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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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Related in: MedlinePlus

Effect of recombinant L. lactis vaccine after clostridium difficile challenge. (A) Cumulative mortalities caused by clindamycin-induced Clostridium difficile-associated diseases (CDAD) in different groups of hamsters; (B) Mortalities caused by clindamycin-induced CDAD in different groups of hamsters; (C) Survival rate (%) in different groups of hamsters; (D) Cumulative morbidities of diarrhea caused by clindamycin-induced CDAD in different groups of hamsters; and (E) Comparison of body weight in different groups of hamsters before and after challenge with Clostridium difficile.
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Figure 2: Effect of recombinant L. lactis vaccine after clostridium difficile challenge. (A) Cumulative mortalities caused by clindamycin-induced Clostridium difficile-associated diseases (CDAD) in different groups of hamsters; (B) Mortalities caused by clindamycin-induced CDAD in different groups of hamsters; (C) Survival rate (%) in different groups of hamsters; (D) Cumulative morbidities of diarrhea caused by clindamycin-induced CDAD in different groups of hamsters; and (E) Comparison of body weight in different groups of hamsters before and after challenge with Clostridium difficile.

Mentions: In the control group, all hamsters had light stool, wet perianal area and tail, less activity with response to stimuli, and a soft abdomen on day 1 after the C. difficile challenge; few animals had bloody stools as observed by naked eye. The majority of animals in the control group had damp claws and lower abdomen, curved or arched bodies with no activity, soft abdomen, disappeared balance, and shrugged fur on day 2 post-challenge; few animals died. All animals in the control group died by the end of day 4 post-challenge (Figure 2A). In the empty plasmid group, animals subject to C. difficile challenge all had light stool and a wet perianal area and tail on day 1 post-challenge; some animals were less active but responded to stimuli; one animal died on day 2 post-challenge (Figure 2A). After L. lactis carrying the empty vector was administered again, diarrhea symptoms were alleviated in the infected animals. Till the end of the experiment, two animals still had diarrhea, but their perianal area and tail were not wet. In the secreted-protein plasmid group, two of the animals subject to C. difficile challenge had diarrhea on day 1, but their perianal area and tail were not wet. After recombinant L. lactis carrying the secreted-protein plasmid was administered again, diarrhea symptoms disappeared in the animals by day 4 post-challenge. In the membrane-anchored group, animals had no symptoms of diarrhea (Table 4).


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)

Effect of recombinant L. lactis vaccine after clostridium difficile challenge. (A) Cumulative mortalities caused by clindamycin-induced Clostridium difficile-associated diseases (CDAD) in different groups of hamsters; (B) Mortalities caused by clindamycin-induced CDAD in different groups of hamsters; (C) Survival rate (%) in different groups of hamsters; (D) Cumulative morbidities of diarrhea caused by clindamycin-induced CDAD in different groups of hamsters; and (E) Comparison of body weight in different groups of hamsters before and after challenge with Clostridium difficile.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Effect of recombinant L. lactis vaccine after clostridium difficile challenge. (A) Cumulative mortalities caused by clindamycin-induced Clostridium difficile-associated diseases (CDAD) in different groups of hamsters; (B) Mortalities caused by clindamycin-induced CDAD in different groups of hamsters; (C) Survival rate (%) in different groups of hamsters; (D) Cumulative morbidities of diarrhea caused by clindamycin-induced CDAD in different groups of hamsters; and (E) Comparison of body weight in different groups of hamsters before and after challenge with Clostridium difficile.
Mentions: In the control group, all hamsters had light stool, wet perianal area and tail, less activity with response to stimuli, and a soft abdomen on day 1 after the C. difficile challenge; few animals had bloody stools as observed by naked eye. The majority of animals in the control group had damp claws and lower abdomen, curved or arched bodies with no activity, soft abdomen, disappeared balance, and shrugged fur on day 2 post-challenge; few animals died. All animals in the control group died by the end of day 4 post-challenge (Figure 2A). In the empty plasmid group, animals subject to C. difficile challenge all had light stool and a wet perianal area and tail on day 1 post-challenge; some animals were less active but responded to stimuli; one animal died on day 2 post-challenge (Figure 2A). After L. lactis carrying the empty vector was administered again, diarrhea symptoms were alleviated in the infected animals. Till the end of the experiment, two animals still had diarrhea, but their perianal area and tail were not wet. In the secreted-protein plasmid group, two of the animals subject to C. difficile challenge had diarrhea on day 1, but their perianal area and tail were not wet. After recombinant L. lactis carrying the secreted-protein plasmid was administered again, diarrhea symptoms disappeared in the animals by day 4 post-challenge. In the membrane-anchored group, animals had no symptoms of diarrhea (Table 4).

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