【作者】 戚昀；

【导师】 陈应华；

【作者基本信息】 清华大学 ， 生物学， 2009， 博士

【摘要】 猪瘟是一种对生猪养殖业具有严重破坏性的猪病,猪瘟疫情的爆发往往会带来巨大的经济损失。因此,世界各国都非常重视对猪瘟疫情的监控和防制。猪瘟病毒是猪瘟的病原体。我国自20世纪50年代成功研制出“54-III”系兔化弱毒疫苗(C株)后,所采取的疫苗免疫政策,在控制我国猪瘟流行方面发挥了重大的作用。该疫苗毒株的安全性和有效性已经为世界所公认。然而,近年来疫苗免疫失败和猪瘟散发性流行呈上升趋势。另外,传统疫苗免疫后的猪只和猪瘟病毒野毒株感染后的猪只在血清学上无法有效地区分。这些正严重影响着我国的生猪养殖业和生猪及猪肉制品贸易的发展。因此,基于我们提出的表位疫苗战略,我们研究一种新型的猪瘟病毒疫苗――猪瘟病毒表位标记疫苗。本研究选取了猪瘟病毒囊膜糖蛋白E2上著名的保守线性中和表位TAVSPTTLR为靶点,制备了一系列基于该表位的重组免疫原。在此基础上我们开展了系统性研究。我们对这些重组免疫原的抗原性和免疫原性进行了研究和分析,确定了满足于候选疫苗基本条件的重组免疫原。我们利用猪瘟病毒兔化弱毒疫苗的兔体模型进行了攻毒/防护试验,发现含有6个TAVSPTTLR表位的重组免疫原可以在模型动物体内诱导抗病毒免疫,使之抵御病毒攻击。进一步地,在病毒体外中和试验中,我们证明该候选疫苗诱导机体产生了具有抗病毒活性的体液免疫反应。这些结果都表明,该候选疫苗具有防护猪瘟病毒感染的能力。本研究主要发现:(1)保守线性中和表位TAVSPTTLR周围序列为该表位特异性中和抗体的诱导提供了结构性信息;(2)基于TAVSPTTLR的表位重组免疫原可代偿该序列周围天然环境对其诱导中和抗体所提供的贡献,诱导产生中和抗体,提供免疫防护。本论文围绕猪瘟病毒E2蛋白上保守线性中和表位TAVSPTTLR开展的一系列工作,加深了对该表位性质的认识,为进一步开发基于该表位的猪瘟病毒标记疫苗提供了夯实的基础。同时,本论文也证明了基于保守线性中和表位的表位疫苗战略是“理论上站得住,实践上有可能”的。更多还原

【Abstract】 Classical swine fever (CSF) is a swine disease having devastating impact on the livestock industry, and the outbreaks of the classical swine fever usually cause great economic losses. Therefore, the surveillance, prophylaxis and control of the CSF outbreaks are treated seriously all over the world. Classical swine fever virus (CSFV) is the etiological agent of classical swine fever. In the 1950s’the“54-III”live attenuated vaccine (the C strain) was successfully developed in China by adaptation of a highly virulent strain of classical swine fever virus to the rabbits, and the immunoprophylaxis strategy using this vaccine has been proved to make great contributions to the control of the CSF epidemics in China. The safety and efficacy of this vaccine have been widely recognized throughout the world. However, the failures in immunization with this vaccine and the sporadic CSF epidemics have been seen on the rise in the recent years. Besides, the vaccinated pigs could hardly be differentiated from those infected by the field strains serologically. All these mentioned above are now severely affected the livestock industry development and the international trading of pigs and pork products. Hence, we aim to develop a new CSFV vaccine– the CSFV epitope marker vaccine based on our epitope-vaccine strategy.In this study, a series of recombinant immunogens targeted on TAVSPTTLR, the well-known conserved linear neutralizing epitope on the glycoprotein E2 of classical swine fever virus, were prepared. Systemic research was performed on the basis of these immunogens. The antigenicity and immunogenicity of these immunogens were analyzed, identifying several immunogens which satisfied the basic requirement of a vaccine candidate. The challenge/protection test was performed on the rabbit model of the live attenuated vaccine to evaluate their potential as vaccine candidates, discovering that the immunogen, which contain 6 copies of the epitope in a single molecule, could provide the susceptible animals protection against virus challenge. We further demonstrated through the in vitro neutralization test that the vaccine candidate managed to induce antiviral humoral immunity. These results demonstrated that the vaccine candidate was able to protect the susceptible animals from CSFV infection. In this work, we discovered: (1) the natural microenvironment of the epitope contributed essential structural information for the induction of the epitope-specific neutralizing antibodies; (2) the immunogens focused on TAVSPTTLR managed to induce neutralizing antibodies and provide protection, compensating the contribution rendered by the epitope context.The work on the TAVSPTTLR epitope made a better understanding of this conserved linear neutralizing epitope on the CSFV E2 glycoprotein, which provided a solid foundation for the further development of the marker CSFV epitope vaccines. Besides, the work was proof-of-concept, demonstrating the epitope vaccine strategy based on the conserved linear neutralizing epitope stood sound not only in theory but also in practice.更多还原