key: cord-0920157-2uvibr2j authors: ul Qamar, Muhammad Tahir; Rehman, Abdur; Ashfaq, Usman Ali; Awan, Muhammad Qasim; Fatima, Israr; Shahid, Farah; Chen, Ling-Ling title: Designing of a next generation multiepitope based vaccine (MEV) against SARS-COV-2: Immunoinformatics and in silico approaches date: 2020-03-22 journal: bioRxiv DOI: 10.1101/2020.02.28.970343 sha: 9c3eb069962650a4696bed5155668d1c5480a3b1 doc_id: 920157 cord_uid: 2uvibr2j Coronavirus disease 2019 (COVID-19) associated pneumonia caused by severe acute respiratory coronavirus 2 (SARS-COV-2) was first reported in Wuhan, China in December 2019. Till date, no vaccine or completely effective drug is available to cure COVID-19. Therefore, an effective vaccine against SARS-COV-2 is crucially needed. This study was conducted to design an effective multiepitope based vaccine (MEV) against SARS-COV-2. Seven antigenic proteins were taken as targets and different epitopes (B-cell, T-cell and IFN-γ inducing) were predicted. Highly antigenic and overlapping epitopes were shortlisted. Selected epitopes indicated significant interactions with the HLA-binding alleles and 99.29% coverage of the world’s population. Finally, 505 amino acids long MEV was designed by connecting sixteen MHC class I and eleven MHC class II epitopes with suitable linkers and adjuvant. Linkers and adjuvant were added to enhance the immunogenicity response of the MEV. The antigenicity, allergenicity, physiochemical properties and structural details of MEV were analyzed in order to ensure safety and immunogenicity. MEV construct was non-allergenic, antigenic, stable and flexible. Molecular docking followed by molecular dynamics (MD) simulation analysis, demonstrated a stable and strong binding affinity of MEV with human pathogenic toll-like receptors (TLR), TLR3 and TLR8. Codon optimization and in silico cloning of MEV ensured increased expression in the Escherichia coli K-12 system. Designed MEV in present study could be a potential candidate for further vaccine production process against COVID-19. However, to ensure its safety and immunogenic profile, the proposed MEV needs to be experimentally validated. Viruses are dangerous pathogens and can cause irreversible losses to human lives and economy. 2 The world hardly learns to deal with a virus when new emerges and threatens the future of 3 humanity. A similar situation arises when a new strain of coronavirus (CoV) not previously 4 identified in humans was reported last year (2019) [1] . Positive-sense RNA viruses called corona 5 viruses belong to the Coronaviridae family that are distributed broadly among human and 6 mammals. In the last two decades there have been more than 10,000 reported infections of two CoV-2 was indicated root cause of COVID-19 through deep sequencing analysis from lower 15 respiratory tract samples of patients [6] . SARS-COV-2 genome sequence is almost 70% similar 16 to SARS-COV, and 40% similar to the MERS-COV [7] . Symptoms of SARS-COV-2 may arise 17 within 2 days or up-to 14 days of exposure. Symptoms such as fever, diarrhea and respiratory 18 disorder are found in infected patients [4] . According to latest research SARS-CoV-2 has an 19 identical genomic organization as of beta-coronaviruses; 5'-untranslated region (UTR), orf1ab 20 (replicas complex), nsps (encoding non-structural proteins), S (spike) protein, E (envelope) 21 protein, M (membrane) protein, Oraf6, orf7a, orf8, N (nucleocapsid) protein, orf10, 3'-UTR and 22 several unknown non-structural open reading frames [3, 8] . 23 There is currently no vaccine or approved treatment for COVID-19. Few traditional 24 Chinese medicine such as Shufengjiedu capsules and Lianhuaqingwen capsules were reported diseases vaccine is the most effective approach. Now a days, availability of genomic 30 information, advance software and immunological data sets could greatly facilitate researchers to 1 identify the effective epitopes from pathogens' proteins that can be used to develop active sub-2 unit vaccines [12] [13] [14] [15] . The subunit vaccine contains the fragments of antigenic proteins of 3 pathogen that can trigger an immune response against the target pathogen [16, 17] [24] with promising results. The in silico methods reduce the number of in vitro experiments and 7 save time, overcome cost obstacles and increase the potential for successful vaccine design [25-8 27] . 