Hepatitis B Virus Surface Antigen Variants Clustered Within Immune Epitopes in Chronic Hepatitis B Carriers from Hormozgan Province, South of Iran

Document Type : Original Article


1 Iranian National Institute for Genetic Engineering and Biotechnology, Tehran, Iran

2 Infectious Diseases and Tropical Medicine Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

3 Digestive Disease Research Center, Shariati Hospital, Tehran, Iran

4 Baqiyatallah University of Medical Sciences, Baqiyatallah Research Centre for Gastroenterology and Liver Disease, Tehran, Iran

5 Hepatitis B Molecular Laboratory,Department of Virology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran


The aim of this study was to characterize the hepatitis B virus surface protein genotypes and sequence variations among hepatitis B virus surface antigen (HBsAg) positive chronic patients in Hormozgan province, south of Iran.
Materials and Methods
A total of 8 patients enrolled in this study. The surface gene was amplified and directly sequenced. Genotypes and nucleotide/amino acid substitutions were identified compared to the sequences obtained from the database.
All strains belonged to genotype D. Overall 77 “mutations” occurred at 45 nucleotide positions, of them, 44 (57.14%) were silent (no amino acid altering) and 33 (42.86%) were missense (amino acid changing). A number of 24 (80%) out of 30 amino acid changes occurred in different immune epitopes within surface protein, of which, 9 (30%) in B cell epitopes in 7 residues (2 occurred in “a” determinant region); 8 (42.1%) in T helper epitopes in 7 residues and 7 (10%) in 4 residues inside CTL epitopes.
Hepatitis B virus genome containing mutated immune epitopes no longer could be recognized by specific T- cells of the host immune surveillance and did not enhance anti-HBs production. This could led to the progression of chronicity of hepatitis B virus infection.


