title: Construction and evaluation of DNA vaccine encoding Hantavirus glycoprotein N-terminal fused with lysosome-associated membrane protein.
abstract: BACKGROUND
  Hantaviral diseases can have a high case fatality rate within the absence of broadly effective antiviral treatments or vaccines. We developed a DNA vaccine targeting the Hantavirus glycoprotein N-terminal (Gn) to major histocompatibility complex class II compartment by fusing the antigen with lysosome-associated membrane protein 1 (LAMP1), which altered antigen presenting pathway and activated the CD4+ T cells.
  
  
  METHODS
  The segments of Gn and LAMP1 were cloned into vector pVAX1, and recombinant plasmid was constructed by inserting Gn sequence into LAMP1, between luminal and the transmembrane/cytoplasmic domains. Subsequently, the protein expression was identified through immunoprecipitation, western blot and Immunofluorescent assay. Adaptive immune responses were assessed by the presence of specific and neutralizing antibodies, interferon (ELISpot results, and cytotoxic T-lymphocyte (CTL) cytotoxicity. Epitope mapping was performed to study the T-cell epitopes. Protective immunity in vivo was evaluated using a novel HTNV-challenging model, and safety evaluation was based on histological and behavioral observations.
  
  
  RESULTS
  Native or LAMP1 targeting HTNV Gn was successfully identified. Humoral immune responses were enhanced, featuring with satisfying titers of specific and neutralizing antibody production. The boosted activities of IFN-γ and CTL cytotoxicity witnessed enhanced cellular immune responses. Effective protection against HTNV in vivo was conferred in all three vaccine groups by the challenge model. Safety was confirmed and one dominant T-cell epitope screened from immunized mice overlapped the specific T-cell hot spot in HFRS patients.
  
  
  CONCLUSIONS
  LAMP1 targeting strategy successfully enhanced the efficacy of HTNV Gn-based vaccine, which is highly immunogenic and safe, showing promise for immunoprophylaxis against HFRS. Further investigations are warranted in the future.
authors.affiliation: Department of Immunology, Fourth Military Medical University, Xi'an 710032, China; Brigade of Cadet, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Dong-Bo
authors.initials: DB
authors.lastname: Jiang
authors.affiliation: Department of Immunology, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Yuan-Jie
authors.initials: YJ
authors.lastname: Sun
authors.affiliation: Department of Microbiology, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Lin-Feng
authors.initials: LF
authors.lastname: Cheng
authors.affiliation: Department of Immunology, Fourth Military Medical University, Xi'an 710032, China; Brigade of Cadet, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Ge-Fei
authors.initials: GF
authors.lastname: Zhang
authors.affiliation: Department of Immunology, Fourth Military Medical University, Xi'an 710032, China; Department of Microbiology, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Dong
authors.affiliation: Department of Immunology, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Bo-Quan
authors.initials: BQ
authors.lastname: Jin
authors.affiliation: Department of Immunology, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Chao-Jun
authors.initials: CJ
authors.lastname: Song
authors.affiliation: Department of Immunology, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Ma
authors.affiliation: Department of Microbiology, Fourth Military Medical University, Xi'an 710032, China. Electronic address: flzhang@fmmu.edu.cn.
authors.forename: Fang-Lin
authors.initials: FL
authors.lastname: Zhang
authors.affiliation: Department of Immunology, Fourth Military Medical University, Xi'an 710032, China. Electronic address: yangkunkun@fmmu.edu.cn.
authors.forename: Kun
authors.initials: K
authors.lastname: Yang
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood; D000914:Antibodies, Viral / Q000097:blood; D003601:Cytotoxicity Tests, Immunologic; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D058501:Enzyme-Linked Immunospot Assay; D005260:Female; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology; D018778:Hantavirus Infections / Q000276:immunology / Q000517:prevention & control; D007372:Interferons / Q000378:metabolism; D051907:Lysosomal Membrane Proteins / Q000235:genetics / Q000276:immunology; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D016019:Survival Analysis; D019444:Vaccines, DNA / Q000008:administration & dosage / Q000009:adverse effects / Q000235:genetics / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000009:adverse effects / Q000235:genetics / Q000276:immunology; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology; D014765:Viral Vaccines / Q000008:administration & dosage / Q000009:adverse effects / Q000235:genetics / Q000276:immunology*
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D006023:Glycoproteins; D051907:Lysosomal Membrane Proteins; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA; D014614:Vaccines, Synthetic; D014764:Viral Proteins; D014765:Viral Vaccines; D007372:Interferons
keywords: Animal model; DNA vaccine; Hantavirus; Immune protection; Immune response; Lysosome-associated membrane protein; Trafficking molecular