9 In present study, SARS-CoV-2 proteome was explored to determine the potent antigenic 10 proteins and their further screening for B-cell, T-cell and IFN-γ inducing epitopes prediction 11 with their MHC (major histocompatibility complex) alleles. Antigenicity, allergenicity and 12 toxicity of predicted epitopes were analyzed. Multiepitope based vaccine (MEV) construct was 13 designed by using the most potential and interacting epitopes, with the addition of suitable 14 linkers and an adjuvant. Adjuvants are generally defined as molecules that may increase or 15 modulate the intrinsic immunogenicity of an antigen [28] . Adjuvants are essential to reduce the 16 amount of antigen and the number of injections, as they help to induce effective and persistent 17 immune responses [29] . Several in silico approaches were utilized to validate the antigenicity, The Expassy Protparam tool was used to determine the physical and chemical properties of 10 selected proteins [31] . To check protein antigenicity, the Vaxijen 2.0 software was used [32] . The threshold value was held at 0.5, and the secondary structure of proteins was predicted by 12 using SOPMA tool [33]. Tertiary stores of most of SARS-CoV-2 proteins are not reported yet. Therefore, combinations of 2 different approaches were employed to predict good quality structures for further analysis. Online tools such as Swiss model which work on homology based modeling algorithms and 4 Raptor X which work on deep learning modules, were primarily used for the tertiary structure In immune system the B-Cell epitope helps to detect viral infection and activities. B-cells Epitopes which show favorable strong binding affinities with a common experimentally 12 validated allele, are good choice to design MEV construct. Therefore, molecular docking 13 between screened epitopes and human allele was performed. Molecular docking is an in silico 14 approach which determined the binning affinity between ligand and its target proteins, and also 15 highlight the important residues involve in the interaction. 3D structures of overlapping, highly 16 antigenic and conserved epitopes with corresponding common alleles were predicted using The selected epitopes for MEV construct should effectively cover major populations across the 24 globe. For population coverage, overlapping, antigenic, conserved and strongly interacting with 25 HLA-B7 allele epitopes were selected and further analysed using the IEDB population coverage 26 analysis tool by maintaining the default analysis parameters [44] . This tool is designed to estimate 27 8 the population coverage of epitopes from diverse counties based on the distribution of their 1 MHC-binding alleles. As SARS-CoV-2 is global pandemic, therefore, worldwide analysis has 2 been performed. 4 To construct a sub-unit vaccine, the epitopes with following properties are usually preferred: (a) As vaccine construct is combination of different epitopes, therefore, the RaptorX server was used 22 to develop MEV 3D tertiary structure. The RaptorX server use a multi-template threading 23 approach to determine the tertiary structure of query protein [34]. 24 25 Galaxy Refine server MD simulation approach was used to refine the MEV predicted 3D 1 MD simulation is an important approach to analyse the stability of the receptor-ligand complex 2 [41, 66] . Complexes of MEV with TLR3 and TLR8 were simulated at 20 ns using GROMACS 23 The amino acid sequences of SARS-CoV-2 important vaccine target proteins (ORF1 Table 1 ) and their secondary structures was predicted using SOPMA tool 8 (Supplementary Table 2 ). The 3D models of selected proteins tertiary structures were predicted using Swiss model tool 10 and Raptor X tool, and in order to select best quality models, predicted structures were further 11 refined by galaxy refine server followed by Ramachandran plot validation. The most of 12 structures predicted using Swiss model were of better quality than Raptor X predicted structures, 13 except for N and Orf7a proteins. Therefore, good quality models were selected for further 14 analysis (Supplementary Figure 1) . There was no suitable structure predicted for ORF10 because 15 of small number of residues. So, its structure was predicted by PEPFOLD server [48] 16 Table 3) . 18 Screened out B-cell epitopes were 100% conserved in all protein sequences and ere highly 19 antigenic. All the target proteins were predicted to have a total 55 linear epitopes (E-4, M-12, 20 ORF6-1, ORF7a-6, ORF8-9, N-22, and ORF10-1) (Supplementary Table 4 ). Moreover, a total 21 of 24 (E-4, M-2, ORF6-3, ORF7a-4, ORF8-4, NC-4, and ORF10-3) conformational epitopes 22 were predicted in all target proteins (Supplementary Table 5 ). As mentioned before, epitopes that can bind to multiple alleles because of their strong 24 defense capabilities are considered the most appropriate epitopes. Therefore, total 31 MHC class Table 7) . 