Characterization of Shigella dysenteriae type 1 hybrid vaccine candidates

Abstract

Shigellae is endemic in many developing countries in the world and a major cause of childhood diarrheal morbidity and mortality. Infections caused by Shigella dysenteriae type 1 are particularly severe. The major virulence factors in Shigella dysenteriae type 1 are the cell envelope lipopolysaccharide, the invasion peptide antigens and the Shiga toxin. Little is known of 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 after introduction of the genes for the Shigella dysenteriae type 1 O-antigen biosynthesis into Escherichia coli K12, aroA mutant Salmonella strains and aroD mutant Shigella flexneri strains. The structural characterization of different E.coli K12/S. dysenteriae type 1 hybrid strains resulted in the following structures in class I, IV and V hybrid strains: E. coli K12 (pSS3) and E. coli K12 (pSS3, pSS9-6; a class I hybrid); alpha-D-Galp(1-->3)beta-D-GlcpNAc(1-->. Class IV hybrids: E. coli K12 (pSS3, pSS9-36); (pSS3, pSS9-107) and (pSS3, pSS9-114); alpha-L-Rhap(1-->2)alpha-D-Galp(1-->3)beta-D-GlcpNAc(1-->. Class V hybrids: E. coli K12 (pSS3, pSS9-78) and (pSS3, pSS9-111); alpha-L-Rhap(1-->3)alpha-L-Rhap(1-->2)alpha-D-Galp(1-->3)bet a-D-GlcpNAc(1-->

The Escherichia coli K12/ Shigella dysenteriae type 1 hybrid strains express parts of the Shigella dysenteriae type 1 O-antigenic tetrasaccharide repeating unit due to Tn 1000 insertions in pSS9, inactivating different rfb genes. To be able to detect different structural elements in the S. dysenteriae type 1 O-antigenic repeating unit, mouse and rat monoclonal antibodies directed against E coli K12/S. dysenteriae type 1 hybrid strains and native S. dysenteriae type 1 strain were produced and characterized. The mAbs produced were found to be specific for epitopes existing in class I, IV and class V hybrid strains: MAEC-SD-1 and MAEC-SD-2 were class IMAEC-SD-II was class IV and MAEC-SD21, MASD-1 and MASD-2 were all class IV specific. The rfp and rfb loci, encoding biosynthesis of the Shigella dysenteriae type 1 O-antigen, were introduced, encoded on a plasmid construct and also by using the Tn 501 mercury resistant transposon into the chromosome, of aroA mutants of Salmonella typhimurium and Salmonella dublin strains. Salmonella typhimurium strain SL3235 and SL3261 expressed both Salmonella typhimurium and Shigella dysenteriae type I O-antigen when the rfp and rfb genes were introduced on a plasmid construct.

For Salmonella dublin strain SL1438 both the plasmid encoded and chromosomally integrated derivatives expressed both Salmonella dublin and Shigella dysenteriae type 1 O-antigen on its surface. For all strains, individual bacteria produced both types of lipopolysaccharides. The Shigella dysenteriae type 1 polysaccharide chains 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, Shigella flexneri serotype 2a SFL1070 and their rough derivatives resulted in bivalent construct 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 polymerized Shigella dysenteriae type 1 O-antigen in the serotype 2a vaccine strain SFL1070. When strains representing other serotypes of Shigella flexneri were tested no polymerized Shigella dysentenae 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 Shigella dysenteriae type 1 O-antigen biosynthesis, it is possible to generate a vaccine candidate which in vitro expresses well the desired O-polysaccharides.