Development of an ancestral DC and TLR4-inducing multi-epitope peptide vaccine against the spike protein of SARS-CoV and SARS-CoV-2 using the advanced immunoinformatics approaches

Abstract

The oldest human coronavirus that started pandemics is severe acute respiratory syndrome virus (SARS-CoV). While SARS-CoV was eradicated, its new version, SARS-CoV2, caused the global pandemic of COVID-19. Evidence highlights the harmful events orchestrated by these viruses are mediated by Spike (S)P protein. Experimental epitopes of the S protein which were overlapping and ancestral between SARS-CoV and SARS-CoV-2 were obtained from the immune epitopes database (IEDB). The epitopes were then assembled in combination with a 50 S ribosomal protein L7/L12 adjuvant, a Mycobacterium tuberculosis-derived element and mediator of dendritic cells (DCs) and toll-like receptor 4 (TLR4). The immunogenic sequence was modeled by the GalaxyWeb server. After the improvement and validation of the protein structure, the physico-chemical properties and immune simulation were performed. To investigate the interaction with TLR3/4, Molecular Dynamics Simulation (MDS) was used. By merging the 17 B- and T-lymphocyte (HTL/CTL) epitopes, the vaccine sequence was created. Also, the Ramachandran plot presented that most of the residues were located in the most favorable and allowed areas. Moreover, SnapGene was successful in cloning the DNA sequence linked to our vaccine in the intended plasmid. A sequence was inserted between the XhoI and SacI position of the pET-28a (+) vector, and simulating the agarose gel revealed the existence of the inserted gene in the cloned plasmid with SARS vaccine (SARSV) construct, which has a 6565 bp in length overall. In terms of cytokines/IgG response, immunological simulation revealed a strong immune response. The stabilized vaccine showed strong interactions with TLR3/4, according to Molecular Dynamics Simulation (MDS) analysis. The present ancestral vaccine targets common sequences which seem to be valuable targets even for the new variant SARS-CoV-2.

Keywords: Computational immunology; Immunoinformatics; Multi-epitope; Reverse vaccinology; SARS-CoV; SARS-CoV-2.

Graphical abstract

Fig. 1

The strategy of designing multi-epitope vaccine for SARS-CoV and SARS-CoV-2.

Fig. 2

Analysis of vaccine population coverage. Population coverage of different country: China 96.29 %, East Africa 91.61 %, Europe: 100 %, India: 97.49 %, Iran: 91.12 %, Japan: 96.33 %, North Africa: 92.37 %, South America: 94.01 %, West Africa 90.87 %.

Fig. 3

MHC I and MHC II cluster analysis. A: MHC class I cluster analysis heatmap B: MHC I cluster analysis advanced tree map C: MHC class II cluster analysis heatmap D: MHC class II cluster analysis advanced tree map.

Fig. 4

Structural analysis A: Prediction of secondary structure with disorder region in PSIPRED web server B: The vaccine's final 3D modeled structure C: Frequency of Secondary structure plot detail of alpha helix, Beta sheet, and super coil in SOPMA server.

Fig. 5

Validation of tertiary structure of SARSV. Variation of tertiary structure before refinement and after refinement with Z-score in ProSA-web server.

Fig. 6

conformational B-cell epitopes predicted in SARSV. The yellow mark showing conformational epitopes. A: 52 residues score: 0.84, B: 65 residues score: 0.74, C: 52 residues score: 0.63, D: 32 residues score: 0.61, E: 4 residues score 0.51. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Docking between peptide and MHC molecules that viewed with UCSF Chimera. A: The interaction of MHC II (HLA-DRB*0101) and KCYGVSA. B: The interaction of MHC I (HLA-A*0201) and SLIDLQELGKYEQYIKW peptide. The Yellow line showed the interaction of MHC and peptide. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

TLR3/4-Vaccine docking. A: The top and high score SARS vaccine construction binding pose with Toll-like receptor-4. The docked complex revealed the contact interaction and the residues in different colors, as well as the hydrogen bonds in blue and other reports. B: The top Binding pose of SARS vaccine (SARSV) construct with Toll-like receptor-3. The docked complex (protein-protein) shown the contact interaction. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 9

Molecular Dynamic simulation of TLR3/4-vaccine complex. Result of different simulation step are demonstrated, including A: Energy during minimization B: Temperature C: Density during the NPT equilibration D: Pressure E: RMSD plot of the c-alpha atoms F: Gyration G: RMSF to detect the stability of complex during the simulation. H: SASA I: H-bond.

Fig. 10

Design of SARSV mRNA. A: Secondary structure of the predicted mRNA, highlighting the 3′ and 5′ terminal that had not hairpin; B: Frequency of single stranded strands in 25 folding; C: The circular plot displays the RNA construct's base pairs; D: The suggested mRNA's energy dot plot.

Fig. 11

Cloning and gel simulation A: In silico cloning of SARSV construct in pET-28a (+) expression vector where the red sequence cloned. B: A vaccine construct built using double separation has been virtual cloned. Line 1: Separated structure of vaccine (SARSV and plasmid) with two enzymes XhoI and SacI; line 2: Separation of two enzymes, plasmid pET-28a (+); line 3: digestion of two enzymes. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 12

Immune simulation profile of SARSV that injected 3 doses. A: B cell population (cells per mm³) B: PLB cell population IgG/IgM (cells per mm³) C: B cell per state (cells per mm³) D: TH cell population (cells per mm³) E: TH cell population per state (cells per mm³) F: Cytokine profile. G: TC cell population (cells per mm³) H: TC cell population per state (cells per mm³) I: NK cell population (cells per mm³) J: Antigen count K: DC population per state (cells per mm³).

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