腸病毒71型次單位疫苗的研發與免疫反應的研究

腸病毒71型1998年於台灣爆發腸病毒大流行，造成78因感染致死的案例，而受到極大重視，往後幾年台灣也有許多兒童受到感染並且會造成手足口症以及其他神經方面嚴重的併發症。由於腸病毒71型感染有如此高的致死率，並且缺少有效的抗病毒藥物，因此開發疫苗在第一道防線提高體內的免疫反應以抵抗外來病毒的入侵，為一首要的研究目標。腸病毒71型是由四種外鞘蛋白組成，分別為VP1、VP2、VP3與VP4，其中VP0為VP2與VP4的前驅蛋白，目前猜測VP1具有最主要的抗原決定位，但VP2與VP3亦可能具有抗原決定位，故利用大腸桿菌表現VP0、VP1與VP3三種抗原蛋白，以期混合此三種病毒蛋白分子作為疫苗致敏小鼠可以刺激免疫反應發生。本研究中發現，VP0、VP1與VP3此三種蛋白分子混合之分子疫苗在鋁鹽的作用下可以產生較VP1分子疫苗更多的腸病毒專一性抗體；而將鋁鹽混合其他TLR 依賴性配體作為佐劑使用可以進一步產生更強烈的後天性免疫反應；此外TLR依賴性配體也可降低因鋁鹽所造成過高的第二型輔助性Ｔ細胞作用，並降低以鋁鹽作為佐劑可能造成過敏的副作用；然而在中和性試驗中發現，分子疫苗致敏小鼠並無法產生抗腸病毒71型之中和性抗體，此外為了研究混合此三種分子是否可以有效中和抗腸病毒的抗體，我們更利用競爭型的中和性測試將抗病毒血清與VP0、VP1與VP3混合之蛋白分子作用，結果顯示只有去活化的腸病毒具有抑制抗體中和病毒的能力。本研究中發現，腸病毒結構蛋白混合分子可以產生較VP1高的專一性體液與細胞免疫反應，然而蛋白分子卻不具有主要的抗原決定位而無法刺激產生中和性抗體;此外合併使用鋁鹽與poly(I:C)可以作為一良好的佐劑。由於腸病毒71型死菌疫苗具有其風險性，故開發分子疫苗仍然為現行疫苗發展之首要目標。

關鍵字

腸病毒71型；分子疫苗；佐劑；抗原決定位

並列摘要

Because of the high mortality rate of enterovirus 71 (EV71), development of an effective vaccine has become the most important priority in terms of control strategies for prevention. Coat proteins of EV71, VP1-VP3 have been suggested to be the neutralizing epitopes. In this study, mice were immunized with recombinant structural proteins, including VP0, VP1 and VP3, which were expressed by Escherichia coli expression system to produce an effective vaccine for the induction of immunity. The results showed that VP0, VP1 and VP3 protein mixture combined with alum could induce higher EV71-specific humoral and cellular immunity than VP1 combined with alum. Combination of subunit vaccine with alum and TLR ligands such as poly(I:C), CpG and imiquimod can induce a stronger immune response against viral infection. Furthermore, TLR ligands could reduce the Th2 response which was induced by alum. However, immunization of subunit vaccine could not produce neutralizing antibody. Pre-incubation of VP0, VP1 and VP3 protein mixture with anti-virus serum, then applied to neutralization test, also did not inhibit the ability of neutralization of anti-virus serum. Maybe the proteins expressed from E.coli system did not have a good neutralizing epitope. In conclusion, EV71 subunit mixture had a higher immunogenicity than VP1 alone but could not produce effective neutralizing Ab. Poly(I:C) can serve a good adjuvant for EV71 subunit vaccination. Based on this study, the development of effective subunit vaccine for the prevention of EV71 infection is still an important issue. We believe the study might shed the light on further developing effective immunity and also understanding the immune response against EV71 infection.

並列關鍵字

enterovirus71 ； subunit vaccine ； adjuvant ； neutralizing determinant

參考文獻

AbuBakar, S., Chee, H.Y., Al-Kobaisi, M.F., Xiaoshan, J., Chua, K.B., and Lam, S.K. (1999). Identification of enterovirus 71 isolates from an outbreak of hand, foot and mouth disease (HFMD) with fatal cases of encephalomyelitis in Malaysia. Virus Res 61, 1-9.

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腸病毒71型次單位疫苗的研發與免疫反應的研究

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