To date, efforts to treat autoimmune diseases have primarily focused on the disease symptoms rather than on the cause of the disease. In large part, this is attributed to not knowing the responsible auto-antigens (auto-Ags) for driving the self-reactivity coupled with the poor success of treating autoimmune diseases using oral tolerance methods. Nonetheless, if tolerogenic approaches or methods that stimulate regulatory T (Treg) cells can be devised, these could subdue autoimmune diseases. To forward such efforts, our approach with colonization factor antigen I (CFA/I) fimbriae is to establish bystander immunity to ultimately drive the development of auto-Ag-specific Treg cells. Using an attenuated Salmonella vaccine expressing CFA/I fimbriae, fimbriae-specific Treg cells were induced without compromising the vaccine's capacity to protect against travelers' diarrhea or salmonellosis. By adapting the vaccine's anti-inflammatory properties, it was found that it could also dampen experimental inflammatory diseases resembling multiple sclerosis (MS) and rheumatoid arthritis. Because of this bystander effect, disease-specific Treg cells are eventually induced to resolve disease. Interestingly, this same vaccine could elicit the required Treg cell subset for each disease. For MS-like disease, conventional CD25+ Treg cells are stimulated, but for arthritis CD39+ Treg cells are induced instead. This review article will examine the potential of treating autoimmune diseases without having previous knowledge of the auto-Ag using an innocuous antigen to stimulate Treg cells via the production of transforming growth factor-beta and interleukin-10.
Animals ; Antigens, Bacterial/*immunology ; Arthritis, Rheumatoid/immunology/prevention & control ; Autoantigens/*immunology ; Fimbriae Proteins/*immunology ; Humans ; Multiple Sclerosis/immunology/prevention & control ; Salmonella/*immunology ; T-Lymphocytes, Regulatory/*immunology ; *Vaccination

In order to amplify pilA gene and ompC gene of avian pathogenic Escherichia coli (APEC) strain, two pairs of primers were designed according to the GenBank sequences, and a 549 bp pilA gene and a 1104 bp ompC gene were obtained by PCR separately. Sequence analysis indicated that the homology of the nucleotide sequence of AEPC strain to those other reference strains was 98.18% of the pilA gene and 97.28% of the ompC gene. Two expression plasmids pETpilA and pETompC were constructed by inserting pilA gene and ompC gene into the prokaryotic expression vector pET-28a. The two plasmids were transformated into E. coli BL21 separately and two recombinant strains BL21 (pETpilA) and BL21 (pETompC) were obtained. The type 1 fimbraie and the out membrane protein were highly expressed when the recombinant strain BL21 (pETpilA) and BL21 (pETompC) were induced by IPTG Two specific proteins were detected by SDS-PAGE and immunogenicity of the expressed protein was confirmed by Western blotting and ELISA. The expressed fimbraie and OmpC were transformed into vaccine. The protective immune response was proved after the mice were immunized with the two vaccines. The results showed that the recombinant strain BL21 (pETpilA) and BL21 (pETompC) could be as candidate vaccine to provide protective immune response against AEPC infection.
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; immunology ; metabolism ; Escherichia coli Proteins ; genetics ; immunology ; metabolism ; Escherichia coli Vaccines ; immunology ; Fimbriae Proteins ; genetics ; immunology ; metabolism ; Gene Expression Regulation, Bacterial ; Genes, Bacterial ; Mice ; Porins ; genetics ; immunology ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

OBJECTIVETo construct a recombinant plasmid containing Lgeionella pneumophila pilE gene, detect its expression in NIH3T3 cells and evaluate its immunogenicity.

METHODSPilE gene (LP) was amplified from Legionella pneumophila DNA by PCR and inserted into pcDNA3.1(+) vector to construct the recombinant plasmid pcDNA3.1-pilE, which as verified by restriction endonuclease digestion, PCR and DNA sequencing analysis. NIH3T3 cells were transfected with the recombinant plasmid with Lipofection strategy. Transient and stable pilE gene products were detected by immunofluorescence and Western blotting, respectively. To evaluate the immunogenicity of pcDNA3.1-pilE, the recombinant plasmid was used as a DNA vaccine to immunize female BALB/c mice intramuscularly and the specific antibodies, lymphocyte proliferation response, interferon (IFN)-gamma production and cytotoxic T-lymphocyte response of the immunized mice were detected and evaluated.

RESULTSThe pilE gene of 429 bp in length was amplified. After transfection of NIH3T3 cells with the recombinant plasmid, strong green fluorescence was observed on the cell membrane and inside the cell. A protein with relative molecular mass of 15.7 kD was detected in the transfected cells with Western blotting, suggesting successful protein expression of pilE gene. pcDNA3.1-pilE resulted in much stronger immune response in the immunized mice than pcDNA3.1(+) (P<0.01).

