Didier Favre

Molecular cloning and characterization of the genetic determinants that express the complete Shigella serotype D (Shigella sonnei) lipopolysaccharide in heterologous live attenuated vaccine strains

The genetic determinants for the complete Shigella sonnei lipopolysaccharide (LPS) have been cloned, characterized by restriction mapping, and expressed in heterologous genetic backgrounds, including Salmonella typhi and Vibrio cholerae live attenuated vaccine strains. The rfb/rfc locus encoding the polymerized serotype‐specific O polysaccharide was mapped within 23 kb of DNA isolated from S. sonnei virulence plasmid pWR105. A highly similar chromosomal DNA sequence was identified by Southern hybridization analysis in Plesiomonas shigelloides known to have the same O serotype specificity as S. sonnei. Expression studies of the rfb/rfc locus have shown that S. sonnei. O polysaccharide is covalently bound to LPS cores of both the K‐12 and RI types, but neither to Salmonella (Ra‐type) nor to V. cholerae O1 cores. In order to express a compatible core structure in the latter organisms, chromosomal rfa loci encoding R1‐type LPS were isolated from both an Escherichia coli R1 strain (rfaR1) and from S. sonnei (rfdsonnei). Restriction mapping and functional analysis of cloned DNA allowed us to localize the rfaR1 locus and to orient it with respect to the neighbouring cysE chromosomal marker. A high degree of sequence similarity was found at the DNA level between rfa loci of enterobacterial species characterized by Ri‐type LPS. Co‐expression studies involving S. sonnei rfb/rfc and rfa loci propagated on compatible plasmids have shown that, at most, 13 to 14 kb of r/api DNA are required for the expression of complete phase‐l‐like S. sonnei LPS in E. coli K‐12 and S. typhi, whereas an adjacent region of about 3.5 kb is needed in the more stringent host, V. cholerae, S. sonnei O antigen expressed in a V. eholerae recombinant vaccine strain is present on the cell surface in a form suitable for the induction of a specific antibody response in vaccinated rabbits.

Expression of Shigella sonnei lipopolysaccharide in Vibrio cholerae

Making use of a newly designed mobilizable suicide vector, the genetic determinants encoding Shigella sonnei lipopolysaccharide (LPS) were stably integrated into the chromosome of the live attenuated Vibrio cholerae vaccine strain CVD103‐HgR. Expression studies showed that the production of complete S. sonnei O‐polysaccharide (O‐PS)‐bearing LPS was limited in bivalent recombinant strains that were also proficient in the synthesis of the host‐encoded Inaba O‐PS. Conversely, high amounts of LPS carrying S. sonnei O‐PS are produced in monovalent Inaba‐deficient derivatives, even in those strains which do not co‐express the compatible R1 LPS core. Thus, the non‐enterobacterial V. cholerae LPS core efficiently acts as a receptor for covalent binding of S. sonnei O‐PS provided that competition with the host O‐PS is avoided. Expression of the R1 core interferes with cell division in recombinant V. cholerae without affecting other physiological properties of vaccine strain CVD103‐HgR. Both monovalent and bivalent strains stimulated high serum‐antibody titres specific for their respective O‐serotype(s) when administered to rabbits. The potential of V. cholerae as an expression carrier for heterologous O‐serotypes is discussed.

Further molecular characterization and stability of the live oral attenuated cholera vaccine strain CVD103-HgR

Vibrio cholerae CVD103-HgR, the first live attenuated vaccine licensed for human use produced by recombinant DNA technology, was genetically compared to its parent strains 569B and CVD103. The genetic stability for both lyophilized vaccine in final container form and for viable organisms shed from vaccinees was determined. Results obtained lead us to conclude: (i) the genetic composition of the examined genes in CVD103-HgR is identical to that of the parent strains except for the alterations induced; (ii) the level of mercury resistance depends on the orientation of the mer operon within hlyA, with the highest level being observed for the orientation found in CVD103-HgR; (iii) no DNA sequences from plasmids used in construction remain in the genome; (iv) the strain is genetically stable; and (v) both CVD103-HgR and its parent strains contain defective lysogenic prophages. We have further confirmed that a certain amount of restriction fragment length polymorphism (RFLP) exists around the chromosomal ctx locus within V. cholerae strains of the classical biotype (detectable on chromosomal DNA restricted by either HindIII or EcoRI, but not PstI).

