key: cord-0823592-fdmbl9j4 authors: Bhattacharya, Manojit; Sharma, Ashish Ranjan; Mallick, Bidyut; Sharma, Garima; Lee, Sang-Soo; Chakraborty, Chiranjib title: Immunoinformatics approach to understand molecular interaction between multi-epitopic regions of SARS-CoV-2 spike-protein with TLR4/MD-2 complex date: 2020-10-08 journal: Infect Genet Evol DOI: 10.1016/j.meegid.2020.104587 sha: 3c395df992008d93b201bcb34a6710d01a55fd02 doc_id: 823592 cord_uid: fdmbl9j4 BACKGROUND: The coronavirus (CoV) spike (S) protein is critical for receptor binding, membrane fusion and internalization of the virus into the human cells. We have tried to search the epitopic component of the S-protein that might be served as crucial targets for the vaccine development and also tried to understand the molecular mechanism of epitopes and TLR4/MD-2 complex for adaptive immunity. MATERIAL AND METHODS: Here we identified the antigenicity and the epitopic divergence of S-protein via immunoinformatics approach. The study was performed to identify the epitopes, composition of amino acids and its distribution in epitopic regions, composition of amino acid between the identified epitopes, secondary structure architecture of epitopes, physicochemical and biochemical parameters and molecular interaction between the identified epitope and TLR4/MD-2 complex. The SARS-CoV-2 can be possibly recognised by TLR4 of host immune cells that are responsible for the adaptive immune response. RESULTS: We identified four SARS-CoV-2 S-protein 9mer antigenic epitopes and observed that they bind with the TLR4/MD-2 complex by varied stable molecular bonding interactions. Molecular interaction between these characterized epitopes with TLR4/MD-2 complex might be indicated the binding affinity and downstream signalling of adaptive immune response. Different physicochemical and biochemical parameters such as O-glycosylation and N-glycosylation, Hydrophobicity, GRAVY were identified within epitopic regions of S-protein. These parameters help to understand the protein-protein interaction between epitopes and TLR4/MD-2 complex. The study also revealed different epitopic binding pockets of TLR4/MD-2 complex. CONCLUSIONS: The identified epitopes impart suitable prospects for the development of novel peptide-based epitopic vaccine for the control of COVID-19 infection. cell pathogenesis and consequently recognised as the potential targets for effective vaccine or drug development. The Toll-like receptor (TLR) proteins identify pathogenic microbial elements and activate the pathway for functioning and regulation of adaptive immune response (Chakraborty et al., 2020c; Iwasaki and Medzhitov, 2004) . Among TLRs family, TLR4 type receptor protein associates with the myeloid differentiation protein 2 (MD2) to form a complex that interacts with viral antigens (Choudhury and Mukherjee, 2020; Fransen et al., 2010) . The TLR4 expressed CD4+ T-cell containing signalling pathway have facilitated the development of safe and effective clinical-grade vaccines in human (Duthie et al., 2011) . Additionally, TLR4 served as the ligand-based adjuvants which are most innovative in the development of commercial vaccines and is important for the specific adaptive responses (Reed et al., 2016) . Current study aim to screen the potential multi-epitopes that are common for B-cells and Tcells from SARS-CoV-2 S-protein sequence. The epitopes are characterised as cytotoxic T lymphocyte (CTL) epitopes, and B-cell epitopes compare to human leukocyte antigen surface marker with potential to elicit cellular and humoral immune responses. Since the whole Sprotein is not responsible to trigger B-cells and T-cells mediated immune response, here, we predicted B-cells and T-cells antigenic epitopes within the full length S-protein of SARS-CoV-2 that can bind to TLR4/MD-2 complex and might activate the adaptive immune response. The complete amino acid sequence (GenBank: QIH45053.1) of SARS-CoV-2 S-protein was retrieved in FASTA format from the National Centre for Biotechnology Information (NCBI) database (Coordinators, 2017) . The obtained amino acid sequence was used for the prediction of antigenic epitopic that can be recognized by T-cells and B-cells. J o u r n a l P r e -p r o o f Journal Pre-proof To identify the location of epitopes in 3D structure of S-protein