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The protective effect of recombinant Lactococcus lactis oral vaccine on a Clostridium difficile-infected animal model.

Yang XQ, Zhao YG, Chen XQ, Jiang B, Sun DY - BMC Gastroenterol (2013)

Bottom Line: 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 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).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gastroenterology, General Hospital of Guangzhou Military Command of People’s Liberation Army, Guangzhou 510010, Guangdong, China.

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.

Conclusion: 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.

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Macropathological observations in all groups. (A&B) Defective cecal mucosa in the control group, with bleeding, edema and disappearing of vascular lakes. Colon mucosa adjacent to the cecum with edema and ulcer; (C) Cecal mucosa of the empty plasmid group, with edema, scattered bleeding points and erosion; (D) Cecal mucosa of the secreted-protein plasmid group; and (E) Cecal mucosa of the membrane-anchored plasmid group.
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Figure 4: Macropathological observations in all groups. (A&B) Defective cecal mucosa in the control group, with bleeding, edema and disappearing of vascular lakes. Colon mucosa adjacent to the cecum with edema and ulcer; (C) Cecal mucosa of the empty plasmid group, with edema, scattered bleeding points and erosion; (D) Cecal mucosa of the secreted-protein plasmid group; and (E) Cecal mucosa of the membrane-anchored plasmid group.

Mentions: After the C. difficile challenge, all animals in the control group had cecal expansion and intestinal bleeding on day 2 (Figures 3A and B). Extensive hemorrhagic lesions were observed in the intestine (Figures 4A and B), some of which occurred as mucosal defects. The worst lesions were in the top segments of the cecum and colon. Slight dilation was detected in the small intestine, which was thicker than the colon. The rectum had slight lesions, with no feces observed in the colon. In the empty plasmid group, one animal died on day 2 post-challenge, with a slightly dilated cecum and intestinal mucosa hyperemia (Figure 3C). In its intestinal cavity, a few hemorrhages and obvious congestion and edema were observed within the intestinal mucosa. Interspersed erosion was visible (Figure 4C). Other animals of the empty plasmid group sacrificed at the end of the experiment had a non-dilated cecum and uncongested intestinal mucosa. Animals of the secreted-protein and membrane-anchored plasmid groups had a non-dilated intestine and formed feces in the colon and rectum (Figures 3D and E), with no congestion or edema in the intestinal mucosa (Figures 4D and E).


The protective effect of recombinant Lactococcus lactis oral vaccine on a Clostridium difficile-infected animal model.

Yang XQ, Zhao YG, Chen XQ, Jiang B, Sun DY - BMC Gastroenterol (2013)

Macropathological observations in all groups. (A&B) Defective cecal mucosa in the control group, with bleeding, edema and disappearing of vascular lakes. Colon mucosa adjacent to the cecum with edema and ulcer; (C) Cecal mucosa of the empty plasmid group, with edema, scattered bleeding points and erosion; (D) Cecal mucosa of the secreted-protein plasmid group; and (E) Cecal mucosa of the membrane-anchored plasmid group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750240&req=5

Figure 4: Macropathological observations in all groups. (A&B) Defective cecal mucosa in the control group, with bleeding, edema and disappearing of vascular lakes. Colon mucosa adjacent to the cecum with edema and ulcer; (C) Cecal mucosa of the empty plasmid group, with edema, scattered bleeding points and erosion; (D) Cecal mucosa of the secreted-protein plasmid group; and (E) Cecal mucosa of the membrane-anchored plasmid group.
Mentions: After the C. difficile challenge, all animals in the control group had cecal expansion and intestinal bleeding on day 2 (Figures 3A and B). Extensive hemorrhagic lesions were observed in the intestine (Figures 4A and B), some of which occurred as mucosal defects. The worst lesions were in the top segments of the cecum and colon. Slight dilation was detected in the small intestine, which was thicker than the colon. The rectum had slight lesions, with no feces observed in the colon. In the empty plasmid group, one animal died on day 2 post-challenge, with a slightly dilated cecum and intestinal mucosa hyperemia (Figure 3C). In its intestinal cavity, a few hemorrhages and obvious congestion and edema were observed within the intestinal mucosa. Interspersed erosion was visible (Figure 4C). Other animals of the empty plasmid group sacrificed at the end of the experiment had a non-dilated cecum and uncongested intestinal mucosa. Animals of the secreted-protein and membrane-anchored plasmid groups had a non-dilated intestine and formed feces in the colon and rectum (Figures 3D and E), with no congestion or edema in the intestinal mucosa (Figures 4D and E).

Bottom Line: 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 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).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gastroenterology, General Hospital of Guangzhou Military Command of People’s Liberation Army, Guangzhou 510010, Guangdong, China.

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus