key: cord-267666-i7uuf3ck authors: Sarkar, Bishajit; Ullah, Md. Asad; Araf, Yusha; Rahman, Mohammad Shahedur title: Engineering a Novel Subunit Vaccine against SARS-CoV-2 by Exploring Immunoformatics Approach date: 2020-11-11 journal: Inform Med Unlocked DOI: 10.1016/j.imu.2020.100478 sha: doc_id: 267666 cord_uid: i7uuf3ck As the number of infections and deaths caused by the recent COVID-19 pandemic is increasing dramatically day-by-day, scientists are rushing towards developing possible countermeasures to fight the deadly virus, SARS-CoV-2. Although many efforts have already been put forward for developing potential vaccines, however, most of them are proved to possess negative consequences. Therefore, in this study, immunoinformatics methods were exploited to design a novel epitope-based subunit vaccine against the SARS-CoV-2, targeting four essential proteins of the virus i.e., spike glycoprotein, nucleocapsid phosphoprotein, membrane glycoprotein, and envelope protein. The highly antigenic, non-allergenic, non-toxic, non-human homolog, and 100% conserved (across other isolates from different regions of the world) epitopes were used for constructing the vaccine. In total, fourteen CTL epitopes and eighteen HTL epitopes were used to construct the vaccine. Thereafter, several in silico validations i.e., the molecular docking, molecular dynamics simulation (including the RMSF and RMSD studies), and immune simulation studies were also performed which predicted that the designed vaccine should be quite safe, effective, and stable within the biological environment. Finally, in silico cloning and codon adaptation studies were also conducted to design an effective mass production strategy of the vaccine. However, more in vitro and in vivo studies are required on the predicted vaccine to finally validate its safety and efficacy. . The step-by-step procedure adapted in the vaccine designing study. (https://blast.ncbi.nlm.nih.gov/Blast.cgi) tool was used in the human homology determination, 251 where Homo sapiens (taxid: 9606) was used as the comparing organism, keeping all other 252 parameter default. An e-value cut-off of 0.05 was set in the experiment and the epitopes that had 253 no hits below the e-value inclusion threshold were selected as non-homologous peptides (Mehla 254 and Ramana, 2006) . and GPGPG), PADRE sequence, adjuvant (human beta-defensin-3), and epitopes (CTL-1, CTL-2, 308 CTL-3, as well as HTL-1, HTL-2, HTL-3, and so on) in sequential and appropriate manner. Figure S1 , 424 we can see that the potential energy of the structure had quickly reduced below the order of 10 6 425 for the protein complex. From this, we inferred that the complex system was stable enough to Temperature) equilibration was done for 100 ps and pressure as well as density were calculated. The crystallized protein structure was also prepared in the same way. Then the resulting The MHC class-I and class-II epitopes were predicted from the target protein sequences for 503 constructing the vaccine. The epitopes that were found to follow all the previously mentioned 504 criteria, were finally selected for vaccine construction considering as the most promising 505 epitopes ( Table 02 ). The 100% conservancy of the epitopes among the selected isolates from IFN-gamma, IL-4, and IL-10 inducing capacity prediction of the HTL epitopes showed that 524 many of the selected HTL epitopes had the capability of inducing at least one of these cytokine. Moreover, all the most promising epitopes were also found to have at least one cytokine 526 production capability (Supplementary Table S1 , S2, S3, and S4). The vaccine has been constructed using the most promising epitopes which could be used to The CV candidate vaccine was predicted to be a potent antigen as well as a non-allergen. In the 553 physicochemical property analysis of the vaccine, a high (basic) theoretical pI, half-life of 1 h in 554 the mammalian cells, and more than 10 h in the E. coli cell culture system were predicted. Moreover, the vaccine construct was also found to be quite stable as well as soluble upon Table S6 and Supplementary Figure S5) . 570 The 3D structure of the CV vaccine construct was predicted by the online server RaptorX. The 571 vaccine had 5 domains with quite low p-value of 3.56e-08, which declared that the quality of the Figure S7) . Since potential disulfide bonds, therefore, it can be considered as a quite stable vaccine construct. Table S7) . ~0.6 nm. The black line of the graph refers to the RMSD relative to the structure present in the 681 minimized, equilibrated system and the red line is the RMSD relative to the crystal structure. Since both these plots are almost highly correlated, so it can be declared that the structure 683 remained quite stable during the experiment. The RMSF and radius of gyration graphs also point 684 to the fact that the CV-TLR-8 complex was quite stable during the experiment (Figure 05b & 685 05c ). Like the complex structure, the single vaccine crystal structure also generated very good 686 results in the MD simulation. In the RMSD graph of Figure 05d , it can be noticed that the 687 structure also pointed towards the fact that the crystallized structure didn't have any significant 690 region with the possibility to undergo deformation, so it can be deduced that the crystalized 691 vaccine CV might be quite stable in the biological environment (Figure 05e & 05f) . So, overall, by these antigen-presenting cells (APCs) (Figure 06j and Figure 06k ). The CV vaccine was also predicted to produce different types of cytokines (Figure 06l) . Henceforth, the overall 746 immune simulation study revealed that, the CV vaccine might be able to stimulate strong 747 immunogenic response after its administration. Finally, the codon adaptation and in silico cloning were performed to design a recombinant 935 plasmid which might be used for the mass production of the CV vaccine in the E. coli strain K12. In the codon adaptation study of the vaccine, the obtained results were found to be satisfactory SARS-CoV-2 vaccines: status report. Immunity FireDock: fast interaction refinement in molecular 1022 docking Codon usage: nature's roadmap to expression and folding of proteins Design of the linkers 1028 which effectively separate domains of a bifunctional fusion protein Designing a fusion protein vaccine against HCV: an in silico approach CD4+ regulatory T cells: mechanisms of 1036 induction and effector function Atypical MHC class II-expressing antigen-presenting 1224 cells: can anything replace a dendritic cell? 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