we developed the 3D structure model of SARS-CoV-2 S-protein. We used I-TASSER server for this model development (Roy et al., 2010) . Simultaneously we have extracted SARS-CoV-2 S-protein from PDB databank (6VYV) for further analysis (Berman et al., 2000) . B-cell epitopes are recognized by lymphocytes and activate B-lymphocytes to secrete antibodies. The Immune Epitope Database (IEDB) prediction server cooperated BepiPred Linear Epitope Prediction algorithm was used to predict the B-cells epitopes present on the SARS-CoV-2 S-protein using the retrieved S-protein amino acid sequence (Jespersen et al., 2017; Vita et al., 2015) . prediction T-cell epitopes are known to trigger the cell-mediated immunity within the host cell. T-cell epitope prediction encompasses the identification of major histocompatibility complex (MHC)-I and MHC-II binding epitopes, consequently activate both the Cytotoxic T lymphocytes (CTL) and helper T-lymphocytes (HTL) mediated immune response. The MHC-I and type II epitopes were searched within B-cell epitopes to identify B-cell derived T-cell epitopes, using ProPred-I and Propred servers to predict the MHC-I and MHC-II binding epitopes, respectively (Singh and Raghava, 2003) . These servers predict epitopes with high efficiency and might propose the interacting with allelic form probability with any J o u r n a l P r e -p r o o f Journal Pre-proof of 47 MHC-I and 51 MHC-II alleles. The 9mer B-cell epitopes are further processed in Proped and Proped-I server to identify T cell as well as known common epitopes. Further, we predict the antigenic score to recognize the antigenicity of the common epitopes (both B-cells and T-cells) within the S-protein of SARS-CoV-2 using VaxiJen 2.0-Drug Design server (default threshold value of 0.45) (Bhattacharya et al., 2020; Doytchinova and Flower, 2007) . To analyse the composition of amino acids distribution in four epitopic regions we have used ProtParam tool-ExPASy and plotted the amino acids distribution (Portal, 2011) . To analyse the secondary structure we retrieved PDBsum database and marked the particular epitopic region into the secondary structure of S-protein (Laskowski et al., 1997). Glycosylation of protein is one type of post translational modification; and it is essential for functional characterization of proteins. Protein Glycosylation has been implicated in folding pattern, transport and interaction with other receptor protein. We have predicted the Oglycosylation and N-glycosylation sites of S-protein using the NetNGlyc 1.0 Server (Hamby and Hirst, 2008; Julenius et al., 2005) . Hydrophobicity of protein plays a crucial role in biomolecular interactions (Xi et al., 2017) . The hydrophobicity of S-protein was predicted based on Kyte-Doolittle algorithm by using ProtScale server (Kyte and Doolittle, 1982) . This web server was also employed to create J o u r n a l P r e -p r o o f Journal Pre-proof plots of percent accessible residues for each S-protein residues in linear weight variation model. For GRAVY analysis we selected each epitopic region of S-protein and as well as the full Sprotein (Kyte and Doolittle, 1982) . To analyse the protein structure hydrophobicity value is important. GRAVY is calculating using hydrophobicity. The sequential linear B-cell epitopes of varying lengths were identified from the IEDB server within SARS-CoV-2 strain. Specific B-cell epitopes were selected based on their positional value, sequence, and length. The epitopic calculation by IEDB applied BepiPred prediction method to support the B-cell epitopic and non-epitopic part with targeted protein chain. T-cell mediated immune response and its cascade impart to interact with peptide-MHC complexes, that is a pivotal factor for specific cellular immunogenicity. As these epitopes are selected from the B-cell epitopic region of S-protein, these can serve as both B-cell and T-cell epitopes. Here, the SARS-CoV-2 S-protein is characterized accordingly of their antigenicity and their binding affinity towards MHC-I and MHC-II alleles. We observed that the S-protein of SARS-CoV-2 consists of our 9mer multi-epitopic regions (VRQIAPGQT: 0.8675, YQAGSTPCN: 0.4992, FQPTNGVGF: 0.5711, ILPDPSKPS: 1.2217) that have high antigenicity score (>threshold score 0.45) and are considered as the probable potent antigen for both B