In-silico Immunomodelling of 2019-nCoV

Background: Novel Corona Virus 2019 (2019-nCoV) is a positive-sense single-strand RNA virus form coronaviridae family, responsible for corona virus infectious disease 2019 (COVID-19) with rapid transmission. The aim of this study is characterization of major viral proteins, prediction of antigen proteasomal cleavage pattern, MHC class I processing and presentation, B-and T-cell epitopes, and anti-inammatory epitopes of 2019-nCoV, compared with SARS-CoV. Methods: The aminoacid sequence of spike surface (S) glycoprotein, membrane (M) glycoprotein, envelop (E) protein and nucleocapsid (N) phosphoprotein were obtained from NCBI. The sequences were aligned by MEGA 7.0 and modeled by SWISS-MODEL. The proteasomal cleavage pattern, MHC class I processing and T-cells epitopes were predicted via IEDB analysis and EPISOFT. The B-cell epitopes were predicted by BepiPred 2.0. Also, prediction of anti-inammatory epitopes was performed by AntiFlam. Results: Two major antigen proteins, S glycoprotein and M glycoprotein of 2019-nCoV, respectively, have 26.57% and 20.59% less e�ciency in proteasomal cleavage and presentation to MHC class I, comparing SARS-CoV. There are less B-cell predicted epitopes in 2019-nCoV, comparing SARS-CoV. The anti-inammatory properties of 2019-nCoV S glycoprotein and N protein is higher than SARS-CoV. Discussion: It seems that the evolution of 2019-nCoV is on the way of de�ciency in antigen presenting to MHC class I and escaping from cellular immunity. Also, the predicted hotspot epitopes potentially can be used to induction of adaptive cellular immunity against 2019-nCoV. In addition, 2019-nCoV appears to be less immunopathogenic than SARS-CoV due to its higher anti-inammatory proteins.


Introduction
At the moment, pandemic of Novel Corona Virus 2019 (2019-nCoV) [responsible for coronavirus infectious disease of 2019 (COVID-19)] is a huge challenge facing to infectious medicine (1).This positive-sense single-strand RNA virus, a member of coronaviridae family, has high invasion power and rapid transmission rate via droplet and aerosol.2019-nCoV has more than 186,000 con rmed cases and 4,500 mortality worldwide, as 22 February 2020 (2).The primary role of the immune system in the face of viral infections is up to cellular immunity.Antigenpresenting cells (APCs) with antigen proteasomal cleavage presents digested peptides to T helper lymphocytes via MHC class I (3).Also, it has been shown that humoral immune system plays a role in adaptive immunity against viruses (4).Despite conventional coronaviruses, e.g.Severe Acute Respiratory Syndrome corona virus (SARS-CoV), 2019-nCoV shows more intensity in transmission and immune response failure.Bioinformatics tools have enabled us to simulate the evolution mechanism of 2019-nCoV in the way of incomplete immune response.
Sequencing of 2019-nCoV genome in the early days of COVID-19 pandemic has enabled humans to predict the probable protein and nucleoprotein structures of 2019-nCoV as a proteomic-based therapeutic and prophylaxis approaches.It is possible that 2019-nCoV has evolved in antigen presentation to adaptive immune system in the way of survival and escaping from immune system.This evolution may be responsible for the alteration of presented

In-silico Characterization of Viral Proteins
After approval of difference in SARS-CoV and 2019-nCoV proteomic sequence, the mentioned proteins were characterized via SWISS-MODEL (Swiss Institute of Bioinformatics, Biozentrum).(5)(6)(7)(8) The comparative 3D-structures, table of comparison with Non-redundant set of PBD structure (adjusted by normalized QMEAN4 score and residual size), and Ramachandran plots were plotted to estimating the difference of the individual protein structure model between SARS-CoV and 2019-nCoV viral proteins.

Prediction of Proteasomal Cleavage Pattern and T Cell-Related Potential Epitope
To prediction of proteasomal cleavage and MHC class I processing of viral antigens, The MHC class I binding predictions were made on 2/26/2020 using the IEDB analysis resource Consensus tool (9) which combines predictions from ANN aka NetMHC (4.0) (10-12), SMM (13) and Comblib (14).The results were categorized by 9-meric peptides, as the biological digestion pattern.For each 9-meric peptide, Proteasome Score, Torsion angle propensity (TAP) Score, MHC Score, Processing Score, Total Score and MHC-50% inhibitory concentration (IC50) were reported.Then, all results were sorted by "Total Score."Top-ten 9meric peptides were inserted to EPISOFT."EPISOFT predicts epitope HLA I (MHC class I) binding pro les and population protection computes (PPC).It also identi es minimal sets of epitopes that reach a target PPC for 5 distinct user-selected ethnic groups," manufacturer says (15).The EPISOFT results report PPC score and MHC class I binding pro le.The statistical analysis was performed by EXCEL 2019.