Morphological and immunocytochemical analysis of Escherichia coli-specific surface antigens in wildtype strains and in recombinant Vibrio cholerae.

Adhesion is the first step in the pathogenesis of enterotoxigenic Escherichia coli infections. The genes encoding the most prevalent adhesion factors CFA/I, CS3 and CS6 were cloned into Vibrio cholerae strain CVD 103–HgR and expression of fimbriae was investigated in wildtype and recombinant strains by transmission electron microscopy in conjunction with immunolabelling and negative staining. Negative staining was effective in revealing CFA/I and CS3, but not CS6. Although morphology of fimbriae differed between wildtype and recombinant strains, corresponding surface antigens were recognized by specific antibodies. The present study provides evidence that ETEC-specific fimbriae can adequately be expressed in an attenuated V. cholerae vaccine strain and that immunoelectron microscopy is a critical tool to validate the surface expression of antigens in view of their possible suitability for recombinant vaccines.

Versatile cosmid vectors for facilitated analysis and subcloning of the insert

A series of cosmid vectors, termed pSSVI215, pSSVI216-1, pSSVI216-2, pSSVI217, and pSSVI218, were constructed in order to facilitate the downstream processing of large inserts. Each vector has dual cos sites as well as a kanamycin resistance (KmR) gene flanked by recognition sites for the very rare cutter I-SceI meganuclease as well as symmetrical NotI and SwaI sites (SCEKAN cassette). Several unique cloning sites, including BamHI, are present on one side of the cassette between the I-SceI and NotI/SwaI sites. The various cosmids differ from each other by one or more of the following features: origin of replication (ori), size, host range, and conjugal transfer capability. Inserts combined with the SCEKAN cassette can be isolated on a NotI or SwaI fragment from any of these vectors and easily subcloned into the vector of choice by selecting for the adjacent KmR gene which can later be removed by I-SceI restriction and self-ligation. In addition, the SCEKAN cassette can be conveniently excised from plasmid pSSVI214 such that any plasmid can easily be fitted with the present system. The subcloning strategy afforded by the new vectors was successfully applied to an approximately 37-kb fragment from the V. cholerae O139 genome carrying the rfb locus which encodes the O-serotype specificity of this organism.

Expression and morphology of enterotoxigenic Escherichia coli surface antigen CS31A in E. coli K12 and Vibrio cholerae

Enterotoxigenic Escherichia coli (ETEC) is known as a worldwide cause of diarrheal disease. The pathogenesis involves the attachment of the microorganisms to the mucosa and the production of enterotoxins. Surface expression of CS31A fimbriae was assessed by Western blots, dot blots, immunofluorescence, and electron microscopy using negative staining and immunogold labeling. These investigations revealed significant differences in both the morphology of the wild-type and recombinant strains and the antigen exposure of CS31A in the wild-type and recombinant strains. In the wild-type ETEC strain, expression of CS31A was subject to phase variation. The recombinant E. coli strain produced CS31A but was prone to epitope shedding. In Vibrio cholerae vaccine strain CVD 103-HgR, the recombinant CS31A antigen was expressed but was only found intracellularly. Thus, E. coli strains seem to lend themselves better to the development of recombinant vaccines expressing ETEC-specific antigens at the cell’s surface than st...Enterotoxigenic Escherichia coli (ETEC) is known as a worldwide cause of diarrheal disease. The pathogenesis involves the attachment of the microorganisms to the mucosa and the production of enterotoxins. Surface expression of CS31A fimbriae was assessed by Western blots, dot blots, immunofluorescence, and electron microscopy using negative staining and immunogold labeling. These investigations revealed significant differences in both the morphology of the wild-type and recombinant strains and the antigen exposure of CS31A in the wild-type and recombinant strains. In the wild-type ETEC strain, expression of CS31A was subject to phase variation. The recombinant E. coli strain produced CS31A but was prone to epitope shedding. In Vibrio cholerae vaccine strain CVD 103-HgR, the recombinant CS31A antigen was expressed but was only found intracellularly. Thus, E. coli strains seem to lend themselves better to the development of recombinant vaccines expressing ETEC-specific antigens at the cells surface than strains from other orders or genera such as V. cholerae.