Escherichia coli heat-labile toxin subunit B fusions with Streptococcus sobrinus antigens expressed by Salmonella typhimurium oral vaccine strains: importance of the linker for antigenicity and biological activities of the hybrid proteins

Abstract

A set of vectors possessing the genes for aspartate semialdehyde dehydrogenase (asd) and the B subunit of the heat-labile enterotoxin of Escherichia coli (LT-B) has been developed. These vectors allow operon or gene fusions of foreign gene epitopes at the C-terminal end of LT-B. Two groups of vectors have been constructed with and without leader sequences to facilitate placing of the foreign antigen in different cell compartments. Two Streptococcus sobrinus genes coding for principal colonization factors, surface protein antigen A (SpaA), and dextranase (Dex), have been fused into the 3' end of the LT-B gene. Resulting protein fusions of approximately 120 to 130 kDa are extremely well recognized by antibodies directed against both SpaA and Dex as well as against LT-B domains and retain the enzymatic activity of dextranase and the biological activity of LT-B in that they bind to GM1 gangliosides. Maximum antigenicity was obtained with the vector possessing an intervening linker of at least six amino acids with two proline residues. Some of the fusion proteins also exhibited another property of LT-B in that they were exported into the periplasm where they oligomerized. LT-B-SpaA and LT-B-Dex hybrid proteins are expressed stably and at a high level in avirulent Salmonella typhimurium vaccine strains which are being used to investigate their immunogenicity and types of induced immune responses. The fusion vectors will also be useful for production and purification of LT-B fusion antigens to be used and evaluated in other vaccine compositions.

Comparative immunological evaluation of recombinant Salmonella Typhimurium strains expressing model antigens as live oral vaccines

“…This strain induced the highest levels of B cell immunogenicity (Table 4 and Figures 2B, 3 and 4). This result is similar to those reported in previous studies, which have found that lowering the plasmid copy number results in enhanced plasmid stability and a reduction in the expression levels of heterologous antigens to non-toxic levels [31][32][33][34].…”

vaccines for use in humans: present and future perspectives

“…It is thought that TetC promotes the immune response against fused antigens by providing additional T cell helper epitopes [131]. In other studies, Streptococcus sobrinus antigens [132] or the E. coli heat-stable enterotoxin [133] were expressed in Salmonella as fusions to the E. coli heat-labile toxin subunit B (LT-B), which is also known to have immune-enhancing e¡ects. LT-B and CT-B, the cholera toxin B subunit, are well-known non-toxic adjuvants which contain structural features that provide adjuvant and immunogenic stimulation in the intestinal mucosa [134].…”

A continuous epitope from transmissible gastroenteritis virus S protein fused to E. coli heat-labile toxin B subunit expressed by attenuated Salmonella induces serum and secretory immunity

“…It was also of interest to express multiple repeats of site D fused to LT-B to increase the immunogenicity of this epitope. Two synthetic complementary oligonucleotides encoding residues 371-400 of TGEV S protein, corresponding to site D sequence, were hybridized, cloned into the asd + LT-B expression plasmid pYA3048 (asd + LT-B +) (Jagusztyn-Krynicka et al, 1993), and electroporated into the restriction negative, modification positive strain S. typhimurium hsdLT hsdSA hsdSB Aasd Z3730 (pYA232) . The vector:insert ratio used in the ligation was 1:10 in order to obtain a collection of recombinant plasmids in which the LT-B genes were fused to a variable number of sequences of site D combined in the two possible orientations.…”

“…The nucleotide sequence of site D that was cloned (5'-ATCTCCT-GTTACACCGTTTCCGACTCCTCCTTCTTCT-CCTACGGTGAAATCCCGTTCGGTGTTAC-CGACGGTCCGCGTTACTGCTACGTT-3') was not exactly the same as that of the S gene from TGEV genome, since some triplets present in the viral sequence were substituted for other synonymous triplets of high frequency of use in Enterobacteriaceae. The fusion of site D sequence to LT-B was done to the carboxyl end of this molecule through a linker coding for 6 amino acids containing two prolines, in order to preserve the molecular properties of LT-B, such as pentamerization and GM1 binding (Jagusztyn- Krynicka et al, 1993).…”

“…LT and its B subunits are potent oral immunogens which can function also as oral adjuvants eliciting high-titer serum and secretory antibodies when administered into the gut (Houghten et al, 1985;Lipscombe et al, 1991). Several asd ÷ LT-B ÷ plasmids allowing fusions of foreign genes to the C terminus of LT-B have been constructed (Jagusztyn-Krynicka et al, 1993). The asd ÷ plasraids ensure stable in vivo maintenance in attenuated Aasd S. typhimurium vaccine strains in the absence of external selection.…”