Non-toxic, and Non-allergic were selected for further screening. The MH Class-1 epitopes were finalized and HLA-B*07:02, HLA-A*68:01, HLA-A*02:06, HLA-A*02:01, and HLA-A*68:01 alleles were recognized as dominant. The epitopes FIAENVRTNV determined the highest antigenic score 1.3369. MHC-2 epitopes were finalized on basis of IC50 value. Out of 100 epitopes for MHC-II, 7 were selected on basis of IC50 values. Epitopes were finalized on basis of toxicity, Allergenicity, and antigenicity for further screening. The epitopes YGLTVSVDM, IKIQEKKVK, and FFIMTTWDK are predicated as a higher binder that interacts with alleles (HLA-DRB5*01:01, HLA-DRB5*01:01, HLA-DRB4*01:01, and HLA-DRB1*07:01).
3.4 Constructed vaccine
B cell epitopes, MHC-1, and MHC-2 epitopes are used for generating vaccine ensembles. For developing vaccine construct, 50S ribosomal protein is considered an adjuvant. Specific immune response produced, when adjuvant and B-cell epitope coupled through EAAAk linker. MHC-1 and B-cell linkage was achieved using CPGPG linkers. For reduction of vaccine size, the B cell epitopes, CTL, and HTL epitopes are combined through the AAY linker Fig. 2. For protein determination and purification, a 6x His tag was inserted in the vaccination sequence (at C-terminus).
3.5 Physical and chemical properties
The characterization of vaccine construct was performed on physical and chemical properties and physiochemical properties of Francisella tularensis demonstrated via ProtParm which recognizes 193 amino acids. A constructed sequence such as non-toxic, antigenic, and non-reactogenic behavior was checked. The molecular formula is C982H1572N286O281S4 and the molecular weight is 22009.27 KDa and the PI value is 9.65. Instability index 36.82 and Aliphatic index 89.38 was. The GRAVY score was − 0.390.
3.5.1 Structural Aspects of the Vaccine
Secondary structure showing 45% alpha-helix 20% beta-sheets and 35% coil structure
Figure. The PROSA 3D server was used to predict the 3D structure of the vaccine sequence, resulting in ten predicted structures for a given query sequence. The fifth model was taken for further investigation Fig. 3. The ERRAT, ProSA-web, and PROCHECK services were used to validate the structure, identifying and correcting any potential mistakes in the projected tertiary structure. The ERRAT server projected the overall quality of the vaccine 3D structure, and the estimated quality score was 90.0%S. The Z-score was calculated to see if the input structure was within the range of similar-sized natural proteins. Figure 3b shows that the computed Z-score for the input structure was − 9.77, indicating that it was outside the normal range for natural proteins of the same size. For Ramachandran analysis, the PROCHECK server computed 92.2% of the residues in the most favored areas, 7.3% in additional allowed regions, 0.5% in generously allowed regions, and 0.0% in residues in disallowed regions.
3.5.2 Molecular Docking
The designed protein was docked with TLR9 (PDB id 3WPF) with help of Cluspro to find interaction. Docking provides multiple complexes and is further analyzed by the Pymol server. PDBePISA and PDBsum have used a representation of different interactions. The H-bond result revealed the interaction formed between the residues are ASP259-SER122, ARG346-ASP127, ARG377-ASP127, PHE402-LYS130, HIS260-SER136, ALA261-LYS152, PRO262-LYS152, ARG482-TYR179, ASP424-TYR179, ARG482-ASP183, ARG482-ASP183, ARG470-ASN189, ARG470-LYS190, GLN399-ARG193, THR395-ARG193, ASN473-ASP204, LYS472-ASP204 and ASN466-LYS219 showing the distance of 2.83, 2.87, 3.15, 2.51, 2.66, 2.69, 2.61, 2.80, 2.90, 2.74, 2.88, 2.77, 2.65, 2.55, 2.67, 2.61, 2.48 and 2.48 respectively Fig. 4.
3.5.3 Immune Simulation
We evaluated the vaccine's ability to trigger an effective immune response in practical use using the C-ImmSim webserver. After the primary reaction, the secondary and tertiary immune responses rose steadily. Antibodies (IgM, IgM + IgG, and IgG1 + IgG2) levels have risen dramatically. Fig represents immune response showed a similar response to other immune responses in the human body. The antibodies IgM and IgG were detected along with memory cell formation Fig. 5a. Similarly, there was a large rise in B-cell counts, with IgG1 and IgM biotypes observed as well as significant memory cell development Fig. 5b. Active T-cells increased dramatically after getting a secondary and tertiary injection dose, although they gradually declined at later stages Fig. 5c, d. an increasing amount of IFN-γ and TH cell population is also noted in Fig. 5e, f.
3.5.4 Optimization of Codons
In E. Coli, codon optimization and reverse translation were determined, which find high expression in vaccines via the Jcat tool. Vaccine construct codons were tailored to E. coli expression system codons using the Jcat web tool. The cDNA sequence generated was 720 nucleotides long. In the vaccine, the GC value was 64.42 and CAI was 0.95, these expressions showing expression was high. The designed construct's 5 and 3 ends were restricted using BanI and TaiI restriction enzymes. The restricted sequence was successfully ligated into the pET28a (+) vector, yielding a 582 bp clone. The cloned map was created with SnapGene Fig. 6.
3.5.5 Analysis of MD Simulation
MD simulation is a popular method for efficiently analyzing protein structural reliability in a simulated environment. To get information about the stability and flexibility of the protein MD simulation was performed via Amber under 100ns to identify RMSD and RMSF analysis. Till 100ns, both RMSD and RMSF were stable. The construct was first energy minimized by using Pymol. Then topology file for both vaccine construct and TLR-9 was generated. To neutralize the system 7 Na+ ions were added to the system. The generated trajectories were evaluated to learn more about the construct's RMSD and RMSF. The calculated RMSD of the backbone atoms reached 8(Å) and oscillated between 4 and 8(Å) throughout the simulation, indicating strong protein stability. The estimated RMSF values also identified flexible regions with peak-like patterns Fig. 7.