ABSTRACT
Acellular whooping cough vaccines are based on pertussis toxoid but their effectiveness may be increased by the addition of other Bordetella pertussis antigens. We expressed the immunogenic outer membrane protein pertactin (P69) from B. pertussis to high levels in multi-copy transformants of the industrial yeast Pichia pastoris. In high-density fermentations, engineered P. pastoris yielded greater than 3 g of the protein per litre of culture. Purified recombinant pertactin was able to stimulate the incomplete protection afforded by toxoid to the level of the whole-cell vaccine, as shown by the Kendrick test, supporting its inclusion in future acellular vaccines.
Subject(s)
Bacterial Outer Membrane Proteins/immunology , Bordetella pertussis/immunology , Pertussis Vaccine/administration & dosage , Recombinant Proteins/biosynthesis , Virulence Factors, Bordetella , Whooping Cough/prevention & control , Bacterial Outer Membrane Proteins/biosynthesis , Base Sequence , Blotting, Western , Fermentation , Gene Expression , Genetic Vectors/genetics , Molecular Sequence Data , Pichia/genetics , Pichia/metabolism , Recombinant Proteins/immunology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Transformation, GeneticABSTRACT
Fragment C is a non-toxic 50 kDa fragment of tetanus toxin which is a candidate subunit vaccine against tetanus. The AT-rich Clostridium tetani DNA encoding fragment C could not be expressed in Saccharomyces cerevisiae due to the presence of several fortuitous polyadenylation sites which gave rise to truncated mRNAs. The polyadenylation sites were eliminated by chemically synthesising the DNA with increased GC-content (from 29% to 47%). Synthesis of the entire gene (1400 base pairs) was necessary to generate full-length transcripts and for protein production in yeast. Using a GAL1 promoter vector, fragment C was expressed to 2-3% of soluble cell protein. Fragment C could also be secreted using the alpha-factor leader peptide as a secretion signal. The protein was present at 5-10 mg/l in the culture medium in two forms: a high molecular mass hyper-glycosylated protein (75-200 kDa) and a core-glycosylated protein (65 kDa). Intracellular fragment C was as effective in vaccinating mice against tetanus authentic fragment C. The glycosylated material was inactive, though it was rendered fully active by de-glycosylation.
Subject(s)
DNA, Fungal/metabolism , Gene Expression , Peptide Fragments/genetics , Poly A/metabolism , Saccharomyces cerevisiae/genetics , Tetanus Toxin/genetics , Adenine/analysis , Animals , Base Sequence , Blotting, Western , Electrophoresis, Agar Gel , Electrophoresis, Polyacrylamide Gel , Genes, Fungal , Glycosylation , Immunization , Mice , Molecular Sequence Data , Peptide Fragments/biosynthesis , Peptide Fragments/immunology , Plasmids , RNA, Fungal/analysis , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Tetanus/prevention & control , Tetanus Toxin/biosynthesis , Tetanus Toxin/immunology , Thymidine/analysis , Transcription, GeneticABSTRACT
Hepatitis B core protein (HBcAg) is a potent antigen that gives both a T-cell-dependent and a T-cell-independent antibody response. It has been shown that a foreign epitope can be fused to the amino terminus of HBcAg without affecting particle integrity, and that the resulting chimaeric cores retain the immunogenicity of the foreign epitope. Here we describe the efficient expression in yeast of two different chimaeric cores, carrying epitopes of Foot and Mouth Disease Virus (FMDV) or human chorionic gonadotrophin (hCG), which are candidates for FMD and contraceptive vaccines, respectively. These cores could not be produced in E. coli in soluble form but were expressed to high levels in yeast. We constructed a yeast expression vector that allows rapid production of different chimaeric cores by cloning in cassettes encoding foreign epitopes. Both FMDV and hCG-cores were shown to present the epitopes at the surface of the particles. The FMDV-cores produced in yeast were efficient inducers of neutralising antibodies in guinea-pigs after one low dose.