scFv6.C4 DNA vaccine with fragment C of Tetanus toxin increases protective immunity against CEA-expressing tumor.

The carcinoembryonic antigen (CEA) is the main tumor-associated antigen of colorectal cancers. Previously, we developed a DNA vaccine using scFv6.C4, a CEA surrogate, against CEA-expressing tumors; 40% of the vaccinated mice were tumor-free after tumor challenge. In order to enhance vaccine efficacy, fragment C of Tetanus Toxin (FrC) was tested as adjuvant. C57BL/6J-CEA2682 mice were electroporated intramuscularly 4 times with uP-PS/scFv6.C4-FrC or uP-PS/scFv6.C4, challenged by s.c. injection of 1 × 105 MC38-CEA cells, and tumor growth was monitored over 100 days. The humoral and cellular immune responses were assessed by ELISA, immunocytochemistry, in-vitro lymphocyte proliferation, and CTL cytotoxicity assays. Immunization with uP-PS/scFv6.C4-FrC or uP-PS/scFv6.C4 induced similar anti-CEA antibody titers. However, immunocytochemistry analysis showed stronger staining with uP-PS/scFv6.C4-FrC-immunized mice sera. When challenged with MC38-CEA cells, 63% of the FrC-vaccinated mice did not develop tumors, half of the rest had a significant tumor growth delay, and the probability of being free of tumors was on average 40% higher than that of scFv6.C4-immunized mice. Addition of the adjuvant led to higher CD4+ and CD8+ proliferative responses and strong CD8+ CTL response against MC38-CEA cells. DNA immunization with scFv6.C4 and FrC increased antitumor effect via induction of high and specific humoral and cellular immune responses to CEA.

Tetanus toxin production is triggered by the transition from amino acid consumption to peptides.

Bacteria produce some of the most potent biomolecules known, of which many cause serious diseases such as tetanus. For prevention, billions of people and countless animals are immunised with the highly effective vaccine, industrially produced by large-scale fermentation. However, toxin production is often hampered by low yields and batch-to-batch variability. Improved productivity has been constrained by a lack of understanding of the molecular mechanisms controlling toxin production. Here we have developed a reproducible experimental framework for screening phenotypic determinants in Clostridium tetani under a process that mimics an industrial setting. We show that amino acid depletion induces production of the tetanus toxin. Using time-course transcriptomics and extracellular metabolomics to generate a 'fermentation atlas' that ascribe growth behaviour, nutrient consumption and gene expression to the fermentation phases, we found a subset of preferred amino acids. Exponential growth is characterised by the consumption of those amino acids followed by a slower exponential growth phase where peptides are consumed, and toxin is produced. The results aim at assisting in fermentation medium design towards the improvement of vaccine production yields and reproducibility. In conclusion, our work not only provides deep fermentation dynamics but represents the foundation for bioprocess design based on C. tetani physiological behaviour under industrial settings.

Transgenic tomatoes express an antigenic polypeptide containing epitopes of the diphtheria, pertussis and tetanus exotoxins, encoded by a synthetic gene.

A current priority of vaccinology is the development of multicomponent vaccines that protect against several pathogens. The diphtheria-pertussis-tetanus (DPT) vaccine prevents the symptoms of three serious and often fatal diseases due to the exotoxins produced by Corynebacterium diphteriae, Bordetella pertussis and Clostridium tetani. We are attempting to develop an edible DPT multicomponent vaccine in plants, based on the fusion of protective exotoxin epitopes encoded by synthetic genes. By means of Agrobacterium mediated transformation we generated transgenic tomatoes with a plant-optimised synthetic gene encoding a novel polypeptide containing two adjuvant and six DPT immunoprotective exotoxin epitopes joined by peptide linkers. In transformed tomato plants, integration of the synthetic DPT (sDPT) gene detected by PCR was confirmed by Southern blot, and specific transcripts of the expected molecular size were detected by RT-PCR. Expression of the putative polypeptide encoded by the sDPT gene was detected by immunoassay with specific antibodies to the diphtheria, pertussis and tetanus exotoxins. The sDPT gene is therefore integrated, transcribed and translated as the expected recombinant sDPT multiepitope polypeptide in transgenic tomatoes that constitute a potential edible vaccine.

Sm14 of Schistosoma mansoni in fusion with tetanus toxin fragment C induces immunoprotection against tetanus and schistosomiasis in mice.

We have constructed vectors that permit the expression in Escherichia coli of Schistosoma mansoni fatty acid-binding protein 14 (Sm14) in fusion with the nontoxic, but highly immunogenic, tetanus toxin fragment C (TTFC). The recombinant six-His-tagged proteins were purified by nickel affinity chromatography and used in immunization and challenge assays. Animals inoculated with TTFC in fusion with or coadministered with Sm14 showed high levels of tetanus toxin antibodies, while animals inoculated with Sm14 in fusion with or coadministered with TTFC showed high levels of Sm14 antibodies. In both cases, there were no changes in the type of immune response (Th2) obtained with the fusion proteins compared to those obtained with the nonfused proteins. Mice immunized with the recombinant proteins (TTFC in fusion with or coadministered with Sm14) survived the challenge with tetanus toxin and did not show any symptoms of the disease. Control animals inoculated with either phosphate-buffered saline (PBS) or Sm14 died with severe symptoms of tetanus after 24 h. Mice immunized with the recombinant proteins (Sm14 in fusion with or coadministered with TTFC) showed a 50% reduction in worm burden when they were challenged with S. mansoni cercariae, while control animals inoculated with either PBS or TTFC were not protected. The results show that the expression of other antigens in fusion at the carboxy terminus of TTFC is feasible for the development of a multivalent recombinant vaccine.

High-level expression of tetanus toxin fragment C-thioredoxin fusion protein in Escherichia coli.

An insert of Clostridium tetani DNA corresponding to fragment C of tetanus toxin was amplified by PCR. This 1.4 kb fragment was cloned into the high-expression vector pET32a, under control of the T7 promoter. Expression of this plasmid in Escherichia coli BL21(DE3) resulted in the production of a fusion protein ( approximately 62 kDa) consisting of 112 amino acids of thioredoxin and approximately 450 amino acids of fragment C. This fusion protein was recognized by anti-tetanus toxoid antiserum in an ELISA and on immunoblots. The recombinant fragment-C-thioredoxin protein was purified significantly in one step by Ni(2+)-chelate Sepharose, the final yield being approximately 35 mg/l. Immunization of animals with the recombinant protein produced antibodies that were able to recognize the tetanus toxin. By using this gene-fusion expression system we produced soluble fragment C of tetanus toxin in a high yield, preventing many problems inherent in the use of other expression systems that produce either insoluble fragment C in inclusion bodies, or a soluble form, but in low yield, using E. coli as the expression host.