利用腺病毒載體研發輪狀病毒之疫苗

Abstract

輪狀病毒(rotavirus)感染會造成嬰幼兒產生腹瀉的症狀，嚴重者甚至會死亡，因此極需要發展有效的疫苗，來對抗此病毒的感染。 輪狀病毒顆粒由內、中、外三層蛋白質殼（capsid）所組成。VP6為構成中層蛋白質殼的主要成份，具有高抗原性及高免疫生成性，保守性較外層蛋白質高，過去的文獻指出，不論是其全長或是C端的部分（第197~397個胺基酸），皆可引發出具有保護性的抗體。另外，NSP4是一種穿膜的非結構性蛋白質，與病毒在細胞內的組裝有關，並被認為是會引發腹瀉的一種腸毒素，其C端的部分（第86∼175個胺基酸）包含了會引發腹瀉的片段，因此NSP4的抗體可能會有效減輕腹瀉的症狀。由於VP6及NSP4在輪狀病毒的結構及功能上極重要，且具有較高的保守性，因此本研究的主要目的，即是發展出針對VP6或是NSP4為目標的疫苗。 腺病毒載體是一種目前被廣泛用於表現外來基因的系統。目前，已有許多病毒疫苗的研發是利用腺病毒當載體，且被認為是有效的。因此，本研究選用腺病毒載體系統，首先分別將輪狀病毒的VP6、VP6197-397、NSP4及NSP486-175基因片段增幅出，擇殖入pShuttle-CMV 載體上，再將其送入已帶有腺病毒基因體的大腸桿菌株BJ5183 ，經由同源重組反應產生重組腺病毒DNA，將其轉染至293A細胞中，產生出帶有不同基因片段的重組腺病毒。完成了帶有VP6、VP6197-397、NSP4及NSP486-175共四個重組腺病毒株，及一個對照腺病毒。之後利用間接免疫螢光染色法，確定了各個重組腺病毒株，其目標蛋白質在293A細胞中的表現情形。再經過三次溶菌斑純化試驗，以得到正確的純種子代病毒株。大量增殖重組腺病毒後，以兩次CsCl密度梯度之超高速離心及透析法純化病毒顆粒，測定病毒力價後，以每一劑1×109 pfu之病毒口服或鼻腔內滴入的方式免疫小鼠兩次，並在最後給予輪狀病毒，觀察其抗體反應。 為了要製備偵測抗體所需之抗原蛋白質，使用重組桿狀病毒感染Sf-9昆蟲細胞，表現VP6蛋白質，以供偵測VP6的抗體。另一方面，使用大腸桿菌系統表現NSP486-175的片段，使用Ni2+ 樹脂管柱純化後，以供偵測NSP486-175的抗體。使用酵素連結免疫吸附試驗，偵測抗體的結果顯示，使用帶有VP6基因的重組腺病毒免疫小鼠後，不論是以口服或鼻腔內滴入的方式，皆能使小鼠血清中產生針對VP6的IgG抗體反應，亦可偵測到力價較低的IgA抗體反應。本研究初步證實，使用腺病毒載體系統表現輪狀病毒蛋白質VP6，可以引發小鼠的抗體免疫反應，具有發展成疫苗的潛力。

Rotavirus infections are the most important cause of severe, even life-threatening dehydrating diarrhea in infants and children. Because of the significant disease burden, vaccines against rotavirus are urgently needed. The viral particle is composed of three layers: inner, middle, and outer capsids. V6 protein is the major component of the middle-layer capsid and is highly antigenic and immunogenic. It is also more conserved than outer capsid proteins. Either full-length or the cytoplasmic domain of VP6 can elicit protective antibody response. In addition, NSP4 protein is a nonstructural transmembrane protein important for viral assembly and is suggested as an enterotoxin. The cytoplasmic domain (amino acid 86-175) include the region that cause diarrhea. Therefore, antibody against NSP4 may effectively reduce the diarrhea symptom. Because of the higher conservation and the importance for viral structure and function, vaccines against VP6 and NSP4 were developed in this research. Adenoviral vector systems were utilized for a variety of animal virus vaccine, and considered to be effective. In this study, we used the adenoviral vector to develop rotavirus vaccine. VP6, VP6197-397, NSP4, and NSP486-175 gene fragments were recovered from rotavirus, then cloned into pShuttle-CMV vector. The vector was used to transform bacteria BJ5183 which carried adenoviral genome to generate recombinant adenoviral DNA through homologous recombination. The recombinant adenoviral DNA was then transfected into 293A cell to generate recombinant adenoviruses carrying each gene fragment. Four recombinant adenoviruses carrying VP6, VP6197-397, NSP4, and NSP486-175 gene were established. Target protein expression in 293A cells could be detected by indirect immunofluorescence assay. In order to get pure viral progeny, three serial plaque purification procedures were carried out. After large scale amplification of recombinant adenoviruses, double CsCl gradient banding and dialysis were performed to purify viral particles. The virus titer was determined, and the mice were immunized with 1×109 pfu viral particles twice via oral or intranasal administration routes. The mice were challenged with rotavirus and the antibody responses were analyzed. In order to prepare antigens to detect specific antibody, the Sf-9 cell were infected with recombinant baculovirus to express VP6 protein. On the other hand, the NSP486-175 were expressed by E.coli system and purified by Ni2+ column, for detection of the anti-NSP486-175 antibody. The ELISA results showed that immunization of mouse with VP6 recombinant adenoviruses via either oral or intranasal route could induce anti-VP6 IgG antibody response. Low level IgA antibody could also be detected in mouse serum. This study showed that using adenoviral vector carrying VP6 gene could induce antibody response in mice. There is potential for the development of rotavirus vaccine.