Prediction of B Cell-Related Potential Epitope
Sequential B-Cell Epitope Predictions were done by BepiPred ver.2.0."The BepiPred-2.0 server predicts Bcell epitopes from a protein sequence, using a Random Forest algorithm trained on epitopes and nonepitope amino acids determined from crystal structures," manufacturer says (16).

Prediction of Anti-in ammatory Peptides
To predict the anti-in ammatory property of 9-meric epitopes on S glycoprotein, M glycoprotein, E protein and N phosphoprotein proteins in SARS-COV and 2019-nCOV protein done via "Prediction of Antiin ammatory Epitopes server (AntiFlam)" [metagenomics.iiserb.ac.in/antiin am]."It allows the users to predict the anti-in ammatory nature of the multiple variants of the query peptide (substitution of each amino acid of the peptide with other amino acids), and thus, is useful in assessing the position-speci c effects of each amino acid in modulating the anti-in ammatory activity of the peptide", manufacturer says.

Minor Differences in Protein Homology of SARS-CoV and 2019-nCoV
There are no major, but Minor differences within the structure of homologous S glycoprotein, M glycoprotein, E protein and N phosphoprotein of SARS-CoV and 2019-nCoV (Figure 1).The lled 3dstructures shows different exposure pattern, implying that sequences of 2019-nCoV viral proteins directly affect on 3D-structure and surface exposure pattern.This result establishes evolution of 2019-nCoV proteome being potentially able to lead to the different behavioral pattern, comparing to SARS-CoV.Minor differences in dots of Ramachandran plots con rm minor, but not major, structural homologous variation of SARS-CoV and 2019-nCoV.
Additionally, comparison of modeled proteins with Non-redundant set of PBD structure ( gure 1-b) con rms the comparative results.

Difference in Proteasomal Cleavage Pattern and MHC Class I Presentation of T-cells Epitopes of 2019-nCoV and SARS-CoV
The IEDB analysis showed the proteasomal cleavage pattern of 9-meric peptides of 2019-nCoV and SARS-CoV are different.The top-ten 9-mer peptides of each viral protein were compared via "total score" index.The S glycoprotein of 2019-nCoV has 26.57% less e ciency in antigen proteasomal cleavage and presentation to MHC class I, compared to SARS-CoV S glycoprotein.Also, the other giant surface-exposed protein, M protein of 2019-nCoV, has 20.59% less e ciency in proteasomal cleavage and presentation to MHC class I compared to SARS-CoV M glycoprotein.(Table 1) Despite above proteins, the E protein and N phosphoprotein of 2019-nCoV and SARS-CoV show the different pattern in antigen proteasomal cleavage and presentation to MHC class I.There is 0.08% more e ciency in the cleavage of E protein of SARS-CoV and its presentation to MHC class I, compared to 2019-nCoV.Also, proteasomal cleavage and MHC class I presentation of N phosphoprotein of SARS-CoV done 15.19% more e cient, compared to 2019-nCoV.

Difference in B-cells Epitopes of 2019-nCoV and SARS-CoV
The results of prediction of potential B-cells epitopes via BepiPred V2.0 show that S glycoprotein and M glycoprotein of 2019-nCoV contain higher-potent B-cell epitopes than SARS-CoV, while E protein and N phosphoprotein of SARS-CoV contain higher-potent B-cell epitopes than 2019-nCoV.(Figure 2) Also, SWISS-MODEL results of structure prediction approved that the predicted epitopes have surface exposure.

