title: The protective effect of recombinant Lactococcus lactis oral vaccine on a Clostridium difficile-infected animal model.
abstract: BACKGROUND
  Oral immunization with vaccines may be an effective strategy for prevention of Clostridium difficile infection (CDI). However, application of previously developed vaccines for preventing CDI has been limited due to various reasons. Here, we developed a recombinant Lactococcus lactis oral vaccine and evaluated its effect on a C. difficile-infected animal model established in golden hamsters in attempt to provide an alternative strategy for CDI prevention.
  
  
  METHODS
  Recombinant L. lactis vaccine was developed using the pTRKH2 plasmid, a high-copy-number Escherichia coli-L. shuttle vector: 1) L. lactis expressing secreted proteins was constructed with recombinant pTRKH2 (secreted-protein plasmid) carrying the Usp45 signal peptide (SPUsp45), nontoxic adjuvanted tetanus toxin fragment C (TETC), and 14 of the 38 C-terminal repeats (14CDTA) of nontoxic C. difficile toxin A (TcdA); and 2) L. lactis expressing secreted and membrane proteins was constructed with recombinant pTRKH2 (membrane-anchored plasmid) carrying SPUsp45, TETC, 14CDTA, and the cell wall-anchored sequence of protein M6 (cwaM6). Then, 32 male Syrian golden hamsters were randomly divided into 4 groups (n = 8 each) for gavage of normal saline (blank control) and L. lactis carrying the empty shuttle vector, secreted-protein plasmid, and membrane-anchored plasmid, respectively. After 1-week gavage of clindamycin, the animals were administered with C. difficile spore suspension. General symptoms and intestinal pathological changes of the animals were examined by naked eye and microscopy, respectively. Protein levels of anti-TcdA IgG/IgA antibodies in intestinal tissue and fluid were analyzed by enzyme-linked immunosorbent assay (ELISA). A cell culture cytotoxicity neutralization assay was done by TcdA treatment with or without anti-TcdA serum pre-incubation or treatment. Apoptosis of intestinal epithelial cells was examined by flow cytometry (FL) assay. Expression of mucosal inflammatory cytokines in the animals was detected by polymer chain reaction (PCR) assay.
  
  
  RESULTS
  After the C. difficile challenge, the animals of control group had severe diarrhea symptoms on day 1 and all died on day 4, indicating that the CDI animal model was established in hamster. Of the 3 immunization groups, secreted-protein and membrane-anchored plasmid groups had significantly lower mortalities, body weight decreases, and pathological scores, with higher survival rate/time than the empty plasmid group (P < 0.05). The tilter of IgG antibody directed against TcdA was significantly higher in serum and intestinal fluid of secreted-protein and membrane-anchored plasmid groups than in the empty plasmid group (P < 0.05) while the corresponding titer of IgA antibody directed against TcdA had no substantial differences (P > 0.05). The anti-TcdA serum of membrane-anchored plasmid group neutralized the cytotoxicity of 200 ng/ml TcdA with the best protective effect achieved by anti-TcdA serum pre-incubation. The incidences of TcdA-induced death and apoptosis of intestinal epithelial cells were significantly reduced by cell pre-incubation or treatment with anti-TcdA serum of membrane-anchored plasmid group (P < 0.05). MCP-1, ICAM-1, IL-6, and Gro-1 mRNA expression levels were the lowest in cecum tissue of the membrane-anchored groups compared to the other groups.
  
  
  CONCLUSIONS
  Recombinant L. lactis live vaccine is effective for preventing CDI in the hamster model, thus providing an alternative for immunization of C. difficile-associated diseases.
authors.affiliation: Department of Gastroenterology, General Hospital of Guangzhou Military Command of People’s Liberation Army, Guangzhou 510010, Guangdong, China.
authors.forename: Xiao-qiang
authors.initials: XQ
authors.lastname: Yang
authors.forename: Ya-gang
authors.initials: YG
authors.lastname: Zhao
authors.forename: Xue-qing
authors.initials: XQ
authors.lastname: Chen
authors.forename: Bo
authors.initials: B
authors.lastname: Jiang
authors.forename: Da-yong
authors.initials: DY
authors.lastname: Sun
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood* / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D001427:Bacterial Toxins / Q000276:immunology*; D001428:Bacterial Vaccines / Q000627:therapeutic use*; D002432:Cecum / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics; D054360:Chemokine CXCL1 / Q000235:genetics; D016360:Clostridioides difficile; D006224:Cricetinae; D004195:Disease Models, Animal; D004761:Enterocolitis, Pseudomembranous / Q000276:immunology* / Q000382:microbiology / Q000517:prevention & control*; D004768:Enterotoxins / Q000276:immunology*; D004847:Epithelial Cells / Q000187:drug effects; D007106:Immune Sera / Q000494:pharmacology; D007070:Immunoglobulin A / Q000097:blood; D007074:Immunoglobulin G / Q000097:blood; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics; D007375:Interleukin-1 / Q000235:genetics; D007413:Intestinal Mucosa / Q000473:pathology; D007422:Intestines / Q000276:immunology; D013294:Lactococcus lactis; D008297:Male; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D014614:Vaccines, Synthetic
chemical_list: D000907:Antibodies, Bacterial; D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D018932:Chemokine CCL2; D054360:Chemokine CXCL1; D004768:Enterotoxins; D007106:Immune Sera; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007375:Interleukin-1; D012333:RNA, Messenger; D014614:Vaccines, Synthetic; C041348:tcdA protein, Clostridium difficile; D018799:Intercellular Adhesion Molecule-1