Neutralizing Anti-Heat-Stable Toxin (STa) Antibodies Derived from Enterotoxigenic Escherichia coli Toxoid Fusions with STa Proteins Containing N12S, L9A/N12S, or N12S/A14T Mutations Show Little Cross-Reactivity with Guanylin or Uroguanylin
Neutralizing Anti-Heat-Stable Toxin (STa) Antibodies Derived from Enterotoxigenic Escherichia coli Toxoid Fusions with STa Proteins Containing N12S, L9A/N12S, or N12S/A14T Mutations Show Little Cross-Reactivity with Guanylin or Uroguanylin
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DOI：
10.1128/aem.01737-17
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发表时间：
2018-01-01
影响因子：
4.4
通讯作者：
Zhang, Weiping
中科院分区：
文献类型：
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作者：
Duan, Qiangde;Huang, Jiachen;Zhang, Weiping
产热稳定毒素（STa）的产肠毒素大肠杆菌（ETEC）菌株是发展中国家儿童中中度至重度腹泻的主要原因，也是旅行者腹泻的常见原因。最近在使用STa类毒素和类毒素融合物诱导中和性抗STa抗体方面取得的进展加速了ETEC疫苗的开发。然而，人们仍然关注的是，衍生的抗sta抗体是否与sta样观音碱和尿观音碱交叉反应，这是两种调节人体肠道和肾脏上皮细胞中液体和电解质运输的鸟苷酸环化酶C （GC-C）配体。为了进一步从观音林和尿观音林中转移STa，并消除STa抗体与观音林和尿观音林的交叉反应性，我们在双突变体STaL9A/N12S、STaL9A/A14H、STaN12S/A14T和STaL9A/N12S/A14H中突变了STa的第9位（白氨酸）、第12位（天冬酰胺）和第14位（丙氨酸）。然后，我们将每个STa突变体（三个拷贝）融合到一个单体热不稳定毒素（LT）突变体（mnLT(R192G/L211A)）中，进行了3个STaL9A/N12S-mnLT（R192G/L211A）、3个STaL9A/A14H-mnLT（R192G/L211A）、3个STaN12S/A14T-mnLT（R192G/L211A）和3个STaL9A/N12S/A14H-mnLT（R192G/L211A）的类毒素融合；检测每个融合体的抗sta免疫原性；并评估衍生抗体对STa毒性的体外中和活性以及与观音碱和尿观音碱的交叉反应性。用每种融合蛋白皮下免疫小鼠可产生抗STa抗体，分别来源于3 × STaN12S- mnlt （R192G/L211A）、3 × STaL9A/N12S-mnLT（R192G/L211A）或3 × STaN12S/A14T-mnLT（R192G/L211A）的抗体可阻止STa对T-84细胞内cGMP的刺激。竞争性酶联免疫吸附试验（elisa）显示，观音苷和尿观音苷几乎不能阻断抗sta抗体与包被sta -卵清蛋白偶联物的结合。结果表明，3 × STaN12S- mnlt （R192G/L211A）、3 × STaL9A/N12S-mnLT（R192G/L211A）和3 × STaN12S/A14T-mnLT（R192G/L211A）的抗体均能中和STa，且与观音和尿观音具有较低的交叉反应性，提示这些类毒素融合物是ETEC疫苗的合适抗原。产肠毒素大肠杆菌（ETEC）菌株是儿童腹泻和旅行者腹泻的主要原因。目前，尚无针对ETEC腹泻的许可疫苗。一个关键的挑战是找到安全的抗原来诱导抗体中和关键的STa，而不与观音碱和尿观音碱发生交叉反应，这两种重要的配体控制着人类肠道和肾脏上皮细胞的稳态。在这项研究中，我们产生了无毒的融合抗原，诱导抗体在体外中和STa的肠毒性，并且不与观音碱或尿观音碱交叉反应。这些融合物已成为开发ETEC疫苗的首选抗原，每年有可能防止数十万幼儿死亡和数亿腹泻病例。
Heat-stable toxin (STa)-producing enterotoxigenic Escherichia coli (ETEC) strains are a top cause of moderate-to-severe diarrhea in children from developing countries and a common cause of travelers' diarrhea. Recent progress in using STa toxoids and toxoid fusions to induce neutralizing anti-STa antibodies has accelerated ETEC vaccine development. However, concern remains regarding whether the derived anti-STa antibodies cross-react with STa-like guanylin and uroguanylin, two guanylate cyclase C (GC-C) ligands regulating fluid and electrolyte transportation in human intestinal and renal epithelial cells. To further divert STa from guanylin and uroguanylin structurally and antigenically and to eliminate anti-STa antibody cross-reactivity with guanylin and uroguanylin, we mutated STa at the 9th (leucine), 12th (asparagine), and 14th (alanine) residues for the double and triple mutants STaL9A/N12S, STaL9A/A14H, STaN12S/A14T, and STaL9A/N12S/A14H. We then fused each STa mutant (three copies) to a monomeric heat-labile toxin (LT) mutant (mnLT(R192G/L211A)) for the toxoid fusions 3 x STaL9A/N12S-mnLT(R192G/L211A), 3 x STaL9A/A14H-mnLT(R192G/L211A), 3 x STaN12S/A14T-mnLT(R192G/L211A), and 3 x STaL9A/N12S/A14H-mnLT(R192G/L211A); examined each fusion for anti-STa immunogenicity; and assessed the derived antibodies for in vitro neutralization activity against STa toxicity and for cross-reactivity with guanylin and uroguanylin. Mice subcutaneously immunized with each fusion protein developed anti-STa antibodies, and the antibodies derived from 3 x STaN12S-mnLT(R192G/L211A), 3 x STaL9A/N12S-mnLT(R192G/L211A), or 3 x STaN12S/A14T-mnLT(R192G/L211A) prevented STa from the stimulation of intracellular cGMP in T-84 cells. Competitive enzyme-linked immunosorbent assays (ELISAs) showed that guanylin and uroguanylin hardly blocked the binding of anti-STa antibodies to the coated STa-ovalbumin conjugate. These results indicated that antibodies derived from 3 x STaN12S-mnLT(R192G/L211A), 3 x STaL9A/N12S-mnLT(R192G/L211A), or 3 x STaN12S/A14T-mnLT(R192G/L211A) neutralized STa and had little cross-reactivity with guanylin and uroguanylin, suggesting that these toxoid fusions are suitable antigens for ETEC vaccines.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children's diarrhea and travelers' diarrhea. Currently, there is no licensed vaccine against ETEC diarrhea. One key challenge is to identify safe antigens to induce antibodies neutralizing the key STa without cross-reacting with guanylin and uroguanylin, two important ligands controlling homeostasis in human intestinal and renal epithelial cells. In this study, we generated nontoxic fusion antigens that induced antibodies that neutralize STa enterotoxicity in vitro and do not cross-react with guanylin or uroguanylin. These fusions have become the preferred antigens for the development of ETEC vaccines to potentially prevent the deaths of hundreds of thousands of young children and hundreds of millions of diarrheal cases each year.