Booster vaccination with a fractional dose of an oral cholera vaccine induces comparable vaccine-specific antibody avidity as a full dose: A randomised clinical trial

Antibody avidity is an important measure of the quality of vaccine-induced immune responses. Murine and human studies suggest that antibody avidity may be augmented by limiting access to antigen. The primary objective of this study was to evaluate in primed Swedish adults if booster vaccination with fractional doses (1/5th and 1/25th) of a model oral vaccine, the cholera vaccine Dukoral®, results in higher avidity antibody responses compared to boosting with a full vaccine dose. We also evaluated if fractional booster vaccination elicited similar magnitudes of antibody response compared to a full dose, and if the previously observed increase in antibody avidity after booster vaccination 1–2 years later occurred when boosting after a shorter interval.To this end, a randomised, open-label, exploratory Phase-II trial was performed. Swedish adults (n = 44), primed with two full doses of Dukoral®, were randomised into three groups and given a booster dose at either full (n = 14), 1/5th (n = 17) or 1/25th (n = 13) dose four months later. Antibody responses to cholera toxin B-subunit (CTB) were measured in serum and mucosal antibody in lymphocyte secretions (ALS).We found that the 1/5th and 1/25th booster doses had similar abilities as the full dose to induce significantly higher avidity anti-CTB antibody responses in both ALS and serum samples, as compared to after priming vaccination. There was a non-significant trend to lower magnitudes of ALS and serum IgA responses after the 1/5th compared to the full booster dose, and responses after the 1/25th dose were significantly lower.Our findings suggest fractional booster doses of Dukoral® four months after priming result in anti-toxoid mucosal antibody responses with increased antibody avidity compared to after priming vaccinations.ISRCTN registry identifier 11806026.     

Bactericidal Effect of Selected Antidiarrhoeal Medicinal Plants on Intracellular Heat-Stable Enterotoxin-Producing Escherichia coli

Diarrhoeal diseases due to enterotoxigenic Escherichia coli continue to be a cause of global concern. Medicinal plants have been gaining popularity as promising antidiarrhoeal agents. In the present study, four antidiarrhoeal plants, viz. Aegle marmelos, Cyperus rotundus, Psidium guajava and Zingiber officinale were screened against a heat-stable toxin-producing enterotoxigenic E. coli strain. Decoctions of these plants were studied for their effect on intracellular killing of the bacterial strain using murine monocytic cell line, J774. [³H] thymidine release assay was used to evaluate the apoptotic/necrotic effect. All plants at concentrations <1% enhanced intracellular killing of the bacteria by J774 cells. However, at higher concentrations, the decoctions induced apoptosis in J774 cells. The study demonstrates that these plants could control diarrhoea caused by heat-stable toxin-producing enterotoxigenic E. coli through their immunomodulatory effect.     

环介导等温基因扩增技术快速检测不耐热型肠毒素大肠杆菌反应条件的优化

[目的]建立环介导等温基因扩增技术快速检测食源性致病菌不耐热型肠毒素大肠杆菌的方法。[方法]基于产肠毒素大肠杆菌的LT基因序列设计1套(共4条)能识别6个区域的特异性LAMP引物,优化引物加入量、反应温度以及反应时间等反应条件,最终评估该方法的特异性和灵敏性。[结果]引物加入量对扩增影响较大;在扩增温度为63℃时扩增最明显;扩增反应进行到45min后即有扩增产物产生。[结论]该方法检测不耐热型肠毒素大肠杆菌具有耗时短、操作简捷、特异性较好、灵敏性高等优点。     

Vi抗原影响产肠毒素大肠杆菌菌毛 在人伤寒沙门菌表面的装配

目的 观察人伤寒沙门菌Vi抗原对大肠杆菌菌毛抗原装配的影响。方法 利用体内、外同源重组系统,构建了VipR基因缺失突变的人伤寒沙门菌菌株,导致其Vi抗原的表达较相应野生菌株偏低。用包含产肠毒素大肠杆菌菌毛抗原基因的表达质料分别转化Vi表达弱化菌株和相应野生菌株,对两者表达的菌毛抗原进行含量分析。结果 产肠毒素大肠杆菌CS3、CFA－I在VipR突变体菌株表面的含量,均比在相应野生菌株表面的含量高。结论 Vi抗原的表达弱化可能有利于菌毛抗原在人伤寒沙门菌表面的装配。本研究结果对于产肠毒素大肠杆菌基因工程疫苗的构建有指导意义。     

