title: Construction of polyomavirus-derived pseudotype virus-like particles displaying a functionally active neutralizing antibody against hepatitis B virus surface antigen.
abstract: BACKGROUND
  Virus-like particles (VLPs) can be efficiently produced by heterologous expression of viral structural proteins in yeast. Due to their repetitive structure, VLPs are extensively used for protein engineering and generation of chimeric VLPs with inserted foreign epitopes. Hamster polyomavirus VP1 represents a promising epitope carrier. However, insertion of large sized protein sequences may interfere with its self-assembly competence. The co-expression of polyomavirus capsid protein VP1 with minor capsid protein VP2 or its fusion protein may result in pseudotype VLPs where an intact VP1 protein mediates VLP formation. In the current study, the capacity of VP1 protein to self-assemble to VLPs and interact with the modified VP2 protein has been exploited to generate pseudotype VLPs displaying large-sized antibody molecules.
  
  
  RESULTS
  Polyomavirus-derived pseudotype VLPs harbouring a surface-exposed functionally active neutralizing antibody specific to hepatitis B virus (HBV) surface antigen (HBsAg) have been generated. The pseudotype VLPs consisting of an intact hamster polyomavirus (HaPyV) major capsid protein VP1 and minor capsid protein VP2 fused with the anti-HBsAg molecule were efficiently produced in yeast Saccharomyces cerevisiae and purified by density-gradient centrifugation. Formation of VLPs was confirmed by electron microscopy. Two types of pseudotype VLPs were generated harbouring either the single-chain fragment variable (scFv) or Fc-engineered scFv on the VLP surface. The antigen-binding activity of the purified pseudotype VLPs was evaluated by ELISA and virus-neutralization assay on HBV-susceptible primary hepatocytes from Tupaia belangeri. Both types of the pseudotype VLPs were functionally active and showed a potent HBV-neutralizing activity comparable to that of the parental monoclonal antibody. The VP2-fused scFv molecules were incorporated into the VLPs with higher efficiency as compared to the VP2-fused Fc-scFv. However, the pseudotype VLPs with displayed VP2-fused Fc-scFv molecule showed higher antigen-binding activity and HBV-neutralizing capacity that might be explained by a better accessibility of the Fc-engineered scFv of the VLP surface.
  
  
  CONCLUSIONS
  Polyomavirus-derived pseudotype VLPs harbouring multiple functionally active antibody molecules with virus-neutralizing capability may represent a novel platform for developing therapeutic tools with a potential application for post-exposure or therapeutic treatment of viral infections.
authors.affiliation: Department of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University, Graciuno 8, LT-02241, Vilnius, Lithuania. milda.pleckaityte@bti.vu.lt.
authors.forename: Milda
authors.initials: M
authors.lastname: Pleckaityte
authors.affiliation: Institute of Medical Virology, National Reference Centre for Hepatitis B and D Viruses, German Centre for Infection Research, Justus-Liebig University of Giessen, Schubertstrasse 81, 35392, Giessen, Germany. Corinna.Bremer@viro.med.uni-giessen.de.
authors.forename: Corinna M
authors.initials: CM
authors.lastname: Bremer
authors.affiliation: Department of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University, Graciuno 8, LT-02241, Vilnius, Lithuania. alma.gedvilaite@bti.vu.lt.
authors.forename: Alma
authors.initials: A
authors.lastname: Gedvilaite
authors.affiliation: Department of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University, Graciuno 8, LT-02241, Vilnius, Lithuania. indre.kodze@bti.vu.lt.
authors.forename: Indre
authors.initials: I
authors.lastname: Kucinskaite-Kodze
authors.affiliation: Institute of Medical Virology, National Reference Centre for Hepatitis B and D Viruses, German Centre for Infection Research, Justus-Liebig University of Giessen, Schubertstrasse 81, 35392, Giessen, Germany. Dieter.Glebe@viro.med.uni-giessen.de.
authors.forename: Dieter
authors.initials: D
authors.lastname: Glebe
authors.affiliation: Department of Immunology and Cell Biology, Institute of Biotechnology, Vilnius University, Graciuno 8, LT-02241, Vilnius, Lithuania. azvirb@ibt.lt.
authors.forename: Aurelija
authors.initials: A
authors.lastname: Zvirbliene
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D002478:Cells, Cultured; D006510:Hepatitis B Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006514:Hepatitis B Surface Antigens / Q000276:immunology*; D017325:Hepatitis B Vaccines / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D022781:Hepatocytes / Q000166:cytology / Q000821:virology; D011120:Polyomavirus / Q000737:chemistry / Q000235:genetics*; D012441:Saccharomyces cerevisiae; D014417:Tupaia; D058425:Vaccines, Virus-Like Particle / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D057134:Antibodies, Neutralizing; D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D017325:Hepatitis B Vaccines; D058425:Vaccines, Virus-Like Particle