1.Wallace LA, Carman WF. Surface gene variation of HBV: Scientific and medical relevance. J Viral Hepatitis 1997; 3:5-16.
2.Honorati MC, Dolzani P, Mariani E, Piacentini A, Lisignoli G, Ferrari C, Facchini A. Epitope specificity of Th0/Th2 CD4+ T-lymphocyte clones induced by vaccination with rHBsAg vaccine. Gastroenterology 1997; 112:2017-2027.
3.Mancini-Bourgine M, Fontaine H, Brechot C, Pol S, Michel ML. Immunogenicity of a hepatitis B DNA vaccine administered to chronic HBV carriers. Vaccine 2006; 24:4482-4489.
4.Ducos J Bianchi, Mondain AM, Pageaux G, Conge AM, Poncet R, Vendrell JP, et al. Hepatitis B virus (HBV)specific in vitro antibody production by peripheral blood mononuclear cells (PBMC) after vaccination by recombinant hepatitis B surface antigen (rHBsAg). Clin Exp Immunol 1996; 103:15-18.
5.Barnaba V, Franco A, Paroli M, Benvenuto R, De Petrillo G, Burgio VL. Selective expansion of cytotoxic T lymphocytes with a CD4+CD56+surface phenotype and a T helper type 1 profile of cytokine secretion in the liver of patients chronically infected with hepatitis B virus. J Immunol 1994; 152:3074-3087.
6.Norder H, Hammas B, Lofdahl S, Courouce AM, Magnius LO. Comparison of the amino acid sequences of nine different serotypes of hepatitis B surface antigen and genomic classification of the corresponding hepatitis B virus strains. J Gen Virol 1992; 73:1201-12018.
7.Norder H, Courouce AM, Magnius LO. Molecular basis of hepatitis B virus serotype variations within the four major subtypes. J Gen Virol 1992; 73:3141-3145.
8.Chisari FV. Immunobiology and pathogenesis of viral hepatitis. Edizioni Minerva Medica, Turin. 1997.
9.Mesenas SJ, Chow WC, Zhao Y, Lim GK, Oon CJ, Ng HS. Wild-type and 'a' epitope variants in chronic hepatitis B virus carriers positive for hepatitis B surface antigen and antibody. J Gastroenterol Hepatol 2002; 17:148-152.
10.Friedt M, Gerner P, Wintermeyer P, Wirth S. Complete hepatitis B virus genome analysis in HBsAg positive mothers and their infants with fulminant hepatitis B. BMC Gastroenterol 2004; 4:11.
11.Ozaslan M, Ozaslan E, Barsgan A, Koruk MJ. Mutations in the S gene region of hepatitis B virus genotype D in Turkish patients. J Genet 2007; 86:195-201.
12.Tabor E. Infections by hepatitis B surface antigen gene mutants in Europe and North America. J Med Virol 2006; 78:S43-47. 
13. Song BC, Kim SH, Kim H, Ying YH, Kim HJ, Kim YJ, et al. Prevalence of naturally occurring surface antigen variants of hepatitis B virus in Korean patients infected chronically. J Med Virol 2005; 76:194-202.
14.Bahramali G, Sadeghizadeh M, Amini-Bavil-Olyaee S, Alavian SM, Behzad-Behbahani A, Adeli A, et al. Clinical, virologic and phylogenetic features of hepatitis B infection in Iranian patients. World J Gastroenterol 2008; 14:5448-5453.
15.Ireland JH, O'Donnell B, Basuni AA, Kean JD, Wallace LA, Lau GK, et al. Reactivity of 13 in vitro expressed hepatitis B surface antigen variants in 7 commercial diagnostic assays. Hepatology 2000; 31:1176-1182.
16.Weber B. Genetic variability of the S gene of hepatitis B virus: clinical and diagnostic impact. J Clin Virol 2005; 32:102-112.
17.Weber B. Diagnostic impact of the genetic variability of the hepatitis B virus surface antigen gene. J Med Virol 2006; 78:S59-65.
18.Hollinger FB. Hepatitis B virus genetic diversity and its impact on diagnostic assays. J Viral Hepat 2007; 1:11-5.
19.Pourkarim MR, Amini-Bavil-Olyaee S, Verbeeck J, Lemey P, Zeller M, Rahman M, et al. Molecular evolutionary analysis and mutational pattern of full-length genomes of hepatitis B virus isolated from Belgian patients with different clinical manifestations. J Med Virol 2010; 82:379-389.
20.Datta S, Panigrahi R, Biswas A, Chandra PK, Banerjee A, Mahapatra PK, et al. Genetic characterization of hepatitis B virus in peripheral blood leukocytes: evidence for selection and compartmentalization of viral variants with the immune escape G145R mutation.J Virol 2009; 83:9983-9992.
21.Sloan RD, Strang AL, Ramsay ME, Teo CG. Genotyping of acute HBV isolates from England, 1997-2001. J Clin Virol 2009; 44:157-160.
22.Salazar M, Deulofeut H, Granja C, Deulofeut R, Yunis DE, Marcus-Bagley D, et al. Normal HBsAG presentation and T-cell defect in the immune response of nonresponders. Immunogenetics 1995; 41:366-374.
23.Bauer T, Weinberger K, Jilg W. Variants of two major T cell epitopes within the hepatitis B surface antigen are not recognized by specific T helper cells of vaccinated individuals . Hepatology 2002; 455-465.
24.Jazayeri and Carman. Virus escape, CTL or B cell epitopes Hepatitis Monthly 2005; 5:133-136.
25.Chen M, Salberg M, Thung SN, Hughes J, Jones J, Milich DR. Modelling the T helper cell response in acute and chronic hepatitis B virus infection using T-cell receptor transgenic mice. Antiviral Res 2001; 52:99-111.
26.Liu CJ, Kao JH, Shau WY, Chen PJ, Lai MY, Chen DS. Naturally occurring hepatitis B surface gene variants in chronic hepatitis B virus infection: correlation with viral serotypes and clinical stages of liver disease. J Med Virol 2002; 68:50-59.
27.Barnaba V, Franco A, Alberti A, Balsano C, Benvenuto R, Balsano F. Recognition of hepatitis B virus envelope proteins by liver-infiltrating T lymphocytes in chronic HBV. Infection. J immunol 1989; 143:2650-2655.
28.Khakoo SI, Ling R, Scott I, Dodi AI, Harrison TJ, Dusheiko GM, et al. Cytotoxic T lymphocyte responses and CTL epitope escape mutation in HBsAg, anti-HBe positive individuals. Gut 2000; 4: 256.
29.Yamauchi K, Nakamura T, Yonemitsu H, Sekiya H, Katoh J, Obata H. Possible role of preS2 peptides presented by MHC class I antigen in the pathogenesis of chronic hepatitis B. J Hepatol 1993; 17:S6-9.
30.Carman WF, Thursz M, Hadziyannis S, McIntyre G, Colman K, Gioustoz A, et al. Hepatitis B e antigen negative chronic active hepatitis: hepatitis B virus core mutations occur predominantly in known antigenic determinants. J Viral Hepat 1995; 2:77-84.
31.Carman WF, Boner W, Fattovich G, Colman K, Dornan ES, Thursz M, et al. Hepatitis B virus core protein mutations are concentrated in B cell epitopes in progressive disease and in T helper cell epitopes during clinical remission. J Infect Dis 1997; 175:1093-10100.
32.Hosono S, Tai PC, Wang W, Ambrose M, Hwang DG, Yuan TT, et al. Core antigen mutations of human hepatitis B virus in hepatomas accumulate in MHC class II-restricted T cell epitopes. Virology 1995; 212:151-162.
33.Rehermann B, Pasquinelli C, Mosier SM, Chisari FV. Hepatitis B virus (HBV) sequence variation of cytotoxic T lymphocyte epitopes is not common in patients with chronic HBV infection. J Clin Invest 1995;
34.Maruyama T, Kuwata S, Koike K, Iino S, Yasuda K, Yotsuyanagi H, et al. Precore wild-type DNA and immune complexes persist in chronic hepatitis B after seroconversion: no association between genome conversion and seroconversion. Hepatology 1998; 27:245-53.
35.Jazayeri SM, Dornan E, Boner W, Waketing M, Hadziyannis S, Fattovich G, et al. Distribution of in vitro expressed HBV core protein from isolates is dependent upon sequence and correlates with disease pattern. J Infect Dis 2004; 189:1634-1645.