title: Protection of mice against a lethal influenza virus challenge after immunization with yeast-derived secreted influenza virus hemagglutinin.
abstract: The A/Victoria/3/75 (H3N2-subtype) hemagglutinin (HA) gene was engineered for expression in Pichia pastoris as a soluble secreted molecule. The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to the Saccharomyces cerevisiae alpha-mating factor secretion signal and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Growth of transformants on methanol-containing medium resulted in the secretion of recombinant non-cleaved soluble hemagglutinin (HA0s). Remarkably, the pH of the induction medium had an important effect on the expression level, the highest level being obtained at pH 8.0. The gel filtration profile and the reactivity against a panel of different HA-conformation specific monoclonal antibodies indicated that HA0s was monomeric. Analysis of the N-linked glycans revealed a typical P. pastoris type of glycosylation, consisting of glycans with 10-12 glycosyl residues. Mice immunized with purified soluble hemagglutinin (HA0s) showed complete protection against a challenge with 10 LD50 of mouse-adapted homologous virus (X47), whereas all control mice succumbed. Heterologous challenge with X31 virus [A/Aichi/2/68 (H3N2-subtype)], resulted in significantly higher survival rates in the immunized group compared with the control group. These results, together with the safety, reliability and economic potential of P. pastoris, as well as the flexibility and fast adaptation of the expression system may allow development of an effective recombinant influenza vaccine.
authors: Department of Molecular Biology, University of Gent, Belgium.
authors: X
authors: X
authors: Saelens
authors: P
authors: P
authors: Vanlandschoot
authors: W
authors: W
authors: Martinet
authors: M
authors: M
authors: Maras
authors: S
authors: S
authors: Neirynck
authors: R
authors: R
authors: Contreras
authors: W
authors: W
authors: Fiers
authors: W M
authors: WM
authors: Jou
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics; D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D006389:Hemagglutinins, Viral / Q000235:genetics / Q000276:immunology*; D007114:Immunization; D007252:Influenza Vaccines / Q000096:biosynthesis / Q000276:immunology*; D000072235:Mating Factor; D051379:Mice; D009975:Orthomyxoviridae / Q000276:immunology*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010455:Peptides / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D006389:Hemagglutinins, Viral; D007252:Influenza Vaccines; D010455:Peptides; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D000072235:Mating Factor; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase