In Silico Analysis of Neutralizing Antibody Epitopes on The Hepatitis C Virus Surface Glycoproteins

Document Type : Original Article


1 Department of Virology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran

2 Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University-TMU, Tehran, Iran

3 Department of Biotechnology, Biotechnology Research Center, Venom and Biotherapeutics Molecules Lab, Pasteur Institute of Iran, Tehran, Iran

4 Department of Pharmacy, Drug Design and Development Research Center, Tehran University of Medical Sciences, Tehran, Iran


Despite of antiviral drugs and successful treatment, an effective vaccine against hepatitis C virus (HCV)
infection is still required. Recently, bioinformatic methods same as prediction algorithms, have greatly contributed to
the use of peptides in the design of immunogenic vaccines. Therefore, finding more conserved sites on the surface
glycoproteins (E1 and E2) of HCV, as major targets to design an effective vaccine against genetically different viruses
in each genotype was the goal of the study.

Materials and Methods:
In this experimental study, 100 entire sequences of E1 and E2 were retrieved from the NCBI
website and analyzed in terms of mutations and critical sites by Bioedit 7.7.9, MEGA X software. Furthermore, HCV-1a
samples were obtained from some infected people in Iran, and reverse transcriptase-polymerase chain reaction (RTPCR)
assay was optimized to amplify their E1 and E2 genes. Moreover, all three-dimensional structures of E1 and
E2 downloaded from the PDB database were analyzed by YASARA. In the next step, three interest areas of humoral
immunity in the E2 glycoprotein were evaluated. OSPREY3.0 protein design software was performed to increase the
affinity to neutralizing antibodies in these areas.

We found the effective in silico binding affinity of residues in three broadly neutralizing epitopes of E2
glycoprotein. First, positions that have substitution capacity were detected in these epitopes. Furthermore, residues
that have high stability for substitution in these situations were indicated. Then, the mutants with the strongest affinity
to neutralize antibodies were predicted. I414M, T416S, I422V, I414M-T416S, and Q412N-I414M-T416S substitutions
theoretically were exhibited as mutants with the best affinity binding.

Using an innovative filtration strategy, the residues of E2 epitopes which have the best in silico binding
affinity to neutralizing antibodies were exhibited and a distinct peptide library platform was designed.


