Construction and Expression of Hepatitis B Surface Antigen Escape Variants within the "a" Determinant by Site Directed Mutagenesis


  • Ali Ahmad Bayat Monoclonal Antibody Research Center
  • Amir Hassan Zarnani Reproductive Immunology Research Center, Avicenna Research Institute, ACECR
  • Azam Roohi Department of Immunology, School of Public Health, Tehran University of Medical Sciences | Department of Molecular Medicine, School of Advanced Medical Technologies, Tehran University of Medical Sciences
  • Fazel Shokri Department of Immunology, School of Public Health, Tehran University of Medical Sciences | Monoclonal Antibody Research Center
  • Forough Golsaz Shirazi Department of Immunology, School of Public Health, Tehran University of Medical Sciences
  • Gholam Ali Kardar Immunology, Asthma and Allergy Research Institute, Tehran University of Medical Sciences, Tehran, Iran
  • Hamed Mohammadi Department of Immunology, School of Public Health, Tehran University of Medical Sciences
  • Jalal Khoshnoodi Department of Immunology, School of Public Health, Tehran University of Medical Sciences
  • Mohammad Mehdi Amiri Department of Immunology, School of Public Health, Tehran University of Medical Sciences
چکیده مقاله:

Background: The antibody response to hepatitis B surface antigen (HBsAg) controls hepatitis B virus infection. The "a" determinant of HBsAg is the most important target for protective antibody response, diagnosis and immunoprophylaxis. Mutations in this area may induce immune escape mutants and affect the performance of HBsAg assays. Objectives: To construct clinically relevant recombinant mutant forms of HBsAg and assessment of their reactivity with anti-HBs monoclonal antibodies (MAbs). Methods: Wild type (wt) and mutant (mt) HBsAg genes were constructed by site directed mutagenesis and SEOing PCR. The amplified genes were inserted into pCMV6-neo plasmid and transfected in CHO cell line. The expression of wt- and mtHBsAg was assessed by commercial ELISA assays and stable cells were established and cloned by limiting dilution. The recombinant mutants were further characterized using a panel of anti-HBs monoclonal antibodies (MAbs) and the pattern of their reactivity was assessed by ELISA. Results: Ten HBsAg mutants having single mutation within the "a" determinant including P120E, T123N, Q129H, M133L, K141E, P142S, D144A, G145R, N146S and C147S together with a wt form were successfully constructed and expressed in CHO cells. Reactivity of anti-HBs MAbs with mtHBsAgs displayed different patterns. The effect of mutations on antibody binding differed depending on the amino acid involved and its location within the ‘‘a’’ determinant. Mutation at amino acids 123 and 145 resulted in either complete loss or significant reduction of binding to all anti-HBs MAbs. Conclusion: Our panel of mtHBsAgs is a valuable tool for assessment of the antibody response to HBV escape mutants and may have substantial implications in HBV immunological diagnostics.

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عنوان ژورنال

دوره 10  شماره 3

صفحات  127- 138

تاریخ انتشار 2013-09-01

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