研究腸病毒七十一型的外鞘蛋白抗原決定位作為腸病毒疫苗開發

Abstract

腸病毒七十一型是腸病毒中最主要造成口手足症以及神經方面嚴重併發症的病毒。腸病毒七十一型和小兒麻痺病毒同屬於微小RNA病毒科（picornaviridae），其外殼蛋白由四種結構蛋白（VP1, VP2, VP3和VP4）組合而成。大部分的腸病毒七十一型的疫苗研究著重在無活性的病毒顆粒跟VP1這個結構蛋白上。雖然VP1被廣泛認為是腸病毒七十一型最重要的抗原決定位，但是卻沒有直接的證據可以證明這一點。除此之外，在疫苗的研發中，只有無活性的病毒顆粒可以保護初生鼠抵抗腸病毒七十一型的感染也降低了受到感染的初生鼠的死亡率，但以VP1結構蛋白當作疫苗則至今都沒有很好的效果。而在小兒麻痺病毒的研究中，已經有三到四個中和抗原決定位被發現，而這些決定位至散佈在三個主要在病毒表面的結構蛋白，VP1-VP3。在本研究中，我們開始研究其他有別於VP1的蛋白，如VP3和VP0（VP0是VP2和VP4的前驅蛋白）。利用大腸桿菌系統，來表現帶有His tag的結構蛋白，VP1、VP3、VP0。我們使用個別的結構蛋白與無活性的病毒顆粒和CFA/IFA等佐劑混合，用腹腔注射的方式致敏老鼠，得到的抗體去測試其中和病毒的能力。抗整個病毒的抗體有較其他單獨純化的結構蛋白有更好的中和效果，而除了VP1蛋白有中和效果外，目前初步的實驗結果也顯示VP0和VP3有中和病毒的效果，但其效果都不及整個病毒所引起的免疫反應。利用ELISA分析病毒特異性抗體，我們發現其中VP1特異性抗體較VP3及VP0特異性抗體多。之後，我們將抗病毒血清與不同的蛋白作用後，再進行中和測試。結果顯示，只有病毒顆粒與抗病毒血清作用後，有明顯抑制血清中和病毒的能力，而其他蛋白只有些微的減低抗病毒血清中和病毒的能力。另一方面，我們以VP1蛋白為主，配合著其他結構蛋白一起致敏老鼠看其引起的免疫反應是否可以達到無活性病毒致敏的免疫反應，而改採用鼻腔給予的方式致敏老鼠，希望在局部黏膜位置產生IgA，可以在第一道防線阻斷病毒的入侵感染。而實驗結果顯示只有無活性病毒致敏可以引發良好的免疫反應，推斷可能其他蛋白給予的量太低而無法達到良好的免疫反應。除此之外，顆粒性抗原、可與M細胞接觸的抗原能夠引發較好的黏膜免疫反應。因此，在未來腸病毒七十一型黏膜疫苗的研究上，應採取整個病毒顆粒去致敏老鼠。而在安全性的考量上，將開發腸病毒七十一型假病毒顆粒來開發黏膜疫苗。

Enterovirus 71 has been the most important enterovirus to cause hand-foot-and-mouth disease accompanied with neurological complication. EV71, like the poliovirus, belongs to the Picornaviridae family and there are four kinds of structural proteins, VP1-VP4, to assemble the virions. It is generally considered that VP1 is the most important antigenic determinant of EV71. Therefore, previous studies on the EV71 vaccine most focused on the inactivated virus and VP1, but inactivated virus vaccine had better protection than VP1 subunit vaccine. In addition, studies on poliovirus showed that three to four neutralizing antigenic sites have been described, involving residues of all three major structural proteins, VP1-VP3. Based on the reasons above, we want to identify neutralizing epitopes of EV71 Caspid, other than VP1, to improve vaccine. We purified other recombinant caspid proteins, other than VP1, to immunize BALB/c mice accompanied with CFA/IFA or cholera toxin as adjuvant. We demonstrated the VP0 (VP0 is the propeptide of VP2 and VP4) and VP3 can induce specific antibodies. In the neutralization test, we also found that anti-VP0 and anti-VP3 serum can protect RD cell against virus infection as well as anti-VP1 serum. Further, serum samples from EV71-immunized mice had the best neutralization effect. About the antigen saturation study, only inactivated virus could block neutralization effect of anti-virus serum. We suggested that denatured VP1, VP3, and VP0 might lose most of conformational epitopes during the purification and had low affinity with anti-virus serum. In the other hand, we estimated the component of anti-virus serum by ELISA and we found that VP1-specific antibody was more than VP3 and VP4-specific antibody. Then, we combined different structural proteins with different ratio to immunize BALB/c mice via nasal route for the reason that induction of mucosal immunity to protect virus infection in the first line of defense. The results showed that only inactivated virus can elicit great magnitude of immune response. In conclusion, antibodies induced by EV71 capsid proteins, other than VP1, had neutralization effect and whole virus could induce the best neutralizing antibodies and mucosal immunity. We estimated components of anti-virus serum by ELISA and considered that VP1 might be more important neutralizing epitopes in virus infection. We considered that intact virus is the proper candidate of the EV71 vaccine development. Thus, we will design enterovirus-like particle for development of mucosal vaccine.