title: Conformational and thermal stability improvements for the large-scale production of yeast-derived rabbit hemorrhagic disease virus-like particles as multipurpose vaccine.
abstract: Recombinant virus-like particles (VLP) antigenically similar to rabbit hemorrhagic disease virus (RHDV) were recently expressed at high levels inside Pichia pastoris cells. Based on the potential of RHDV VLP as platform for diverse vaccination purposes we undertook the design, development and scale-up of a production process. Conformational and stability issues were addressed to improve process control and optimization. Analyses on the structure, morphology and antigenicity of these multimers were carried out at different pH values during cell disruption and purification by size-exclusion chromatography. Process steps and environmental stresses in which aggregation or conformational instability can be detected were included. These analyses revealed higher stability and recoveries of properly assembled high-purity capsids at acidic and neutral pH in phosphate buffer. The use of stabilizers during long-term storage in solution showed that sucrose, sorbitol, trehalose and glycerol acted as useful aggregation-reducing agents. The VLP emulsified in an oil-based adjuvant were subjected to accelerated thermal stress treatments. None to slight variations were detected in the stability of formulations and in the structure of recovered capsids. A comprehensive analysis on scale-up strategies was accomplished and a nine steps large-scale production process was established. VLP produced after chromatographic separation protected rabbits against a lethal challenge. The minimum protective dose was identified. Stabilized particles were ultimately assayed as carriers of a foreign viral epitope from another pathogen affecting a larger animal species. For that purpose, a linear protective B-cell epitope from Classical Swine Fever Virus (CSFV) E2 envelope protein was chemically coupled to RHDV VLP. Conjugates were able to present the E2 peptide fragment for immune recognition and significantly enhanced the peptide-specific antibody response in vaccinated pigs. Overall these results allowed establishing improved conditions regarding conformational stability and recovery of these multimers for their production at large-scale and potential use on different animal species or humans.
authors: Division of Animal Biotechnology, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors: Erlinda
authors: E
authors: Fernández
authors: Jorge R
authors: JR
authors: Toledo
authors: Lídice
authors: L
authors: Méndez
authors: Nemecio
authors: N
authors: González
authors: Francisco
authors: F
authors: Parra
authors: José M
authors: JM
authors: Martín-Alonso
authors: Miladys
authors: M
authors: Limonta
authors: Kosara
authors: K
authors: Sánchez
authors: Ania
authors: A
authors: Cabrales
authors: Mario P
authors: MP
authors: Estrada
authors: Alina
authors: A
authors: Rodríguez-Mallón
authors: Omar
authors: O
authors: Farnós
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002021:Buffers; D017250:Caliciviridae Infections / Q000276:immunology / Q000517:prevention & control*; D002850:Chromatography, Gel; D006691:Classical Swine Fever / Q000276:immunology / Q000517:prevention & control; D006692:Classical Swine Fever Virus / Q000276:immunology; D018869:Heat-Shock Response; D006384:Hemagglutination; D017929:Hemorrhagic Disease Virus, Rabbit / Q000276:immunology*; D006863:Hydrogen-Ion Concentration; D007114:Immunization; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D010843:Pichia / Q000378:metabolism*; D011817:Rabbits; D012685:Sepharose; D013552:Swine; D013696:Temperature; D014765:Viral Vaccines / Q000096:biosynthesis*; D014771:Virion / Q000276:immunology* / Q000648:ultrastructure; D014783:Viscosity
chemical_list: D002021:Buffers; D010455:Peptides; D014765:Viral Vaccines; D012685:Sepharose