and T cell (Figure 2A , B, C, D Table S1 ). Secondary structure architecture plays an important role in protein-protein interaction and surface accessibility (Kringelum et al., 2013) . Therefore, secondary structure has a significant role in interaction between epitopic regions of S-protein and TLR4/MD-2 complex. Component of secondary structure architecture and motif structure such as α helix, β hairpin, β turn as well as γ turn were recorded in all four epitopic region ( Figure 4A , B, C,D). Hydrophobicity is considered as a vital property of protein and we have presented the pattern of hydrophobicity of S-protein residues. The pattern of S-protein hydrophobicity was also found to confirmed higher peak in epitope ILPDPSKPS (0.5-1.0) above the -0.0‖ midline as compared with the hydrophobicity score of others epitopes. Even though, epitope VRQIAPGQT and FQPTNGVGF shown positive hydrophobicity score ( Figure 5B ). ). Subsequently, four amino acid residues of TLR4 and one from MD-2 involved to the epitope VRQIAPGQT ( Figure 6A , Table 1 ). The molecular interaction between the epitope YQAGSTPCN and TLR4/MD-2 complex was established based on the minimal ACE value of protein-protein docked composite structure. The ACE value of epitope YQAGSTPCN-TLR4/MD-2 complex was -184.83Kcal/mol. The epitope: YQAGSTPCN and TLR4/MD-2 complex formed only one hydrogen bond (bond length 3.557Å). Within this molecular interaction the single amino acid residue of TLR4 involved into the H-bond of epitope VRQIAPGQT ( Figure 6B , Table 1 ). The epitope FQPTNGVGF and TLR4/MD-2 complex was complementarily interacting to require the minimal ACE value. The ACE value of epitope FQPTNGVGF and TLR4/MD-2 complex was -432.73Kcal/mol. The epitope: FQPTNGVGF and TLR4/MD-2 complex shaped only one hydrogen bond (bond length 3.272Å). Inside this molecular interaction the single amino acid residue of MD-2 was involved to the H-bond with FQPTNGVGF epitope ( Figure 6C , Table 1 ). The epitope ILPDPSKPS and TLR4/MD-2 complex was bind with lowest ACE value -291.68Kcal/mol. The docked complex also formed one hydrogen bond (bond length 2.691 Å), and within molecular interaction the single amino acid residue of MD-2 was involved to the H-bond of ILPDPSKPS epitope ( Figure 6D , Table 1 ). The molecular interface area was 941.10Å 2 of epitope VRQIAPGQT and TLR4/MD-2 complex binding pocket, and transformation score of rotational angles of ligand was 1.11, -0.75, 2.16. Conversely, the molecular interface area score of epitope FQPTNGVGF was 783.10Å 2 and MD-2 receptor of TLR4/MD-2 complex binding pocket, and transformation score of rotational angles of ligand was 1.55, 0.57, 0.95. In case of epitope ILPDPSKPS and MD-2 receptor of TLR4/MD-2 complex binding pocket the molecular interface area was 912.90Å 2 , and transformation score of rotational angles of ligand was 1.79, 0.49, 2.45. Table 1 The four multi-epitopes of S-protein and TLR4/MD-2 complex intermolecular interactions including hydrophobic and hydrogen bonding interactions on the most representative structure obtained from cluster analysis was performed by Ligplot ( Figure S1 , A-D). J o u r n a l P r e -p r o o f Here, we identified multi-epitopes of SARS-CoV-2 S-protein that are common of both Bcells and T cells. Our observation indicated that the multi-epitopes derived from SARS-CoV-2 S-protein bind to human TLR4/MD2 complex. It is possible that the interaction of SARS-CoV-2 S-protein with TLR4/MD2 complex might induce TLR4-mediated signaling cascade that is responsible for adaptive immune response in host body after SARS-CoV-2 infection. Therefore, we studied the interaction of identified multi-epitopic region of S-protein with human TLR4/MD2 complex, which might be responsible for adaptive immune response associated with SARS-CoV-2 infection. It is known that TLR4 can recognize the viral proteins (Lester and Li, 2014) . The interaction of TLR4 viral epitopes has been associated with the viral infection and immune responses (Olejnik et al., 2018) . Geogel et al. (2007) demonstrated that vesicular stomatitis virus glycoprotein induces CD14/TLR4 dependent signalling pathway (Georgel et al., 2007) . It has been shown that the envelop glycoprotein of mouse mammary tumor virus (MMTV) binds to and activate TLR4 (Burzyn et al., 2004) . In addition, it has been