Background: Oral immunization with vaccines may be an effective strategy for prevention of Clostridium difficile\r\ninfection (CDI). However, application of previously developed vaccines for preventing CDI has been limited due\r\nto various reasons. Here, we developed a recombinant Lactococcus lactis oral vaccine and evaluated its effect on\r\na C. difficile-infected animal model established in golden hamsters in attempt to provide an alternative strategy\r\nfor CDI prevention.\r\nMethods: Recombinant L. lactis vaccine was developed using the pTRKH2 plasmid, a high-copy-number\r\nEscherichia coli-L. shuttle vector: 1) L. lactis expressing secreted proteins was constructed with recombinant\r\npTRKH2 (secreted-protein plasmid) carrying the Usp45 signal peptide (SPUsp45), nontoxic adjuvanted tetanus\r\ntoxin fragment C (TETC), and 14 of the 38 C-terminal repeats (14CDTA) of nontoxic C. difficile toxin A (TcdA); and\r\n2) L. lactis expressing secreted and membrane proteins was constructed with recombinant pTRKH2 (membrane-anchored\r\nplasmid) carrying SPUsp45, TETC, 14CDTA, and the cell wall-anchored sequence of protein M6 (cwaM6). Then, 32 male\r\nSyrian golden hamsters were randomly divided into 4 groups (n = 8 each) for gavage of normal saline (blank control) and\r\nL. lactis carrying the empty shuttle vector, secreted-protein plasmid, and membrane-anchored plasmid, respectively. After\r\n1-week gavage of clindamycin, the animals were administered with C. difficile spore suspension. General symptoms and\r\nintestinal pathological changes of the animals were examined by naked eye and microscopy, respectively. Protein levels\r\nof anti-TcdA IgG/IgA antibodies in intestinal tissue and fluid were analyzed by enzyme-linked immunosorbent assay\r\n(ELISA). A cell culture cytotoxicity neutralization assay was done by TcdA treatment with or without anti-TcdA serum\r\npre-incubation or treatment. Apoptosis of intestinal epithelial cells was examined by flow cytometry (FL) assay.\r\nExpression of mucosal inflammatory cytokines in the animals was detected by polymer chain reaction (PCR) assay.
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