口服免疫无融合标签VP1蛋白纳米颗粒疫苗诱生CVB3特异性粘膜免疫应答及其预防病毒性心肌炎的作用及机制研究

Viral myocarditis is a common clinical cardiovascular disease mainly induced by coxsackievirus B3 (CVB3) with no effective therapeutic measures. Induction of efficient mucosal immune responses is very critical against CVB3-induced myocarditis. The VP1 is a major capsid protein of CVB3, which contains many antigentic epitopes of B and T cells and can induce effective immune responses in mice. So, VP1 is the best vaccine candidate antigens. However, the peptide-based vaccines generate a weaker immune response, and the inclusion of adjuvants and/or the use of vaccine delivery systems is highly needed. The use of nanoparticles in vaccine formulations allows not only improved antigen stability and immunogenicity, but also targeted delivery and slow release. A number of nanoparticle vaccines varying in composition, size, shape, and surface properties have been approved for human use and the number of candidates is increasing. In this context, Inclusion bodies (IBs) are protein-based nanoparticles formed in Escherichia coli through stereospecific aggregation processes during the overexpression of recombinant proteins. In the last years, it has been shown that IBs can be used as nanostructured biomaterials to stimulate mammalian cell attachment, proliferation, and differentiation. In addition, these nanoparticles have also been explored as natural delivery systems for protein replacement therapies. So production of VP1 as bacterial inclusion body as a protein-based nanoparticle vaccine may improve its stability and immunogenicity. However VP1 is very difficult to express in E.coli, and a fusion tag protein is needed to jointly to achieve efficient protein expression. The presence of fusion tag in purified IBs samples can be a major drawback when using these nanoparticles for biomedical and industrial applications, as fusion tags can interfere in various physicochemical properties and immunogenicity of the recombinant proteins. Although many proteases and self-cleaving tags have been developed to cleave fusion tags, these protocols can only play a role on soluble proteins, removing fusion tag from inclusion body particles had not been reported until now. In this study we have explored a tag-free inclusion body particles production protocol though a truncated Ssp DnaX mini-intein (Ic) spontaneous C-cleavage in vivo. Following oral delivery of the purified tag-free VP1 inclusion body particles to explore a novel protein nanoparticle vaccine for generating strong CVB3-specific mucosal immunity and effective preventing of CVB3-induced myocarditis.

由于多肽疫苗在体内易降解等缺陷，因此直接免疫VP1蛋白引起的免疫应答较弱，不能起到很好的免疫保护效果。而纳米多肽疫苗可以对抗原起保护作用，延长体内存留时间，提高免疫效应，而且纳米疫苗可被DC有效吞噬和递呈，同时纳米载体本身可以充当免疫佐剂。因此，可将VP1制备成多肽纳米疫苗来提高机体免疫应答从而起到更好的免疫保护效果。在众多的纳米材料中，细菌的包涵体蛋白是无毒且具有完全生物相容性的纳米粒子，近年来在生物材料及组织工程领域中具有广泛应用。然而VP1在E.coli中难以表达，只有在融合蛋白的帮助下才能有效表达且以包涵体的形式存在。而融合标签的存在可能会因为机体针对融合标签的免疫应答，从而限制了其作为纳米多肽疫苗的应用。因此，本项目提供了一种利用intein体内自发断裂活性成功制备无融合标签VP1多肽纳米疫苗，并将其以口服方式免疫小鼠，可有效引起粘膜免疫应答，预防CVB3型心肌炎的发生。

疫苗是人类预防疾病最主要的手段之一，然而有效安全的疫苗仍然是疫苗研究面临的重要难题。多肽疫苗具有成分单一，生物安全性高的优点，但是直接免疫多肽疫苗引起的免疫应答较弱，因此通常需要加入佐剂或者采用递送系统。而纳米颗粒疫苗可以对抗原起保护作用，延长体内存留时间，从而被DC有效摄取和递呈提高免疫效果。病毒性心肌炎是指病毒感染引起的心肌局限性或弥漫性的急性或慢性炎症病变，属于感染性心肌疾病，近年来已经成为青少年猝死、慢性扩张性心肌症、心衰等的主要原因之一 ，然而目前尚无有效的预防和治疗手段。因此，在本研究中我们将柯萨奇病毒3型（CVB3）的主要抗原VP1制备成纳米颗粒，研究其引起的免疫应答和免疫保护效果，为CVB3诱导的病毒性心肌炎开发安全有效的口服多肽疫苗。我们将VP1蛋白与蛋白内含子（intein）融合表达，之后通过intein的自发断裂制备了无融合标签的VP1蛋白纳米颗粒疫苗，并以口服方式免疫小鼠。我们的研究结果表明：1）通过与intein融合表达以及其体内自发断裂的特性，成功制备无融合标签的VP1蛋白纳米颗粒；2）VP1蛋白所形成的纳米颗粒大小均一、直径在500-700nm之间且具有生物安全性；3）在体外可以被BMDC高效摄取并诱导T细胞增殖；4）口服免疫小鼠之后可以引起较强的黏膜免疫应答和有效的免疫保护效果。本项目利用蛋白质内含子的体内自发断裂活性，以大肠杆菌为宿主，通过生物化学方法成功制备VP1 蛋白纳米颗粒疫苗，为预防CVB3诱导的病毒性心肌炎提供了一种有效的多肽疫苗，同时为新型多肽疫苗的设计提供了一种思路。

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