Vaccinomic approach for novel multi epitopes vaccine against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)

Abstract

Background: The spread of a novel coronavirus termed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in China and other countries is of great concern worldwide with no effective vaccine. This study aimed to design a novel vaccine construct against SARS-CoV-2 from the spike S protein and orf1ab polyprotein using immunoinformatics tools. The vaccine was designed from conserved epitopes interacted against B and T lymphocytes by the combination of highly immunogenic epitopes with suitable adjuvant and linkers.

Results: The proposed vaccine composed of 526 amino acids and was shown to be antigenic in Vaxigen server (0.6194) and nonallergenic in Allertop server. The physiochemical properties of the vaccine showed isoelectric point of 10.19. The instability index (II) was 31.25 classifying the vaccine as stable. Aliphatic index was 84.39 and the grand average of hydropathicity (GRAVY) was - 0.049 classifying the vaccine as hydrophilic. Vaccine tertiary structure was predicted, refined and validated to assess the stability of the vaccine via Ramachandran plot and ProSA-web servers. Moreover, solubility of the vaccine construct was greater than the average solubility provided by protein sol and SOLpro servers indicating the solubility of the vaccine construct. Disulfide engineering was performed to reduce the high mobile regions in the vaccine to enhance stability. Docking of the vaccine construct with TLR4 demonstrated efficient binding energy with attractive binding energy of - 338.68 kcal/mol and - 346.89 kcal/mol for TLR4 chain A and chain B respectively. Immune simulation significantly provided high levels of immunoglobulins, T-helper cells, T-cytotoxic cells and INF-γ. Upon cloning, the vaccine protein was reverse transcribed into DNA sequence and cloned into pET28a(+) vector to ensure translational potency and microbial expression.

Conclusion: A unique vaccine construct from spike S protein and orf1ab polyprotein against B and T lymphocytes was generated with potential protection against the pandemic. The present study might assist in developing a suitable therapeutics protocol to combat SARSCoV-2 infection.

Keywords: B-lymphocytes; Multiepitopes vaccine; SARS CoV-2; Spike S protein; T-lymphocytes; orf1ab polyprotein.

Fig. 1

Multiple sequence alignment (MSA) of the retrieved strains of a spike S protein and b orf1ab polyprotein using Bioedit software and ClustalW. Letters within the rectangular indicated the non-conserved areas and dots indicated the conserve regions

Fig. 2

Multi-epitope vaccine design. T helper epitopes (blue colour) and B cell epitopes (red colour) from both spike S protein and orf1ab polyprotein were linked by the short peptide linker KK, while T cytotoxic epitopes (purple color) were linked by GPGPG linker. Human β-defensin-3 (green color) was used as an adjuvant at N and C-terminals and linked by the short peptide EAAAK linkers. C-terminal 6-his was added as his-tag

Fig. 3

Cluster analysis of the HLA alleles in heat map representation. The red areas indicated strong interaction between HLA alleles while the yellow areas indicated weak interaction

Fig. 4

Secondary structure prediction plot of the vaccine construct. Alpha Helices were shown in blue color, while extended strands and beta turns were shown by red and green colours, respectively. The visualization of the prediction (a) and the score curves for each predicted state (b) were shown

Fig. 5

a The 3D model of the vaccine construct obtained after homology modelling on I-TASSER. b The 3D model was refined in modrefiner and galaxyrefiner and c the validated refined model was assessed by Ramachandran plot analysis that demonstrated 91.2%, 5.3% and 3.4% of protein residues in favoured, allowed, and disallowed (outlier) regions respectively. d ProSA-server, giving a Z-score of − 3.6

Fig. 6

Solubility of the vaccine construct as obtained by protein sol server. The solubility of the vaccine construct was shown to be 0.571 compared to 0.45 of the population average solubility of E. coli

Fig. 7

Stability of the vaccine construct by disulfide bond engineering in a the original form and b the mutant form. Six disulfide bond regions were shown in golden sticky forms indicated by white arrows in the mutant form

Fig. 8

molecular docking between the vaccine construct and the TLR4 chains. The yellow colour represents the vaccine construct while the brown color represents the TLR4 chains. a A cartoon structure of the vaccine construct docked with chain A of TLR4 while b represents the ball structure. c A cartoon structure of the vaccine construct docked with chain B of TLR4 while d represents the ball structure

Fig. 9

The cytokine levels induced by two injections of the vaccine construct given in interval of 30 days as simulated by C-ImmSim server. The main plot provided the concentration of cytokines and interleukins after the injections. The insert plot showed danger signal together with leukocyte growth factor IL-2 with the Simpson index, D (diversity) shown by the dotted line. The smaller the D value, the lower the diversity

Fig. 10

The immune simulation with vaccine construct using C-immsim server. a Immunoglobulins production increased in response to exposure to antigen injections with marked decrease in the antigen concentration observed. b Showed the B-cell populations with marked increase in the memory and non-memory immunoglobulins. Figure c and d Showed increased level in the populations of the active T helper and T cytotoxic cells per state after the injections, respectively. The resting state provided cells not exposed to antigen while the anergic state provided tolerance of the T-cells to the antigen exposures

Fig. 11

In silico cloning of the final vaccine construct sequence into the pET30a (+) expression vector. The vector was shown in black color, while the red color provided the gene coding for the vaccine construct protein. The DNA sequence of the vaccine construct was typically cloned in the MCS of the vector between BamH1 and Xho1 cutting sites