Enhancement of the specific mucosal IgA response in vivo by interleukin-5 expressed by an attenuated strain of Salmonella serotype Dublin.

It has been shown that cytokines have potential as therapeutic adjuvants in vaccination. Interleukin-5 (IL-5) is a cytokine that regulates antibody production, in particular enhancing IgA production by activated mucosal B cells. This study examined the expression of a cloned cytokine gene encoding murine IL-5 (mIL-5) by an attenuated aroA strain (SL5631) of Salmonella serotype Dublin. The resulting strain, SL5631(pTRXFUS-mIL-5), expressed mIL-5 as a fusion with thioredoxin as demonstrated by immunological and biological assays. When strain SL5631(pTRXFUS-mIL-5) was used as a live vaccine in BALB/c mice, it colonised and multiplied at higher levels in spleens and livers than the strain carrying the empty plasmid. A reduction in invasiveness of SL5631(pTRXFUS-mIL-5) was observed in in-vitro invasion assays. Enhanced IgA response against salmonella LPS in mucosal secretions and enhanced IgA and IgG responses were detected by ELISA and ELISPOT methods in sera of mice immunised with the strain expressing mIL-5. Results with IL-5-deficient mice showed that the enhanced IgA response was due to Salmonella-expressed mIL-5 rather than endogenous mIL-5.

The immune response to a B-cell epitope delivered by Salmonella is enhanced by prior immunological experience.

Attenuated, heterologous strains of Salmonella have shown potential as live, recombinant vaccines against foreign pathogens. Studies in animal models have demonstrated that immunization with these heterologous vaccines is an effective way to induce both cellular and humoral immune responses against Salmonella and the foreign antigen. We studied the consequence of priming mice with Salmonella dublin 3-6 months before intraperitoneal administration with the same strain carrying a model B-cell epitope. Mice primed with the carrier strain demonstrated enhanced serum Ig titres against the foreign antigen. This immune enhancement was observed up to approximately 6 months after priming. These findings suggest that previous immunological experience with Salmonella does not limit the immune response to a foreign antigen carried by the same organism. In fact, prior exposure to Salmonella appears to enhance the response to the foreign antigen.

Shigella flexneri type-specific antigen V: cloning, sequencing and characterization of the glucosyl transferase gene of temperate bacteriophage SfV.

With lysogeny by bacteriophage SfV, Shigella flexneri serotype Y is converted to serotype 5a. The glucosyl transferase gene (gtr) from bacteriophage SfV of S. flexneri, involved in serotype-specific conversion, was cloned and characterized. The DNA sequence of a 3.7 kb EcoRI-BamHI fragment of bacteriophage SfV which includes the gtr gene was determined. This gene, encoding a polypeptide of 417 aa with 47.67 kDa molecular mass, caused partial serotype conversion of S. flexneri from serotype Y to type V antigen as demonstrated by Western blotting and the sensitivity of the hybrid strain to phage Sf6. The deduced protein of the partially sequenced open reading frame upstream of the gtr showed similarity to various glycosyl transferases of other bacteria. Orf3, separated from the gtr by a non-coding region and transcribed convergently, codes for a 167 aa (18.8 kDa) protein found to have homology with tail fibre genes of phage lambda and P2.

Molecular characterization of the genes involved in O-antigen modification, attachment, integration and excision in Shigella flexneri bacteriophage SfV.

Bacteriophage SfV is a temperate phage of Shigella flexneri responsible for converting serotype Y (3,4) to serotype 5a (V; 3,4) through its glucosyl transferase gene. The glucosyl transferase (gtr) gene of SfV has been cloned and shown to partially convert S. flexneri serotype Y to serotype 5a. In this study, we found that the serotype-converting region of SfV was approximately 2.5 kb in length containing three continuous ORFs. The recombinant strain carrying the three complete ORFs expressed the type V and group antigen 3,4, both indistinguishable from that of S. flexneri 5a wild-type strain. The interruption of orf5 or orf6 gave partial conversion in the S. flexneri recombinant strain indicated by the incomplete replacement of group antigen 3,4. The region adjacent to the serotype-conversion genes was found to be identical to the attP-int-xis region of phage P22. Altogether, an approximately 2.2-kb sequence covering a portion of the serotype-conversion (approximately 500 nt)-attP-int-xis regions of SfV was remarkably similar to that of P22.

Immune response to a Murray Valley encephalitis virus epitope expressed in the flagellin of an attenuated strain of Salmonella.

Recent developments in vaccine construction include the use of attenuated, avirulent strains of Salmonella as carriers of foreign antigens. These recombinant strains can elicit a heterologous immune response when injected into animals, demonstrating potential for their use in the construction of many vaccines. In the present study, a B-cell epitope of Murray Valley encephalitis virus (MVE) was identified and expressed in a Salmonella strain to evaluate its potential to induce a specific immune response to MVE. A synthetic oligonucleotide encoding the B-cell epitope (residues E201-224) of the envelope protein of MVE was inserted into the cloned flagellin gene of the Salmonella strain. The construct was sequenced to ensure correct orientation of the epitope. Expression of the epitope was demonstrated by Western blot analysis and immunogold electron microscopy with monoclonal antibody specific to the epitope. Electron microscopy analysis revealed multiple copies of the epitope along the flagella. The recombinant Salmonella carrying the hybrid flagellin gene elicited an immune response to the MVE epitope in a mouse model. The MVE-specific antibodies partially neutralised the virus in vitro. The significance of this system for engineering vaccines for other medically important flaviviruses is discussed.