Differences in Anti-in ammatory Properties of 2019-nCOV and SARS-COV Epitopes
The Protein scan of four major viral proteins of 2019-nCoV and SARS-COV, e.g. S glycoprotein, M glycoprotein, E protein and N phosphoprotein proteins, show that the 9-meric epitopes of 2019-nCOV have higher anti-in ammatory properties in S glycoprotein (264 9-meric epitopes in 2019-nCOV [Top-ten mean score = 4.50 ± 0.10] , comparing 77 9-meric epitopes in SARS-CoV [Top-ten mean score = 4.34 ± 0.36] ) and N phosphoprotein (224 9-meric epitopes in 2019-nCOV, comparing 54 9-meric epitopes in SARS-CoV).Despite two above proteins, analysis of the anti-in ammatory property of the 9-meric M glycoprotein epitopes in 2019-nCOV and SARS-COV shows that this protein has the slightly more anti-in ammatory property SARS-CoV (29 9-meric anti-in ammatory epitopes), comparing 2019-nCoV (23 9-meric antiin ammatory epitopes).The analysis of E protein in these two viruses also showed the same result (7 9meric anti-in ammatory epitopes).(Table 2) Discussion 2019 novel corona virus (2019-nCoV), which is responsible for corona virus infectious disease 2019 (COVID-19), evolved to higher transmission potential comparing to Severe Acute Respiratory Syndrome corona virus (SARS-CoV), which introduced this virus as a global concern.Bioinformatic tools equipped us to realize the molecular evolution pattern of 2019-nCoV.
After the virus enters the host body, the antigen presenting cells (APCs) capture them and start proteasomal cleavage process of viral proteins.After antigen digestion, 9-meric peptides present to Thelper lymphocytes in the cellular immunity system, via major histocompatibility complex (MHC) class I. Now, cellular immunity leads to eliminate infectious cells from host body.Different pattern of viral protein digestion leads to different MHC class I presenting, and nally, different immune responses to viruses.Spike (S) surface glycoprotein, membrane (M) glycoprotein, envelop (E) protein and nucleocapsid (N) phosphoprotein are major structural proteins in 2019-nCoV.Our results authenticate that there is different in proteasomal cleavage pattern of S glycoprotein, M glycoprotein, E protein and N phosphoprotein in 2019-nCoV comparing to SARS-CoV.The results show that two major antigen proteins of 2019-nCoV, S glycoprotein and M glycoprotein, have 26.57% and 20.59% fewer e ciency in antigen proteasomal cleavage and presentation to MHC class I respectively, comparing to SARS-CoV.Thus, cellular immune system is more powerless in the elimination of 2019-nCoV, in comparison with SARS-CoV.
Our results show that there is minor difference homology in row alignment of S glycoprotein, M glycoprotein, E protein and N phosphoprotein.Also, SWISS-MODEL results reveal the minor difference in row homology leads to minor changes in 3D-structure of these viral proteins.In another hand, various researches established the role of humoral immunity against viral infections.Thus, it is possible that 2019-nCoV has evolved to escape from humoral immune system.The exposed B-cell epitopes of 2019-nCoV are predicted by BepiPred, and were compared to SARS-CoV.The results showed that S glycoprotein and M glycoprotein (most exposed proteins) of SARS-CoV contain higher potent epitopes.So, it seems that probably, humoral immune system is more involved in response to SARS-CoV compared to 2019-nCoV.
Control of in ammatory response of the immune system to these two viruses is of great importance in controlling the complications, since the basis of the pathogenesis in these two viruses is immunopathogenic.According to the analysis results of the anti-in ammatory properties of 9-meric epitopes on the four main proteins of 2019-nCoV and SARS-CoV, 2019-nCoV appears to be less pathogenic than SARS-CoV due to its higher anti-in ammatory proteins and better control of the in ammatory response of immune system.The less mortality of 2019-nCoV con rms this.The immune response should be controlled to the extent that it prevents both the proliferation of the virus and does not lead to in ammation and its complications.Therefore, it is better to prioritize the design of the drug and vaccine to achieve this goal.
proteomic homology, proteasomal cleavage pattern, Major histocompatibility complex (MHC) class I presenting, and B-and T-cells epitopes of SARS-CoV and 2019-nCoV with different bioinformatics tools, in four major viral proteins, including spike (S) surface glycoprotein, membrane (M) glycoprotein, envelop (E) protein and nucleocapsid (N) phosphoprotein.Methods 2.1 Proteome Sequences and Alignment The amino acid sequences of S glycoprotein, M glycoprotein, E protein and N phosphoprotein in SARS-CoV (Accession Number NC_004718) and 2019-nCoV (Accession Number MT106053) were obtained from "Nucleotide" database [National Center for biotechnology information (NCBI)].The open reading frame of S glycoprotein, M glycoprotein, E protein and N phosphoprotein were aligned by MEGA ver.7.0 for investigation of row homology.

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