29 The HTLs helps to activate CTLs together with other immune cells upon various types of Table 8) . 6 As stated before, to construct a sub-unit vaccine, the chosen epitopes should be 100% conserved, 7 overlapping and antigenic. Therefore, total 50 conserved/antigenic epitopes from selected 14 Docking binding energy scores together with their detail information is mentioned in Table 1 . All the 27 selected epitopes ensured their binding efficiency as well as their suitability to be used 16 in multiple epitope-based vaccine construct. 1 The distribution and expression of HLA alleles vary by ethnic groups and regions of the world. Epitopes were merge together based on their interaction's compatibility in sequential manner 7 with AAY and GPGPG linkers respectively. AAY and GPGPG prevents the generation of 8 junctional epitopes, which is a major concern in the design of multiepitope vaccines; On the 1 First, Blast-p analysis was performed against Homo sapiens proteome with default parameters to 2 validate that MEV is non-homologous. Protein with less than 37% identity generally considered 3 as non-homologous protein. However, MEV showed no similarity (higher or equal to 37%) with 4 the human proteins. Next, allergenicity, antigenicity and toxicity of the vaccine construct were evaluated. 6 Results described that MEV is highly antigenic (0.6741 at 0.5% threshold), non-allergenic and 7 non-toxic. Figure 4) . 25 To determine the tertiary structure of vaccine RaptorX server was used. Structure was refined 26 by Galaxy refine server (Figure 4) show that the refined model is of good quality. 3 B-lymphocytes besides secreting cytokines, also produce antigens, which in return provide 4 humoral immunity [77] . Therefore, MEV ideally should have B-cell epitopes with its domains. 9 The in silico immune system simulation against MEV showed significant activity of B-cells and 8 An appropriate association between immune receptor molecules and the antigen molecule is 9 essential to activate an immune responsiveness. HADDOCK server has thus been used to shown in the orange color, while the MEV is shown in the blue color, respectively, in Figure 6A . In case of TLR8, it is shown in the grey color, while the MEV is shown in the blue color, 11 respectively, in Figure 7A . It was observed that MEV made 9 hydrogen bond interactions within 12 range of 3.00 Å with TLR8 Figure 6B -C. Similar to TLR3, MEV interacting amino acids with 13 hydrogen bonding to TLR8 shown in green color stick representation, while TLR8 amino acids 14 interacting through hydrogen bonding with MEV shown in red color stick representation. These 15 results indicated that this MEV is best suitable candidate for vaccine production. 11 MD simulation is a common approach used to analyse the micro-interactions between 12 ligand/vaccine and protein/receptor structures [66, 78] . In order to further assess MEV dynamics 13 and stability, its docking complexes with TLR3 and TLR8 were simulated by 20 ns MD followed showing the high possibility of positive protein expression and reliability. In next step, buffer 1 compatible restriction enzymes XhoI and HindIII restriction sites were added to the both ends of 2 MEV optimized nucleotide sequence to aid the cloning/purification process. Finally, refined 3 MEV sequence was cloned between XhoI and HindIII restriction sites at the multiple cloning site 4 of pET28a (+) vector (Figure 9 ). The total length of the clone was 6.875 kbp. none have yet been declared as clinically approved anti-COVID-19 therapeutic. In this study, a 21 successful attempt was made to design a sub-unit MEV against SARS-COV-2. Immunoinformatics and in silico approaches were used to develop a potential and safe MEV that 23 could trigger three types of the immune responses: humoral, innate and cellular. A highly 24 immunogenic, safe, stable and strongly interacting with human receptors, MEV has been 25 reported in present study that could be a potential candidate for vaccine production. However, Table 1 of main text. with yellow color dotted lines. Numbering 1-11 is consistent with the Table 1 of main text. 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