demonstrated that TLR4 can also recognise the respiratory syncytial viral protein (Kurt-Jones et al., 2000) . be able to induce adaptive immune responses (Singh and Raghava, 2001) . We observed that the identified multi-epitopic regions of SARS-CoV-2 S-protein can bind with the TLR4/MD-2 complex. The binding interaction between epitope: VRQIAPGQT and TLR4/MD-2 complex formed five H-bonds and had higher molecular interface area compared to other recognised three epitopic regions. The highest value of molecular interface area between VRQIAPGQT and TLR4/MD-2 complex binding pocket, and maximum transformation score of ligand rotational angles implies that the strength of VRQIAPGQT and TLR4/MD-2 complex molecular docked interface is the most stabilised structure compared to other identified epitopic regions. On the contrary the epitopes: FQPTNGVGF and ILPDPSKPS were not able to form any molecular interaction with TLR4, however these epitopes bind with the MD-2 region of TLR4/MD-2 complex. Both the epitope shown lower value of molecular interface area with the MD-2 receptor of TLR4/MD-2 complex. The O-glycosylation and N-glycosylation sites prediction of S-protein showed no such significant consequences of studied multi-epitopic regions, except ILPDPSKPS epitope, that may play a critical role in folding of S-protein and molecular interactions with receptor molecules of TLR4/MD-2 complex. Moreover, the residues of S-protein epitope ILPDPSKPS showed strong hydrophobic interactions with the residues of TLR4/MD-2 complex compared to the epitopes VRQIAPGQT and FQPTNGVGF. In current study, we intended to identify novel multi-epitopic regions of S-protein (SARS-CoV-2) containing more effective antigenic epitopes-rich domains. The molecular binding activity of these short protein chains were assessed using bioinformatics tools to identify the stable molecular interaction between the epitopes candidate and the immunoreceptors (TLR4) that might elicits adaptive immune response. Finally we have also Figure-7 A, B, C, D) . We also projected a mechanism for the probable activation of adaptive immune response through the epitopic interaction with TLR4/MD-2 complex (Figure 8) . Therefore, our analysis support that the recognised four multi-epitope from the SARS-CoV-2 S-protein might serve as potent peptide vaccine candidate against COVID-19, by using the techniques of reverse vaccinology in platforms of immunoinformatics. The 2019-nCoV is a novel pathogenic virus that becomes a severe public health emergency of global concern. Our computational analysis of S-protein sequence of SARS-CoV-2 predicted the level of antigenicity and numbers of epitopes for B and T cells. The four multiepitopic part of SARS-CoV-2 S-protein has been found to bind with TLR4/MD-2 complex. Hence, the stable molecular interaction of S-protein 9mer multi-epitopic parts and TLR4/MD-2 possibly develop adaptive immune response. Understanding the interaction of TLR4/MD-2 complex with the screened epitopes present within S-protein of SARS-CoV2 as immunogenic target might be beneficial in the development of multi-epitopic based peptide vaccine by inducing the cell-mediated immune response. Thus, we suggest that these recognised multi-epitopes might be exploited as peptide based vaccine against SARS-CoV-2 infection. However, the proposed B and T cell epitopes-based peptide vaccine quickly needs to validate clinically that certify its safety and immunogenic profile to support on stopping this epidemic before it leads to devastating global outbreaks. All of the authors declare that there is no competing interest in this work. Table Table S1 : Common B cell and T cell epitopes, position of epitopic regions and antigenicity score of SARS-CoV-2 S-protein. Highlights:  Identification and characterization of antigenic multi-epitopic region of SARS-CoV-2 Spike (S) protein.  Molecular interaction study of SARS-CoV-2 S-protein antigenic epitopes and the human TLR4/MD-2 complex through molecular docking.  Binding with TLR4/MD-2 complex with four identified epitopes from SARS-CoV-2 Sprotein might be inducing adaptive immune response. 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Cell Discovery 6, 14 All authors conceived of the research, designed the study, interpreted data and wrote, edited the final manuscript.Appendix.