Genome based evolutionary lineage of SARS-CoV-2 towards the development of novel chimeric vaccine

Abstract

The present study aimed to predict a novel chimeric vaccine by simultaneously targeting four major structural proteins via the establishment of ancestral relationship among different strains of coronaviruses. Conserved regions from the homologous protein sets of spike glycoprotein, membrane protein, envelope protein and nucleocapsid protein were identified through multiple sequence alignment. The phylogeny analyses of whole genome stated that four proteins reflected the close ancestral relation of SARS-CoV-2 to SARS-COV-1 and bat coronavirus. Numerous immunogenic epitopes (both T cell and B cell) were generated from the common fragments which were further ranked on the basis of antigenicity, transmembrane topology, conservancy level, toxicity and allergenicity pattern and population coverage analysis. Top putative epitopes were combined with appropriate adjuvants and linkers to construct a novel multiepitope subunit vaccine against COVID-19. The designed constructs were characterized based on physicochemical properties, allergenicity, antigenicity and solubility which revealed the superiority of construct V3 in terms safety and efficacy. Essential molecular dynamics and normal mode analysis confirmed minimal deformability of the refined model at molecular level. In addition, disulfide engineering was investigated to accelerate the stability of the protein. Molecular docking study ensured high binding affinity between construct V3 and HLA cells, as well as with different host receptors. Microbial expression and translational efficacy of the constructs were checked using pET28a(+) vector of E. coli strain K12. However, the in vivo and in vitro validation of suggested vaccine molecule might be ensured with wet lab trials using model animals for the implementation of the presented data.

Keywords: COVID-19; Chimeric vaccine; Evolutionary relationship; Molecular docking; Normal mode analysis; Restriction cloning; SARS-CoV-2.

Graphical abstract

Fig. 1

Flow chart summarizing the protocol of multi-epitope subunit vaccine design against SARS-CoV-2 through reverse vaccinology approach.

Fig. 2

Phylogeny of 61 species of coronaviruses. Seven pathogenic human coronaviruses have been represented by blue star and the IDs have been made bold. COVID-19 clade has been shown with red colour. SARS-CoV-1 and MERS virus have been represented by orange and blue colors, respectively. The genera have been represented on the left coloured labels. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Phylogeny of spike glycoprotein of coronavirus. The sub-genera have been labeled in the left table. The filled and unfilled circles show the presence and absence of the domains labeled on the top.

Fig. 4

Phylogeny of envelope proteins of coronaviruses. The sub-genera under three different genera have been shown on the left labels. The star signs represent the COVID-19 virus. 5 different pathogenic human corona viruses have been shown in bold form, including the MERS virus (blue) and SARS-CoV-1 (yellow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Phylogeny of membrane proteins of coronavirus. The sub-genera under three different genera have been shown on the left labels. The blue and orange star represent the SARS-CoV1 and MERS virus, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Phylogeny of nucleocapsid proteins of coronavirus. The sub-genera under three different genera have been shown on the left labels. The filled and unfilled circles show the presence and absence of the domains labeled on the top. COVID-19, SARS-CoV1 and MERS viruses are clades are labeled with blue, green and red colors, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Population coverage analysis of spike protein (A), envelope protein (B), membrane protein (C) and nucleocapsid proteins (D).

Fig. 8

Homology modeling, structure validation and solubility prediction of construct V3 (A: Cartoon structure, B: Surface structure, C: Ramachandran Plot analysis, D: Quality factor analysis, E: Solubility analysis).

Fig. 9

Predicted conformational epitopes (A and B) and linear epitopes (C and D) within construct V3.

Fig. 10

Normal Mode Analysis (NMA) of vaccine protein V3. The directions of each residues are given by arrows and the length of the line represented the degree of mobility in the 3D model (A and B). The main-chain deformability derived from high deformability regions indicated by hinges in the chain which are negligible (C). The experimental B-factor was taken from the corresponding PDB field and calculated from NMA (D). The eigenvalue represents the motion stiffness and directly related to the energy required to deform the structure (E). The variance associated to each normal mode is inversely related to the eigenvalue. Coloured bars show the individual (red) and cummulative (green) variances (F). The covariance matrix indicates coupling between pairs of residues, where they may be associated with correlated, uncorrelated or anti-correlated motions, indicated by red, white and blue colors respectively (G). The elastic network model identifies the pairs of atoms connected via springs. Each dot in the diagram is coloured based on extent of stiffness between the corresponding pair of atoms. The darker the greys, the stiffer the springs (H). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 11

Docked complex of vaccine construct V3 with human ACE 2 (A), TLR- (B), DPP4 (C) and APN (D).

Fig. 12

In silico restriction cloning of the gene sequence of final vaccine construct V3 into pET28a(+) expression vector (A: Restriction digestion of the vector pET28a(+) and construct V3 with BglII and BglI, B: Inserted desired fragment (V3 Construct) between BglII (401) and BglI (1943) indicated in red colour. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. S1

Secondary structure prediction of constructed vaccine protein V3.

Fig. S2

3D modelled structure of vaccine protein V1 and V2.

Fig. S3

Disulfide engineering of vaccine protein V3 (A: Initial form, B: Mutant form).