title: Generation of recombinant bacillus Calmette-Guérin and Mycobacterium smegmatis expressing BfpA and intimin as vaccine vectors against enteropathogenic Escherichia coli.
abstract: Enteropathogenic Escherichia coli (EPEC) is an important cause of diarrhea in children. EPEC adheres to the intestinal epithelium and causes attaching and effacing (A/E) lesions. Recombinant Mycobacterium smegmatis (Smeg) and Mycobacterium bovis BCG strains were constructed to express either BfpA or intimin. The entire bfpA gene and a portion of the intimin gene were amplified by PCR from EPEC genomic DNA and inserted into the pMIP12 vector at the BamHI/KpnI sites. The pMIP_bfpA and pMIP_intimin vectors were introduced separately into Smeg and BCG. Recombinant clones were selected based on kanamycin resistance and designated rSmeg_pMIP_(bfpA or intimin) and rBCG_pMIP_(bfpA or intimin). The expression of bfpA and intimin was detected by Immunoblotting using polyclonal anti-BfpA and anti-intimin antibodies. The immunogenicity of these proteins was assessed in C57BL/6 mice by assaying the feces and serum for the presence of anti-BfpA and anti-intimin IgA and IgG antibodies. TNF-α and INF-γ were produced in vitro by spleen cells from mice immunized with recombinant BfpA, whereas TNF-γ was produced in mice immunized with recombinant intimin. The adhesion of EPEC (E2348/69) to HEp-2 target cells was blocked by IgA or IgG antibodies from mice immunized with recombinant BfpA or intimin but not by antibodies from non-immunized mice. Immunogenic non-infectious vectors containing relevant EPEC virulence genes may be promising vaccine candidates.
authors: Laboratory of Immunochemistry, Butantan Institute, São Paulo, Brazil.
authors: Halyka Luzorio Franzotti
authors: HL
authors: Vasconcellos
authors: Karina
authors: K
authors: Scaramuzzi
authors: Ivan Pereira
authors: IP
authors: Nascimento
authors: Jorge M
authors: JM
authors: Da Costa Ferreira
authors: Cecilia M
authors: CM
authors: Abe
authors: Roxane M F
authors: RM
authors: Piazza
authors: Andre
authors: A
authors: Kipnis
authors: Wilmar
authors: W
authors: Dias da Silva
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics / Q000276:immunology*; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001500:BCG Vaccine / Q000008:administration & dosage* / Q000276:immunology; D002460:Cell Line; D054308:Enteropathogenic Escherichia coli / Q000276:immunology / Q000472:pathogenicity; D004847:Epithelial Cells / Q000166:cytology / Q000382:microbiology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000276:immunology*; D005260:Female; D036781:Fimbriae Proteins / Q000235:genetics / Q000276:immunology*; D005822:Genetic Vectors; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D009163:Mycobacterium bovis / Q000235:genetics / Q000276:immunology; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000276:immunology*; D013154:Spleen / Q000166:cytology / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000276:immunology
chemical_list: D018829:Adhesins, Bacterial; D000907:Antibodies, Bacterial; D001500:BCG Vaccine; C104901:BfpA protein, E coli; D029968:Escherichia coli Proteins; D014409:Tumor Necrosis Factor-alpha; D014614:Vaccines, Synthetic; C073819:eaeA protein, E coli; D036781:Fimbriae Proteins; D007371:Interferon-gamma