Connaught Laboratories Limited

Other

Washington University

Raafat Emil Fahim

Michel Henri Klein

Pele Chong

Gail E Jackson

Larry U Tan

John Vose

Yan-Ping Yang

Robert S Munson, Jr

Patrick McVerry

Mary E Ewasyshyn

Ali Kandil

George A Cates

Michel H Klein

Lucy Gisonni

Susan Grass

Dwo Yan Charles Sia

Robert Tolan

Andrew Herbert

Luis Barreto

John Thipphawong

Alison L Symington

John R Vose

Pertactin (formerly 69 kDa protein) is recovered in stable biologically pure form having no detectable adenylate cyclase activity from fermentation broth from the fermentation of as well as from the cells. The broth is processed to selectively remove pertussis toxin (PT) and filamentous haemagglutinin (FHA), the pertactin is precipitated by ammonium sulphate and the precipitate is dissolved in buffer at pH 6.0 to 8.5, the solution then is passed through hydroxyapatite and ion-exchange chromatograph columns before final ultrafiltration. Cells are extracted with urea and the extract ultrafiltered and diafiltered. The pertactin is precipitated from the extract and the precipitate processed as above. In a variation, the broth is contacted with ammonium sulphate to precipitate pertactin, PT and FHA, the precipitate is dissolved and the PT and FHA selectively removed, before the solution is passed to the chromatograph columns.

Purification of a pertussis outer membrane protein

Parainfluenza virus glycoproteins and vaccines

The gene for outer membrane protein P1 of Haemophilus influenzae b is expressed in E. coli. Methods for expression and demonstration of the immunogenicity of recombinant P1 and portions thereof are disclosed, along with an improved method for the purification of P1. The nucleotide sequence of the P1 gene and the derived amino acid sequence of the P1 protein of Haemophilus influenzae type b are disclosed and the methods used to determine the same. Also disclosed are the methods used to clone and express the P1 gene as well as the purification protocol for the P1 gene products (recombinant P1 and P1 fusion proteins). Fourteen peptides are synthesized corresponding to specific sequences of the mature P1 protein. The use of the P1 protein as n immunogens for immunization against the disease caused by Haemophilus influenzae type b and the use of the protein as a carrier for conjugation with an oligosaccharide derived from Haemophilus to generate a potentially efficacious vaccine against the disease, are described. Also disclosed is the use of P1 peptide-conjugates as immunizing agents to elicit anti-Haemophilus influenzae type b antibodies.

Outer membrane protein P1 and peptides of Haemophilus influenzae type B

The nucleotide sequence of the P2 gene and the derived amino acid sequence of the P2 protein of Haemophilus influenzae type b are disclosed and the methods used to determine the same. Also disclosed are the methods used to clone and express the P2 gene as well as the purification protocol for the gene and protein. Also disclosed is the synthesis of peptides corresponding to the N-terminal and C-terminal ends of the P2 protein. Also disclosed is the use of the P2 protein as a protective agent for the disease caused by Haemophilus influenzae type b and the use of the protein as a carrier for conjugation with an oligosaccharide derived from Haemophilus to generate a potentially efficacious vaccine against the disease. Also disclosed is the use of P2 peptide-conjugates as immunising agents.

Membrane proteins and peptides of haemophilus influenzae type B

A fimbrial agglutinogen preparation is prepared from a bordetella strain, particularly a B. pertussis strain, by a multiple step procedure involving extraction of the fimbrial agglutinogens from cell paste and concentrating and purifying the extracted material. The fimbrial agglutinogen preparation may be used to prepare acellular pertussis vaccines with other pertussis antigens, including pertussis toxin or toxoid thereof, the 69 kDa protein and filamentous hemagglutinin and other Bordetella antigens.

Acellular pertussis vaccines and methods of preparing thereof

Immunogenic conjugate molecules comprising at least a portion of a capsular polysaccharide of a Streptococcus strain linked to at least a portion of an outer membrane protein of a Haemophilus strain are provided in which the immunogenicity of the capsular polysaccharide is increased. Particularly capsular polysaccharide from Streptococcus pneumoniae are linked to an outer membrane protein of a Haemophilus influenzae strain, which protein may be the P1, P2 or particularly the P6 outer membrane protein. Conjugate molecules comprising the P6 protein linked to a capsular polysaccharide from an encapsulated pathogen other than Streptococcus also are described, in which the immunogenicity of the capsular polysaccharide is enhanced. Such conjugate molecules may be incorporated into immunogenic compositions for protecting a host against disease caused by the Streptococcus strain and preferably also the Haemophilus strain. The conjugate molecules and antibodies specific for the capsular polysaccharide or specific for the outer membrane protein may be employed in diagnostic procedures and kits. A process for individually isolating P1, P2 and P6 outer membrane proteins from a Haemophilus strain also is provided.

Immunogenic conjugate molecules

Pertactin (formerly 69 kDa protein) is recovered in stable biologically pure form having no detectable adenylate cyclase activity from fermentation broth from the fermentation of Bordetella pertussis as well as from the cells. The broth is processed to selectively remove pertussis toxin (PT) and filamentous haemagglutinin (FHA), the pertactin is precipitated by ammonium sulphate and the precipitate is dissolved in buffer at pH 6.0 to 8.5, the solution then is passed through hydroxyapatite and ion-exchange chromatograph columns before final ultrafiltration. Cells are extracted with urea and the extract ultrafiltered and diafiltered. The pertactin is precipitated from the extract and the precipitate processed as above. In a variation, the broth is contacted with ammonium sulphate to precipitate pertactin, PT and FHA, the precipitate is dissolved and the PT and FHA selectively removed, before the solution is passed to the chromatograph columns.

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Mississauga, Canada

Raafat Emil Fahim