A novel hepatitis B virus mutant with A-to-G at nt551 in the surface antigen gene

Abstract

Aim: Hepatitis B surface antigen (HBsAg) mutant of hepatitis B virus (HBV) is one of the important factors that result in immune escape and cause failure of immunization. In this study we reported and characterized a novel HBV mutant with A-to-G at nt551 and intended to provide theoretical data for prevention of HBV infection in China.

Methods: A methodology comprising polymerase chain reaction (PCR) amplifying, M13 bacteriophage cloning and nucleotide sequencing was used to analyze the sera of the pediatric patient who was hepatitis B (HB) immune failure. Expression plasmids containing the mutant S gene and a wild-type (adr) S gene were constructed respectively and the recombinant HBsAg were expressed in COS-7 cells under the regulation of SV40 early promoter. The recombinant proteins were investigated for their immunological reactivity with different monoclonal antibodies (mAb) against "a" determinant and vaccine-raised human neutralizing antibodies.

Results: It was found that there was a new point mutation at nt551 of the HBV (adr) genome from A to G, leading to a substitution of methionine (Met) to valine (Val) at position 133 in the "a" determinant of HBsAg. Compared to the wild-type HBsAg, the binding activity of the mutant HBsAg to mAbs (A6, A11 and S17) and to vaccine-raised human anti-hepatitis B surface antibody (anti-HBs) decreased significantly.

Conclusion: According to the facts that the patient has been immunized with HB vaccine and that the serum is anti-HBs positive and HBsAg negative, and based on the nucleotide sequence analysis of the mutant HBV S gene and its alteration of antigenicity, the HBV is considered to be a new vaccine-induced immune escape mutant different from the known ones.

Figure 1

The complete nucleotide sequence of the mutant S gene. The A-to-G mutation site at nt551 of HBV genome is in bold letter. The underlined are the EcoRI-like site, the initiation codon of HBsAg and the amino acid codon (ATG to GTG) respectively. The first C of the EcoRI-like site (GAACTC) is counted as nt1. The GenBank accession number of the sequence is AF052576.

Figure 2

Comparative analysis of the HBsAg 'a' determinant coding regions of different HBV genomes. HBsAg 'a' determinant is a conformational epitope which has a special two-loop construction kept by the disulfide bonds between Cys124 and Cys137, Cys139 and Cys147, respectively. 48 HBV genome sequences were downloaded from National Center for Biotechnology Information (NCBI), USA (http://www.ncbi.nlm.nih.gov). “Cene names” are their names in the original gene databases. Here the ※ labeled ones are from DDBJ, the labeled are from EMBL, and the § labeled are from GenBank.