Humoral Immunigenecity Of Deglycosylated DNA Vaccine Encoded Middle Protein Of Hepatitis B Virus Surface Antigen

Background:Chronic hepatitis B is one of the most prevalent infectious diseases which caused a grave threat to the public health. HBsAg is one of main antigen expressed by hepatitis B virus antigen. Anti-HBs Antibodies triggered by HBsAg, as exclusive neutralization antibody, play a critical role in the process of completely eliminating HBV. The emergence of anti-HBs is an important marker of recovery from acute HBV infection. It is not well known the reasons of HBsAg immune tolerance without anti-HBs production in chronic hepatitis B patients. It is believed that the immune systems of young children, particularly infants who infected in the way of vertical transmission, is not fully mature and easy to be induced immune tolerance by HBsAg. It is very important to break immune tolerance of HBsAg and generate anti-HBs for recovery of HBV of infection.HBsAg are glycoproteins with multiply glycosylation sites. Little is known about roles of glycon in humoral immunity. Glycosylation of the HIV and SIV envelope proteins limits their immunogenicity and in addition restricts the binding of certain antibodies to their epitopes on the virion surface. Deletion of glycosylation sites in HIV GP120 results in a higher susceptibility of these viruses to the neutralizing activity of antibodies. It is useful to construct an new plasmid encoded deglycosylation middle protein of hepatitis B virus surface antigen (MHBs) by gene cloning technique for investigating the role of deglycosylation in humoral immunity to MHBs. It is essential for device the coming vaccine against hepatitis B virus with stronger immunogenicity to induce more valid neutralization antibodies.Objective To investigate the role of deglycosylation of MHBs on the sites of 146 (NET) and 59-(NHS) potential N-linked glycan in humoral immunogenecity.Methods Deglycosylation MHBs DNA vaccine (Dg23) was construct by gene cloning technique based on PCR from wild strains MHBs DNA vaccine pSW3891/MHBs/adr (adr). The Balb/c mice were immuned with DNA vaccine Dg23, adr and pSW3891 vcetor. Anti-HBs in sera of mice was tested by ELISA (enzyme linked immune sorbent array). McAb cell lines was developed by means of fusing immunized mice spleen cells with myeloma cells and screening the hybridoma cells with HAT culture medium. McAb cell lines were selected by detection of antibodies against synthetic peptides located around potential N-linked glycan sites in the supernatant. Antigenic binding specificity of monoclonal antibodies was verified by Western blot. The neutralization ability of McAb and sera from immunized mice were comfirmed by blocking test.Results There was no significant difference in titres of anti-HBs in the Sera of mice immunized with Dg23 and adr DNA vaccine. All the sera from both groups can block HBV infection in vitra effectively. Two (1-9A, 2-5F) hybridoma cell lines secreting monoclonal anti-HBs antibodies were obtained. The ending titers of ascites produced by 2 mAb cell lines were 1:3.6×10⁴ (1-9A) and 1:1.2×10⁴ (2-5F) respectly. The relative affinity were 2.6×10^-5(1-9A) and 1.25×10^-4 (2-5F) respectively. It was shown that both two mAbs can recognize deglycosylation MHBs (33KD) but not glycosylation MHBs (36KD) by SDS-PAGE and Western blot analysis. Monoclone antibody 2-5F can block HBV infection to Hep G2 cells but 1-9A cann't.Conclusions Deglycosylation does not affect the titre of antibodies induced by MHBs DNA vaccine in Balb/c mice but may change the species of antibodies. Two mAbs showed different ability of blocking HBV infection suggested that two mAbs combine with different B-cell epitopes. It is speculated that two mAbs combine with different B-cell epitopes result in different change in tertiary structure which change HBV infectivity. The role of glycosylation should be considered during the design of HBV vaccine. The monoclonal antibody (2-5F) obtained with a good blocking effect of HBV infection would be used as a potential therapeutic antibody.