CONCLUSIONThe recombinant plasmid containing Lgeionella pneumophila pilE gene constructed in this study is capable of expression in NIH3T3 cells, and can induce specific humoral and cellular immune responses in mice.

Animals ; Blotting, Western ; Cell Proliferation ; Fimbriae Proteins ; genetics ; immunology ; metabolism ; Fimbriae, Bacterial ; Fluorescent Antibody Technique ; Humans ; Immunization ; methods ; Injections, Intramuscular ; Interferon-gamma ; metabolism ; Legionella pneumophila ; genetics ; immunology ; metabolism ; Lymphocytes ; cytology ; immunology ; metabolism ; Mice ; Mice, Inbred BALB C ; NIH 3T3 Cells ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Transfection ; Vaccines, DNA ; administration & dosage ; genetics ; immunology

A pair primer was designed by Oligo 6.0 according to the pilA gene sequence of E. coli isolated from human in GenBank. The pilA Gene was obtained by PCR with the enteropathogenic E. coli isolated from ducks as template and cloned into pMD18-T vector. It was identified by PCR, restriction endonuclease analysis, DNA sequencing and then subcloned into BamH I/Hind III site of prokaryotic expression vector pET-32a(+) and recombinant expression plasmid pET-32a-pilA was constructed successfully. The plasmid was transformed into Eschericha coli BL21 (DE3) and 36kD pilA recombinant protein was expressed be induced with IPTG. The protein was purified by Ni-agarose affinity chromatograghy and was prepared as vaccine with Freund' s adjuvant. The ducklings were immunized with the vaccine at 1 and 8-day-old respectively. Two weeks after last immunized, the antibody titer of duck serum was detected by ELISA and the ducklings were challenged with 10(9) PFU enteropathogenic E. coli GH1.2 virulent strain. The immunoprotection effect of pilA recombinant protein vaccine was evaluated according to the mortality, re-isolated rate of E. coli, and grades of pathological changes. The results show that the antibody titer are 1:12800, but 1:200 were detected from ducklings immunized with homologous whole cells E. coli inactivated vaccine. The mortality, re-isolated rate of E. coli, degree of pathological changes of immunized ducklings is lower than that of the control ducklings and showed significant or extremely significant differences (P < 0.01 or P < 0.05), but non-significant difference compared to the ducklings which immunized with homologous whole cells E. coli inactivated vaccine (P > 0.05). The results show that pilA recombinant protein has some immunoprotection effect with the challenging of virulent strains of E. coli GH1.2.
Animals ; Animals, Newborn ; Antibodies, Bacterial ; blood ; immunology ; Ducks ; microbiology ; Electrophoresis, Polyacrylamide Gel ; Enteropathogenic Escherichia coli ; genetics ; immunology ; pathogenicity ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli Proteins ; genetics ; immunology ; metabolism ; Fimbriae Proteins ; genetics ; immunology ; metabolism ; Humans ; Immunization ; Polymerase Chain Reaction ; Poultry Diseases ; immunology ; mortality ; prevention & control ; Recombinant Proteins ; administration & dosage ; immunology ; metabolism ; Survival Rate ; Virulence

Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and possible adverse effects on the oral administration of obtained tobacco seeds were evaluated in a mouse model. Tobacco was transformed via Agrobacteium tumefaciens with chimeric constructs containing structural parts of the major subunit FedA of the F18 adhesive fimbriae and VT2e B-subunit genes under control of a seed specific GLOB promoter. We showed that the foreign Vt2e-B and F18 genes were stably accumulated in storage tissue by the immunostaining method. In addition, Balb-C mice receiving transgenic tobacco seeds via the oral route showed a significant increase in IgA-positive plasma cell presence in tunica propria when compared to the control group with no observed adverse effects. Our findings encourage future studies focusing on swine for evaluation of the protective effects of transformed tobacco seeds against E. coli infection.
Administration, Oral ; Agrobacterium tumefaciens ; Animals ; Antigens, Bacterial/genetics/metabolism ; Bacterial Vaccines/administration & dosage/adverse effects/*pharmacology ; Edema Disease of Swine/*immunology/microbiology ; Escherichia coli Infections/immunology/microbiology/*veterinary ; Escherichia coli Proteins/*genetics/metabolism ; Female ; Fimbriae Proteins/genetics/metabolism ; Genetic Engineering ; Intestines/immunology/microbiology/pathology ; Mice ; Mice, Inbred BALB C ; Models, Animal ; Plants, Genetically Modified/*genetics/metabolism ; Seeds/genetics/metabolism ; Shiga Toxin 2/genetics/metabolism ; Shiga-Toxigenic Escherichia coli/genetics/immunology/*pathogenicity ; Swine ; Tobacco/*genetics/metabolism ; Virulence Factors/genetics/metabolism