肠毒素性大肠杆菌CS3菌毛呈现载体的构建

目的 构建大肠杆菌ＣＳ３菌毛呈现载体,实现外源表位在细菌表面的呈现。方法 通过对ＣＳ３亚基蛋白二级结构、抗原表位、亲水性及柔韧性的预测分析,确定外源表位的插入位点,重叠延伸ＰＣＲ方法进行定点突变,将口蹄疫病毒ＶＰＩ插入到ＣＳ３中以验证表现呈现能力;用重组菌腹腔注射免疫小鼠以探讨其抗原性。结果 在大肠杆菌ＣＳ３的１３６位氨基酸残在后突变插入ＢａｍＨⅠ酶切点构建成呈现载体,全细胞ＥＬＩＳＡ、电镜和免疫     

产肠毒素大肠杆菌耐热性肠毒素(ST)单克隆抗体研制及应用

本研究用小分子量大肠杆菌耐热性肠毒素(ST)经戊二醛聚合成功地免疫了Balb/c小鼠,小鼠脾细胞与SP2/O小鼠骨髓瘤细胞融合,获得1株产生特异性ST单克隆抗体(STMcAb)的杂交瘤细胞株并用特的STMcAb建立了检测ETEc ST的竞争性ELISA。该方法检测ST的敏感性较乳鼠灌胃法(SMA)高30倍。用两种方法对330株腹泻监测点的大肠杆菌做平行试验,发现12个相同的菌株两种试验ST均阳性,其余菌株两种试验均阴性。     

三对引物同时PCR检测大肠杆菌的方法及在分子流行病学研究中的应用

在大肠杆菌（ETEC）肠毒素基因内合成三对引物，建立了三对引物同时PCR检测ETEC的方法，一次PCR即可扩增出627bp(LTh),240bp(STIa)和169bp(STIb)三种肠毒素基因片段，可同时鉴别LTh,STIa,STIb,LTh-STIa,LTh-STIb五种基因型的ETEC，与非ETEC对照菌无交叉反应，最小检出量为10cfu，显示了很高的特异性和敏感性，摸索出一种简便的腹泻粪便标本处理方法，并应用于山东省六县市ETEC感染的初步调查，为国内ETEC腹泻的分子流行病学研究提供了基本数据和较先进的诊断工具。     

3种食源性致病菌的多重PCR快速检测方法的建立

目的:建立快速检测食源性致病菌的多重PCR方法。方法:本研究根据肠毒性大肠埃希菌(enterotoxigenicE.coli,ETEC)的大肠杆菌不耐热毒素(Heat labileenterotoxin,LT)的B亚单位(LTB)、伤寒沙门菌(Salmonella typhi)的侵袭蛋白A(invasion protein A,invA)、福氏志贺菌(Shigella flexneri)侵袭性质粒抗原H基因(invasion plasmid antigen H,ipaH),分别设计了三对引物,预计PCR扩增的目的基因片段为194、279和435 bp。对单个基因PCR和单管多重PCR扩增的特异性、敏感性分析以及建立L16(43)正交试验对单管多重PCR扩增条件如引物浓度、dNTP浓度和Tm值等的优化,建立了快速检测肠毒性大肠埃希菌、伤寒沙门菌和福氏志贺菌的稳定的单管多重PCR方法。结果:该方法检测的灵敏度分别为:145 pg/ml肠毒性大肠埃希菌的基因组DNA、100 ng/ml伤寒沙门菌的基因组DNA、7 ng/ml福氏志贺菌的基因组DNA,与单基因PCR检测的灵敏度相同。并且模拟检测食品中的细菌,结果很稳定。结论:该方法操作简单、检验周期短、特异性和灵敏度高,能够快速地实现对食品中的多种致病菌的诊检和监控。     

产毒性大肠杆菌对豚鼠致病性的实验研究

目的 试图通过产毒性大肠杆菌（ＥＴＥＣ）对豚鼠致病性的实验研究,进一步探讨该菌的致病机理。方法 利用人源性ＥＴＥＣ菌株Ｅ４４８１３、Ｅ４４８１５、Ｅ４４８２２和Ｅ４４８２３攻击豚鼠,造成豚鼠ＥＴＥＣ感染模型,观察豚鼠ＥＴＥＣ感染的病变特点。结果 ４株ＥＴＥＣ菌株均能造成豚鼠发病,以Ｅ４８１３致病性最大。ＥＴＥＣ能定植于豚鼠肠道至少达１４天。发病豚鼠肠肠明显充血、肿胀、以回肠为最严重。敏感组无发病症