The protective effect of recombinant Lactococcus lactis oral vaccine on a Clostridium difficile-infected animal model.
The protective effect of recombinant Lactococcus lactis oral vaccine on a Clostridium difficile-infected animal model.
复制标题
重组乳酸乳球菌口服疫苗对艰难梭菌感染动物模型的保护作用
DOI：
10.1186/1471-230x-13-117
复制
发表时间：
2013-07-17
影响因子：
2.4
通讯作者：
Sun DY
中科院分区：
文献类型：
--
作者：
Yang XQ;Zhao YG;Chen XQ;Jiang B;Sun DY
背景
口服疫苗免疫可能是预防艰难梭菌感染（CDI）的有效策略。然而，由于各种原因，先前开发的用于预防CDI的疫苗的应用受到限制。本研究以乳酸乳球菌为研究对象，研制了重组乳酸乳球菌口服疫苗，并评价了其对一株C. difficile感染的动物模型建立在金黄色仓鼠，试图提供一种替代的策略CDI预防。
方法
重组L.乳酸菌疫苗是使用pTRKH 2质粒，一种高拷贝数的大肠杆菌-L.穿梭载体：1）L.用携带Usp 45信号肽（SPUsp 45）、无毒佐剂破伤风毒素片段C（TETC）和无毒C末端重复序列中的14个（14 CDTA）的重组pTRKH 2（分泌蛋白质粒）构建了表达分泌蛋白的乳酸杆菌。艰难梭菌毒素A（TcdA）;用含有SPUsp45、TETC、14-CDTA和M6蛋白的细胞壁锚定序列（cwaM6）的重组pTRKH 2（膜锚定质粒）构建了表达分泌蛋白和膜蛋白的乳酸菌。将32只雄性叙利亚金黄地鼠随机分为4组，每组8只，分别灌胃生理盐水（空白对照）和L.分别携带空穿梭载体、分泌蛋白质质粒和膜锚定质粒的乳酸菌。克林霉素灌胃1周后，用C.艰难梭菌孢子悬液。肉眼观察动物的一般症状，显微镜下观察动物肠道的病理变化。采用酶联免疫吸附试验（ELISA）检测肠组织和肠液中抗TcdA IgG/IgA抗体的蛋白水平。细胞培养细胞毒性中和测定通过在有或没有抗TcdA血清预孵育或处理的情况下进行TcdA处理来进行。用流式细胞术（FL）检测肠上皮细胞凋亡。采用聚合物链反应（PCR）法检测黏膜炎性细胞因子的表达。
结果
在C.艰难梭菌攻毒后，对照组动物第1天出现严重腹泻症状，第4天全部死亡，表明仓鼠CDI动物模型建立成功。在3个免疫组中，分泌蛋白和膜锚定质粒组的死亡率、体重下降和病理评分显著低于空质粒组，存活率/时间高于空质粒组（P 0.05）。分泌蛋白组和膜锚定质粒组血清和肠液中抗TcdA的IgG抗体滴度均显著高于空质粒组（P0.05），而相应的抗TcdA的IgA抗体滴度无显著差异（P0.05）。膜锚定质粒组的抗TcdA血清可中和200 ng/ml TcdA的细胞毒性，其中抗TcdA血清预孵育的保护效果最好。膜锚定质粒组的细胞预孵育或抗TcdA血清处理均能显著降低TcdA诱导的肠上皮细胞死亡和凋亡发生率（P0.05）。MCP-1、ICAM-1、IL-6和Gro-1 mRNA表达水平在膜锚定组的盲肠组织中最低。
结论
重组L.乳酸梭菌活疫苗能有效预防仓鼠CDI，为免疫接种提供了一种新的途径。艰难相关疾病。
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.