title: Structural basis for the development of avian virus capsids that display influenza virus proteins and induce protective immunity.
abstract: Bioengineering of viruses and virus-like particles (VLPs) is a well-established approach in the development of new and improved vaccines against viral and bacterial pathogens. We report here that the capsid of a major avian pathogen, infectious bursal disease virus (IBDV), can accommodate heterologous proteins to induce protective immunity. The structural units of the ~70-nm-diameter T=13 IBDV capsid are trimers of VP2, which is made as a precursor (pVP2). The pVP2 C-terminal domain has an amphipathic α helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, 466-residue pVP2 intermediates bearing this α helix assemble into genuine VLPs only when expressed with an N-terminal His6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for protein insertion, as they are large enough (cargo space, ~78,000 nm(3)) and are assembled from a single protein. We explored HT-VP2-466-based chimeric capsids initially using enhanced green fluorescent protein (EGFP). The VLP assembly yield was efficient when we coexpressed EGFP-HT-VP2-466 and HT-VP2-466 from two recombinant baculoviruses. The native EGFP structure (~240 copies/virion) was successfully inserted in a functional form, as VLPs were fluorescent, and three-dimensional cryo-electron microscopy showed that the EGFP molecules incorporated at the inner capsid surface. Immunization of mice with purified EGFP-VLPs elicited anti-EGFP antibodies. We also inserted hemagglutinin (HA) and matrix (M2) protein epitopes derived from the mouse-adapted A/PR/8/34 influenza virus and engineered several HA- and M2-derived chimeric capsids. Mice immunized with VLPs containing the HA stalk, an M2 fragment, or both antigens developed full protection against viral challenge.
  
  
  OBJECTIVE
  Virus-like particles (VLPs) are multimeric protein cages that mimic the infectious virus capsid and are potential candidates as nonliving vaccines that induce long-lasting protection. Chimeric VLPs can display or include foreign antigens, which could be a conserved epitope to elicit broadly neutralizing antibodies or several variable epitopes effective against a large number of viral strains. We report the biochemical, structural, and immunological characterization of chimeric VLPs derived from infectious bursal disease virus (IBDV), an important poultry pathogen. To test the potential of IBDV VLPs as a vaccine vehicle, we used the enhanced green fluorescent protein and two fragments derived from the hemagglutinin and the M2 matrix protein of the human murine-adapted influenza virus. The IBDV capsid protein fused to influenza virus peptides formed assemblies able to protect mice against viral challenge. Our studies establish the basis for a new generation of multivalent IBDV-based vaccines.
authors.affiliation: Department of Structure of Macromolecules, Centro Nacional de Biotecnología/CSIC, Cantoblanco, Madrid, Spain.
authors.forename: Elena
authors.initials: E
authors.lastname: Pascual
authors.affiliation: Department of Structure of Macromolecules, Centro Nacional de Biotecnología/CSIC, Cantoblanco, Madrid, Spain.
authors.forename: Carlos P
authors.initials: CP
authors.lastname: Mata
authors.affiliation: Department of Structure of Macromolecules, Centro Nacional de Biotecnología/CSIC, Cantoblanco, Madrid, Spain.
authors.forename: Josué
authors.initials: J
authors.lastname: Gómez-Blanco
authors.affiliation: Centro de Investigación en Sanidad Animal (INIA-CISA), Valdeolmos, Madrid, Spain.
authors.forename: Noelia
authors.initials: N
authors.lastname: Moreno
authors.affiliation: Centro de Investigación en Sanidad Animal (INIA-CISA), Valdeolmos, Madrid, Spain.
authors.forename: Juan
authors.initials: J
authors.lastname: Bárcena
authors.affiliation: Centro de Investigación en Sanidad Animal (INIA-CISA), Valdeolmos, Madrid, Spain.
authors.forename: Esther
authors.initials: E
authors.lastname: Blanco
authors.affiliation: Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología/CSIC, Cantoblanco, Madrid, Spain CIBER de Enfermedades Respiratorias, Mallorca, Illes Balears, Spain.
authors.forename: Ariel
authors.initials: A
authors.lastname: Rodríguez-Frandsen
authors.affiliation: Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología/CSIC, Cantoblanco, Madrid, Spain CIBER de Enfermedades Respiratorias, Mallorca, Illes Balears, Spain.
authors.forename: Amelia
authors.initials: A
authors.lastname: Nieto
authors.affiliation: Department of Structure of Macromolecules, Centro Nacional de Biotecnología/CSIC, Cantoblanco, Madrid, Spain jlcarras@cnb.csic.es jrcaston@cnb.csic.es.
authors.forename: José L
authors.initials: JL
authors.lastname: Carrascosa
authors.affiliation: Department of Structure of Macromolecules, Centro Nacional de Biotecnología/CSIC, Cantoblanco, Madrid, Spain jlcarras@cnb.csic.es jrcaston@cnb.csic.es.
authors.forename: José R
authors.initials: JR
authors.lastname: Castón
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology*; D002213:Capsid / Q000276:immunology* / Q000648:ultrastructure; D020285:Cryoelectron Microscopy; D004195:Disease Models, Animal; D004337:Drug Carriers; D017930:Genes, Reporter / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics / Q000276:immunology; D007243:Infectious bursal disease virus / Q000235:genetics*; D009980:Influenza A virus / Q000235:genetics / Q000276:immunology*; D007252:Influenza Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D008807:Mice, Inbred BALB C; D009976:Orthomyxoviridae Infections / Q000276:immunology / Q000517:prevention & control; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology; D058425:Vaccines, Virus-Like Particle / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology* / Q000648:ultrastructure; D014763:Viral Matrix Proteins / Q000235:genetics / Q000276:immunology; D000086082:Viroporin Proteins
chemical_list: D000956:Antigens, Viral; D004337:Drug Carriers; D049452:Green Fluorescent Proteins; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; D058425:Vaccines, Virus-Like Particle; D014763:Viral Matrix Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins; C485184:enhanced green fluorescent protein