肠道病毒71型（EV71）与柯萨奇病毒A组16型（CA16）单克隆抗体治疗的可行性评价

发布时间：2018-05-19 10:15

本文选题：肠道病毒71型 + 柯萨奇病毒A组16型　；参考：《厦门大学》2014年硕士论文

【摘要】：肠道病毒71型(EV71)与柯萨奇病毒A组16型(CA16)是引起儿童手足口病(HFMD)的主要病原体。近些年来,手足口病在我国的发病率逐年上升,由手足口病导致的重症感染和死亡人数也明显增加,严重危害了儿童的生命安全,因此急需有效的治疗性药物来治疗并控制其感染与流行。单克隆抗体由于具有毒性低、特异性高等优点,是治疗病毒性感染的良好的候选药物。本研究的目的为优化EV71的小鼠攻毒模型同时建立稳定的CA16小鼠感染模型,并利用动物模型筛选和评价具有治疗潜力的EV71及CA16治疗性单抗,为开展EV71及CA16治疗性药物研制提供基础。本研究首先对原有的EV71动物模型的攻毒方式和攻毒剂量进行了优化,使该模型能够达到稳定的100%致死率。同时利用13株CA16临床分离株对1日龄新生鼠进行攻毒评价,结果发现大多数CA16病毒对小鼠表现出了强毒力,攻毒小鼠表现出瘫痪和肢体麻痹等神经症状。病理学检测显示攻毒小鼠的大脑和脊髓等部位出现了严重的坏死与炎症,与CA16在人体内的感染情况极为相似。基于上述结果,本研究建立了通过灌胃注射CA16感染1日龄BALB/c小鼠的CA16小鼠感染模型,为开展后续研究提供了重要基础。本研究应用所建立的CA16攻毒模型对CA16中和单抗的体内治疗效果进行评价,并根据体内外实验的结果确立了一株候选的CA16中和单抗14B10。通过评价感染CA16后不同治疗时间点及治疗策略的效果发现,14B10在攻毒后2天内进行单针治疗能够达到超过90%的治疗存活率,而利用三针治疗方案则能够在攻毒后第3天、即小鼠开始发病前1天有效治疗小鼠,从而证实了14B10对CA16的感染具有良好的体内治疗能力。本研究进一步应用EV71攻毒模型对13株EV71中和单抗的体内治疗能力进行筛选评价,从而确立了一株EV71中和单抗CT11F9。通过对攻毒EV71后不同治疗时间和治疗策略的评价发现,CT11F9在攻毒后三天内进行单针治疗能够达到100%的存活率及健康率,而利用三针治疗方案则能够在攻毒后第4天、即小鼠开始发病时有效提高小鼠的存活率,且对瘫痪小鼠具有一定的治疗能力。通过与人血清的阻断ELISA实验发现,CT11F9识别的表位在人血清中是免疫优势的。至此证实了CT11F9对EV71感染具有良好的体内治疗能力。综上所述,本研究优化了原有的EV71动物模型的攻毒参数并建立了稳定的CA16小鼠感染模型,利用该模型成功筛选并获得了对EV71或CA16感染具有良好治疗能力的中和单抗。这些研究为研发EV71及CA16治疗性抗体提供了重要的数据支持与理论基础。
[Abstract]:Enterovirus 71 (EV71) and Coxsackie virus group A (CA16) are the main pathogens of HFMDs in children. In recent years, the incidence of HFMD in China has increased year by year, and the number of severe infections and deaths caused by HFMD has also increased significantly, which seriously endangers the lives of children. Therefore, effective therapeutic drugs are urgently needed to treat and control their infection and prevalence. Because of its low toxicity and high specificity, monoclonal antibody is a good candidate for the treatment of viral infection. The aim of this study was to optimize the mice model of EV71 attack and to establish a stable CA16 mouse model of infection, and to screen and evaluate the therapeutic potential of EV71 and CA16 monoclonal antibody by animal model. To provide a basis for the development of EV71 and CA16 therapeutic drugs. In this study, we first optimized the attack mode and dose of EV71 animal model, so that the model can achieve a stable mortality of 100%. At the same time, 13 clinical isolates of CA16 were used to evaluate the toxicity of 1 day old newborn mice. The results showed that most of the CA16 viruses showed strong virulence to mice, and the attacking mice showed paralysis and limb paralysis. Pathological examination showed that severe necrosis and inflammation occurred in the brain and spinal cord of the mice, which was similar to the infection of CA16 in human body. Based on the above results, the model of CA16 mice infected by intragastric injection of CA16 for 1 day in BALB/c mice was established, which provided an important basis for further study. In this study, the CA16 model was used to evaluate the therapeutic effect of CA16 neutralizing monoclonal antibody in vivo, and a candidate CA16 neutralizing monoclonal antibody 14B10 was established according to the results of in vivo and in vitro experiments. By evaluating the effects of different treatment time points and treatment strategies after CA16 infection, it was found that single needle therapy could achieve more than 90% survival rate within 2 days after drug attack, while three-needle therapy regimen could achieve the survival rate of more than 90% on the third day after drug attack. That is to say, one day before the onset of the disease, mice were effectively treated with 14B10, which proved that 14B10 had a good in vivo therapeutic ability for CA16 infection. In this study, EV71 model was used to screen and evaluate the therapeutic ability of 13 EV71 neutralizing monoclonal antibodies in vivo, and a EV71 neutralizing monoclonal antibody CT11F9 was established. Through the evaluation of different treatment time and treatment strategy after EV71 attack, it was found that the survival rate and health rate of CT11F9 could reach 100% within three days after drug attack, while the treatment with three needles could achieve 100% survival rate and health rate on the 4th day after drug attack. That is to say, the survival rate of the mice was improved effectively when the mice began to develop the disease, and the palsy mice had certain therapeutic ability. By blocking ELISA with human serum, it was found that the epitope recognized by CT11F9 was immune dominant in human serum. It has been proved that CT11F9 has good in vivo treatment of EV71 infection. To sum up, we optimized the parameters of the original EV71 animal model and established a stable CA16 mouse model of infection. Using this model, we successfully screened and obtained neutralized McAbs with good ability to treat EV71 or CA16 infection. These studies provide important data support and theoretical basis for the development of EV71 and CA16 therapeutic antibodies.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.5

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