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.

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.

Figure 1

Schematic diagram of Clostridium difficile challenge experiment.

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.

Figure 3

Changes of intestine loop in all groups at the end of experiment. (A&B) Enlarged and bleeding cecum and upper colon without formed stool in the control group; (C) Slightly dilated cecum in the empty plasmid group, with high tension, hydropsia, congestion and no formed stool; and (D) Cecum of the secreted-protein and membrane-anchored plasmid groups, with formed stool and no sign of congestion, bleeding, or dilation (E).

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.

Figure 5

Histopathological observations in all groups. (A&B) Section of cecal mucosa in the control group. (C&D) Section of cecal mucosa in the empty plasmid group. (E) Section of cecal mucosa in the secreted-protein plasmid group. (F) Section of cecal mucosa in the membrane-anchored plasmid group.

Figure 6

Comparison of macropathological and histopathological scores among different groups of hamsters. The macropathological and histopathological scores are higher in the control group than in the 3 immunization groups.

Figure 7

Anti-toxin A (TcdA) IgG and IgA antibodies in serum and intestinal fluid of different groups of hamsters. The anti-TcdA IgA titer in serum of secreted-protein and membrane-anchored plasmid groups was higher than that of the control group (P < 0.05). There was no significant difference in anti-TcdA IgA titer of intestinal fluid among all groups.

Figure 8

Results of cytotoxicity neutralization assay. (A) Control: nucleus was normal. (B)Clostridium difficile toxin A (TcdA) treatment: condensation, fragmentation and dissolution of nuclei were found. (C) Anti-TcdA serum pre-treatment: cells became slightly round but still spindly. (D) Anti-TcdA serum treatment: cells became round and narrow; nuclei appeared condensed, but some cells were still spindly (×200 magnification).

Figure 9

Effect of anti-Clostridium difficile toxin A (TcdA) sera against the cytotoxicity of TcdA assayed by flow cytometry. (A) Control; (B) TcdA treatment; (C) Anti-TcdA serum pre-treatment; and (D) Anti-TcdA serum treatment.

Figure 10

Relative values of the grayscale mRNA levels of mucosal inflammatory cytokines in different groups of hamsters.