title: N-Linked Glycosylation at an Appropriate Position in the Pre-S2 Domain Is Critical for Cellular and Humoral Immunity against Middle HBV Surface Antigen.
abstract: Infection with hepatitis B virus (HBV) remains a worldwide health problem, and DNA-based vaccines against HBV have been tested for therapeutic applications. HBV possesses three envelope lipoproteins that are translated from a single reading-frame: large, middle, and small HBV surface antigens. Among these envelope proteins, the middle HBV surface antigen (MHBs) contains a constitutive N-linked glycosylation site at position 4 (Asn4) in the amino-terminal portion (MQWNSTTFHQ) of pre-S2 domain. Asn4 (shown in bold) is essential for secretion of viral particles and conserved among all serotypes of HBV, but its influence on the immunogenicity of MHBs remains unknown. Here, we constructed four MHBs genes carrying mutations, underlined, in the amino-terminal portion of pre-S2 domain. One mutant protein contains Q at position 4 (MQWQSTTFHQ). In addition, each of three mutant MHBs proteins contains a N-linked glycosylation site (N-X-S/T), relocated to position 5 (MQWQNTTFHQ), 6 (MQWQSNTSHQ) or 7 (MQWQSTNFTQ) in pre-S2 domain. The expression and immunogenic properties of mutant DNA vaccines were examined in 293T human renal epithelial cells and in BALB/c mice, respectively. We showed that Asn4 was critical for secretion and immunogenicity of MHBs. Moreover, the MHBs protein that carries a N-linked glycosylation site at position 5 or 7 retained the properties similar to wild-type MHBs. In contrast, the secretion-defective mutant protein carrying Asn at position 6 induced only marginal humoral and cellular immune responses in mice, despite the N-linked glycosylation. In conclusion, N-linked glycosylation at an appropriate position in pre-S2 domain is an essential requirement for DNA vaccine expressing MHBs.
authors.affiliation: Department of Infectious Diseases, The First Affiliated Hospital of Nanjing Medical University, 2)  Department of Infectious Diseases, Nanjing Children's Hospital, Affiliated to Nanjing Medical University, Nanjing, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Liu
authors.forename: Shixia
authors.initials: S
authors.lastname: Wang
authors.forename: Yiqiong
authors.initials: Y
authors.lastname: Jia
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Zuhu
authors.initials: Z
authors.lastname: Huang
authors.forename: Shan
authors.initials: S
authors.lastname: Lu
authors.forename: Yiping
authors.initials: Y
authors.lastname: Xing
mesh_terms: D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006031:Glycosylation; D006514:Hepatitis B Surface Antigens / Q000235:genetics / Q000276:immunology*; D017325:Hepatitis B Vaccines / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007111:Immunity, Cellular / Q000276:immunology*; D056724:Immunity, Humoral / Q000276:immunology*; D007371:Interferon-gamma / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation / Q000235:genetics / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology; D019444:Vaccines, DNA / Q000235:genetics / Q000276:immunology*
chemical_list: D006514:Hepatitis B Surface Antigens; D017325:Hepatitis B Vaccines; D019444:Vaccines, DNA; D007371:Interferon-gamma