Characterization of Shigella dysenteriae type 1 hybrid vaccine candidates

Sammanfattning: Shigellae is endemic in many developing countries in the world and a major cause of childhooddiarrheal morbidity and mortality. Infections caused by Shigella dysenteriae type 1 areparticularly severe. The major virulence factors in Shigella dysenteriae type 1 are the cellenvelope lipopolysaccharide, the invasion peptide antigens and the Shiga toxin. Little is knownof the relative importance of each of the virulence factors and their role in protective immunity.In this study, the expression of Shigella dysenteriae type 1 O-antigen has been studied afterintroduction of the genes for the Shigella dysenteriae type 1 O-antigen biosynthesis intoEscherichia coli K12, aroA mutant Salmonella strains and aroD mutant Shigella flexneristrains .The structural characterization of different E.coli K12/S. dysenteriae type 1 hybrid strainsresulted in the following structures in class I, IV and V hybrid strains:S. dysenteriae type I O-antigen | E. coli K12 core | a-L-Rha-( l-3)-a-L-Rha-(1-2)-a-D-Gal-(1-|-3)-b-D-GlcNAc( l-6)-a-D-Glc- ________________________________Class I ______________________________________________Class IV _____________________________________________________________Class VThe Escherichia coli K12/ Shigella dysenteriae type I hybrid strains express parts of theShigella dysenteriae type I O-antigenic tetrasaccharide repeating unit due to TnlOOO insertionsin pSS9, inactivating different rfb genes. To be able to detect different structural elements in theS dysenteriae type I O-antigenic repeating unit, mouse and rat monoclonal antibodies directedagainst E coli K12/S. dysenteriae type I hyhrid strains and native S. dysenteriae typc I strainwere produced and characterized. The mAbs produced were found to be specific for epitopesexisting in class I, IV and class V hybrid strains: MAEC-SD-I and MAEC-SD-2 were class IMAEC-SD-II was class IV and MAEC-SD21, MASD-I and MASD-2 were all class IVspecific.The rfp and rfb loci, encoding biosynthesis of the Shigella dysenteriae type I O-antigen, wereintroduced, encoded on a plasmid construct and also by using the Tn 501 mercury resistanttransposon into the chromosome, of aroA mutants of Salmonella typhimurium and Salmonelladublin strains. Salmonella typhimurium strain SL3235 and SL3261 expressed both Salmonellatyphimurium and Shigella dysenteriae type I O-antigen when the rfp and rfb genes wereintroduced on a plasmid construct. For Salmonella dublin strain SL1438 both the plasmidencoded and chromosomally integrated derivatives expressed both Salmonella dublin andShigella dysenteriae type 1 O-antigen on its surface. For all strains, individual bacteriaproduced both types of lipopolysaccharides. The Shigella dysenteriae type 1 polysaccharidechains were linked to position 0-4 of the subterminal D-glucose residue of the Salmonella core.The introduction of the genetic determinants rfp and rfb into Shigella flexneri serotype YSFL124, Shigellaflexneri serotype 2a SFL1070 and their rough derivatives resulted in bivalentconstruct of SFL124 expressing both Shigella flexneri and Shigella dysenteriae type 1 O-antigen and the monovalent construct expressing only Shigella dysenteriae type 1 O-antigen.The polysaccharide chains were shown to be linked to position 0-4 of the subterminal D-glucose residue of the Shigella flexneri core. There was no expression of the polymerizedShigella dysenteriae type 1 O-antigen in the serotype 2a vaccine strain SFL1070. When strainsrepresenting other serotypes of Shigella flexneri were tested no polymerized Shigelladysentenae type 1 O-antigen were seen in some of the serotypes.This study has shown that by introducing the rfp and rfb loci, encoding for the Shigelladysenteriae type 1 O-antigen biosynthesis, it is possible to generate a vaccine candidate whichin vitro expresses well the desired O-polysaccharides.ISBN 91-628-1796-5