Iranian Journal of Immunology

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

Document Type : Original Article

Authors

Forough Golsaz Shirazi 1
Mohammad Mehdi Amiri 1
Hamed Mohammadi 1
Ali Ahmad Bayat 2
Azam Roohi 1, 3
Jalal Khoshnoodi 1
Amir Hassan Zarnani 4
Mahmood Jeddi-Tehrani 2
Gholam Ali Kardar 5
Fazel Shokri 1, 2

1 Department of Immunology, School of Public Health, Tehran University of Medical Sciences

2 Monoclonal Antibody Research Center

3 Department of Molecular Medicine, School of Advanced Medical Technologies, Tehran University of Medical Sciences

4 Reproductive Immunology Research Center, Avicenna Research Institute, ACECR

5 Immunology, Asthma and Allergy Research Institute, Tehran University of Medical Sciences, Tehran, Iran

https://doi.org/10.22034/iji.2013.16821
Abstract
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.

Keywords

"a" Determinant
HBs Ag
Monoclonal antibody
Iranian Journal of Immunology
Volume 10, Issue 3
Summer 2013
Pages 127-138

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