1. Lindenbach BD, Rice CM. The ins and outs of hepatitis C virus entry and assembly. Nat Rev Microbiol. 2013;11(10): 688-700.
2. Freedman H, Logan MR, Law JL, Houghton M. Structure and function of the hepatitis C virus envelope glycoproteins E1 and E2: antiviral and vaccine targets. ACS Infect Dis. 2016; 2(11): 749-762.
3. Shimizu YK, Hijikata M, Iwamoto A, Alter HJ, Purcell RH, Yoshikura H. Neutralizing antibodies against hepatitis C virus and the emergence of neutralization escape mutant viruses. J Virol. 1994; 68(3): 1494-1500.
4. Law M, Maruyama T, Lewis J, Giang E, Tarr AW, Stamataki Z, et al. Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. Nat Med. 2008; 14(1): 25-27.
5. Perotti M, Mancini N, Diotti RA, Tarr AW, Ball JK, Owsianka A, et al. Identification of a broadly cross-reacting and neutralizing human monoclonal antibody directed against the hepatitis C virus E2 protein. J Virol. 2008; 82(2): 1047-1052.
6. Keck ZY, Saha A, Xia J, Wang Y, Lau P, Krey T, et al. Mapping a region of hepatitis C virus E2 that is responsible for escape from neutralizing antibodies and a core CD81-binding region that does not tolerate neutralization escape mutations. J Virol. 2011; 85(20): 10451-10463.
7. Keck ZY, Xia J, Cai Z, Li TK, Owsianka AM, Patel AH, et al. Immunogenic and functional organization of hepatitis C virus (HCV) glycoprotein E2 on infectious HCV virions. J Virol. 2007; 81(2): 1043-1047.
8. Owsianka AM, Tarr AW, Keck ZY, Li TK, Witteveldt J, Adair R, et al. Broadly neutralizing human monoclonal antibodies to the hepatitis C virus E2 glycoprotein. J Gen Virol. 2008; 89(Pt 3): 653-659.
9. Keck ZY, Sung VM, Perkins S, Rowe J, Paul S, Liang TJ, et al. Human monoclonal antibody to hepatitis C virus E1 glycoprotein that blocks virus attachment and viral infectivity. J Virol. 2004; 78(13): 7257-7263.
10. Kong L, Kadam RU, Giang E, Ruwona TB, Nieusma T, Culhane JC, et al. Structure of hepatitis C virus envelope glycoprotein E1 antigenic site 314-324 in complex with antibody IGH526. J Mol Biol. 2015; 427(16): 2617-2628.
11. Keck ZY, Xia J, Wang Y, Wang W, Krey T, Prentoe J, Carlsen T, Li AY, Patel AH, Lemon SM, Bukh J, Rey FA, Foung SK. Human monoclonal antibodies to a novel cluster of conformational epitopes on HCV E2 with resistance to neutralization escape in a genotype 2a isolate. PLoS Pathog. 2012; 8(4): e1002653.
12. Potter JA, Owsianka AM, Jeffery N, Matthews DJ, Keck ZY, Lau P, et al. Toward a hepatitis C virus vaccine: the structural basis of hepatitis C virus neutralization by AP33, a broadly neutralizing antibody. J Virol. 2012; 86(23): 12923-12932.
13. Sautto G, Tarr AW, Mancini N, Clementi M. Structural and antigenic definition of hepatitis C virus E2 glycoprotein epitopes targeted by monoclonal antibodies. Clin Dev Immunol. 2013; 2013: 450963.
14. Bhardwaj VK, Singh R, Sharma J, Das P, Purohit R. Structural based study to identify new potential inhibitors for dual specificity tyrosine-phosphorylation- regulated kinase. Comput Methods Programs Biomed. 2020; 194: 105494.
15. Gopalakrishnan C, Kamaraj B, Purohit R. Mutations in microRNA binding sites of CEP genes involved in cancer. Cell Biochem Biophys. 2014; 70(3): 1933-1942.
16. Singh R, Bhardwaj VK, Purohit R. Potential of turmeric-derivedcompounds against RNA-dependent RNA polymerase of SARSCoV-2: an in-silico approach. Comput Biol Med. 2021; 139: 104965.
17. Tanwar G, Purohit R. Gain of native conformation of Aurora A S155R mutant by small molecules. J Cell Biochem. 2019; 120(7): 11104-11114.
18. Rajendran V, Gopalakrishnan C, Purohit R. Impact of point mutation P29S in RAC1 on tumorigenesis. Tumour Biol. 2016; 37(11): 15293-15304.
19. Rajendran V, Gopalakrishnan C, Sethumadhavan R. Pathological role of a point mutation (T315I) in BCR-ABL1 protein-A computational insight. J Cell Biochem. 2018; 119(1): 918-925.
20. De Berardinis P, Sartorius R, Fanutti C, Perham RN, Del Pozzo G, Guardiola J. Phage display of peptide epitopes from HIV-1 elicits strong cytolytic responses. Nat Biotechnol. 2000; 18(8): 873-876.
21. Li W, Li L, Sun T, He Y, Liu G, Xiao Z, et al. Spike protein-based epitopes predicted against SARS-CoV-2 through literature mining. Med Nov Technol Devices. 2020; 8: 100048.
22. Bozovičar K, Bratkovič T. Evolving a peptide: library platforms and diversification strategies. Int J Mol Sci. 2019; 21(1): 215.
23. Mink MA, Benichou S, Madaule P, Tiollais P, Prince AM, Inchauspe G. Characterization and mapping of a B-cell immunogenic domain in hepatitis C virus E2 glycoprotein using a yeast peptide library. Virology. 1994; 200(1): 246-255.
24. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018; 35(6): 1547-1549.
25. Yechezkel I, Law M, Tzarum N. From structural studies to HCV vaccine design. Viruses. 2021; 13(5): 833.
26. Land H, Humble MS. YASARA: a tool to obtain structural guidance in biocatalytic investigations. Methods Mol Biol. 2018; 1685: 43-67.
27. Vita R, Mahajan S, Overton JA, Dhanda SK, Martini S, Cantrell JR, et al. The immune epitope database (IEDB): 2018 update. Nucleic Acids Res. 2019; 47(D1): D339-D343.
28. Hallen MA, Martin JW, Ojewole A, Jou JD, Lowegard AU, Frenkel MS, et al. OSPREY 3.0: open-source protein redesign for you, with powerful new features. J Comput Chem. 2018; 39(30): 2494-2507.
29. Bazmara S, Shadmani M, Ghasemnejad A, Aghazadeh H, Pooshang Bagheri K. In silico rational design of a novel tetraepitope tetanus vaccine with complete population coverage using developed immunoinformatics and surface epitope mapping approaches. Med Hypotheses. 2019; 130: 109267.
30. Pierce BG, Keck ZY, Lau P, Fauvelle C, Gowthaman R, Baumert TF, et al. Global mapping of antibody recognition of the hepatitis C virus E2 glycoprotein: implications for vaccine design. Proc Natl Acad Sci USA. 2016; 113(45): E6946-E6954.
31. Kong L, Giang E, Nieusma T, Kadam RU, Cogburn KE, Hua Y, et al. Hepatitis C virus E2 envelope glycoprotein core structure. Science. 2013; 342(6162): 1090-1094.
32. Kong L, Giang E, Robbins JB, Stanfield RL, Burton DR, Wilson IA, et al. Structural basis of hepatitis C virus neutralization by broadly neutralizing antibody HCV1. Proc Natl Acad Sci USA. 2012; 109(24): 9499-9504.
33. Keck ZY, Olson O, Gal-Tanamy M, Xia J, Patel AH, Dreux M, et al. A point mutation leading to hepatitis C virus escape from neutralization by a monoclonal antibody to a conserved conformational epitope. J Virol. 2008; 82(12): 6067-6072.
34. El-Diwany R, Cohen VJ, Mankowski MC, Wasilewski LN, Brady JK, Snider AE, et al. Extra-epitopic hepatitis C virus polymorphisms confer resistance to broadly neutralizing antibodies by modulating binding to scavenger receptor B1. PLoS Pathog. 2017; 13(2): e1006235.
35. Rodrigo C, Walker MR, Leung P, Eltahla AA, Grebely J, Dore GJ, et al. Limited naturally occurring escape in broadly neutralizing antibody epitopes in hepatitis C glycoprotein E2 and constrained sequence usage in acute infection. Infect Genet Evol. 2017; 49: 88-96.
36. Keck Zy, Angus AG, Wang W, Lau P, Wang Y, Gatherer D, et al. Non-random escape pathways from a broadly neutralizing human monoclonal antibody map to a highly conserved region on the hepatitis C virus E2 glycoprotein encompassing amino acids 412-423. PLoS Pathogens. 2014; 10(8): e1004297.