Doctor of Philosophy
Department of Biological Sciences
Smith, Adam M., Analysis and expression of important vaccine antigens of Bordetella pertussis, Doctor of Philosophy thesis, Department of Biological Sciences, University of Wollongong, 1999. http://ro.uow.edu.au/theses/1035
Bordetella pertussis is the causative agent of whooping cough, a contagious childhood respiratory disease. Increasing public concern over the safety of current whole-cell vaccines has led to decreased immunisation rates and a subsequent increase in the incidence of the disease. The preparation of safer vaccines is at present concentrated on the production of detoxified virulence factors such as pertussis toxin (PT), filamentous haemagglutinin (FHA), pertactin and serotype specific fimbriae for inclusion in acellular vaccine preparations. This study analyses the expression of several of these antigens in different genetic backgrounds.
A permanently avirulent Bordetella bronchiseptica strain was previously engineered to constitutively produce P T (Walker et al., 1991). An in vivo cloning technique, based on the principles of conjugal mating and chromosome transfer was employed to transfer the PT expression locus of this strain to virulent and avirulent strains of B. bronchiseptica. This transfer was confirmed by Southern hybridisation. An analysis of PT secretion in isogenic virulent and avirulent strains of B. bronchiseptica revealed that the PT produced was cell-associated, and not secreted to the growth medium. This evidence further suggests that B. bronchiseptica does not possess functional PT secretion (ptl) genes. Therefore, to achieve a PT expression and secretion system suitable for vaccine purposes in Bordetella bronchiseptica, functional ptl genes of B. pertussis are also required.
The use of "natural mutants" of Bordetella pertussis overexpressing immunogenic proteins as whooping cough vaccine strains may have a higher level of public acceptance than both the traditional whole cell preparation and the genetically engineered recombinant acellular vaccines currently under formulation. Mutants naturally overexpressing various candidate vaccine components have been created by Commonwealth Serum Laboratories (CSL Ltd.) via continuous subculture. These three strains (CSL 127S, CSL 137S and CSL 1237S) were genetically characterised by amplifying relevant regions of the genome, cloning these fragments into plasmid vectors and performing DNA sequencing analyses. B. pertussis CSL 137S, producing large amounts of type 3 fimbriae was found to possess an extra cytosine residue in the "fimbrial C stretch" of the promoter when compared to its parental strain. B. pertussis CSL 127S, which overexpresses type 2 fimbriae as well as F H A also has an altered C stretch. It contains one less cytosine than its parental strain. The upregulation in F H A could not be explained in terms of promoter differences. When the parental strain and CSL 127S were compared, the promoter regions of the F H A gene and the FHA/fimbrial accessory gene cluster were genetically identical. The third candidate vaccine strain (CSL 1237S) presented a more puzzling expression pattern. A moderate increase in the expression of most virulence factors analysed compared to its parental strain suggested a possible favourable mutation in the BvgAS promoter region. This however was not the case, with no differences being observed at any of the promoters analysed.
The two strategies for vaccine antigen expression investigated here may eventually lead to the production of readily manufactured and therefore more affordable whooping cough vaccine preparations.