用固定化金屬離子親合力層析法單步驟純化似病毒粒子當作雞華氏囊病毒之疫苗

本計畫長遠的目標是利用昆蟲細胞/桿狀病毒表達系統（Insect cellculture/baculovirus expression system）來生產似病毒粒子 (Virus-like particles,簡稱VLPs) 當作防治雞傳染性華氏囊病毒 (Infectious Bursal Disease Virus,簡稱IBDV)的疫苗。之所以利用似病毒粒子而不用單一次單元蛋白的原因是似病毒粒子通常才能保有真正病毒原本之空間立體結構且誘發高 titer 的中和抗體，是比較好的疫苗；而且這些似病毒粒子本身又不帶有 DNA 或 RNA 等遺傳性物質，就此點而言，發展似病毒粒子當作疫苗，又比用馴化活毒疫苗或死毒疫苗來得安全。在去年計畫中，我們利用 IBDV 之大段RNA (大小約 3.4kb) 當作template 來合成 dsDNA，轉殖此段 dsDNA gene，定其序列，並且將其放入桿狀病毒(Baculovirus) 基因體內以構築成重組桿狀病毒，來感染培養之昆蟲細胞以同時表達 IBDV 之 VP2、 VP3、 VP4 重組蛋白以形成似病毒粒子，其中 VP2 及VP3均是構成 IBDV 之 virion 的最主要結構蛋白。因目前利用蔗糖梯度離心來純化似病毒粒子之方法，並不適用於工業化大量生產，所以本計畫將利用蛋白質工程之方法，讓似病毒粒子之表面能擁有可以鍵結上金屬離子 (如 Zn+2, Ni+2 等)之部位，如此我們便可利用如固定化金屬離子親和層析法，單步驟來純化似病毒粒子，而且這純化流程很容易Scale-up，應可大量純化得似病毒粒子，以降低疫苗之生產成本。以似病毒粒子當作疫苗是未來疫苗發展的一重要方向，本計畫是一連串有系統兼定量性研究發展似病毒粒子之生產，及如何形成、如何簡易其純化步驟中的重要一步。IBDV 是影響全世界養雞工業經濟效益的主要病源體，所以它是一個非常好的模型系統，以後我們會朝其他疫苗發展，以增強台灣疫苗開發及生產能力

開發雞傳染性華氏囊炎病毒結構蛋白VP2所形成之似病毒粒子為疫苗載體(II)

傳染性華氏囊病毒(Infectious Bursal Disease Virus, 簡稱 IBDV)是影響全世界養雞產業經濟效益的主要病原體。而具有誘發高力價之中和抗體之rVP2H似病毒粒子(rVP2H virus-like particles, 簡稱 rVP2H particles)是由IBDV之宿主保護疫原結構蛋白(host-protective immunogen)VP2於C端加上六個組胺酸(Hisitine)之chimeric蛋白所組裝而成。因VP2蛋白之C端所接上六個組胺酸(Histidine)並不會影響似病毒粒子之組裝，而且固定化金屬親合力層析法(immobilized metal-ion affinity chromatography, IMAC)之方法可有效地純化rVP2H particles，這代表VP2蛋白之C端可以接上外來之胺基酸序列或其他之epitopes，並不會破壞其立體結構及其誘發anti-IBDV中和抗體之能力。所以本計畫延續去年計畫的目標，要將rVP2H particles改造成能攜帶其他epitopes的似病毒粒子，最終之目的即利用rVP2H particles為載體平台，以開發多價載體疫苗或具其他功能性枝生物製劑。目前，我們已在VP2 蛋白之C端接入外來之勝.序列，初步證實 IBDV VP2 virus-like particles可開發為一載體平台。為擴展本載體平台之應用性，本計畫預計在VP2 蛋白之C端，加入其他三類之勝.序列，第一即加入具T-cell epitopes之勝.序列以開發不需佐劑之傳染性華氏囊炎疫苗，第二是加入具抗新城雞病病毒(Newcastle disease virus, NDV)之neutralizing epitopes，以開發雞傳染性華氏囊炎及新城雞病兩用疫苗；第三是加入具抗雞傳染性支氣管炎病毒(infectious bronchitis virus, IBV)之epitopes，以開發傳染性華氏囊炎及傳染性支氣管炎兩用疫苗。本計畫預計完成這三類chimeric VP2似病毒粒子的表現、生產、純化及初步之動物試驗，作為將來疫苗開發之準備工作

Regulatory Mechanisms of Chicken Il-12 in the Immune Responses to Infectious Bursal Disease Virus Vp2 and Newcastle Disease Virus Hn Proteins

在我們先前的試驗中發現以重組禽痘毒，rFPV/chIL-12，表現的鷄細胞素chIL-12可促進由重組禽痘毒，rFPV/VP2，製造的VP2 引起雞隻體液及細胞性免疫反應，但卻會抑制由重組禽痘毒，rFPV/HN，製造的HN 蛋白質引起的抗體反應，雖然它也會促進雞隻對HN 蛋白質所引起的細胞性免疫。顯然相同細胞素chIL-12 對於不同抗原存在著不同的調控機制。因此，本計畫擬利用重組禽痘毒表現的VP2及HN 蛋白質分別和chIL-12 或油質佐劑做不同搭配組合，並於培養的細胞 (invitro) 及免疫雞隻 (in vivo)，分析和免疫反應相關基因mRNA 表現之消長，從而探討chIL-12 對VP2 及HN 蛋白質免疫反應調控機制。擬分三年進行。第一年擬從三種重組禽痘毒感染DF-1 細胞製備之溶解液中分別純化IBDV VP2、NDV HN及chIL-12 蛋白質並測試其生物活性；同時進行雞DC 細胞特異性標記蛋白質CD83 基因選殖，CD83 蛋白質表現純化。鑑定後，用以免疫小白鼠製備多價及單源抗體。第二年著重於脾臟DC 細胞、巨噬細胞及淋巴球的分離培養。接著利用不同組合的蛋白質/佐劑和細胞共同培養，並利用real-time RT-PCR 檢測受刺激後的細胞和免疫反應相關基因的mRNA 表現情形以及對Dextran-FITC 吞噬能力是否被激化。第三年將不同組合的蛋白質/佐劑和DC 細胞及淋巴球共同培養，再以ELISPOT 分析淋巴球分泌抗體數目是否增加。接著將不同搭組合之蛋白質/佐劑免疫雞隻，取脾臟分析DC 細胞、淋巴球，再依in vitro 方式進行DC 細胞和免疫反應相關基因mRNA的表現及ELISPOT 分析產生抗體淋巴球數目是否增加等。如此，比較分析細胞培養或體內試驗結果，預期可以找出chIL-12 於雞隻對VP2 及HN 蛋白質免疫反應的調控機制，對於更有效疫苗的研發或許可以進一步提供有用的資訊。Our previous studies have demonstrated that recombinant rFPV/chIL-12 expressedchIL-12 enhanced both humoral and cell mediated immune responses against IBDVVP2 protein expressed by a recombinant rFPV/VP2; whereas, chIL-12 inhibitedhumoral immune response (HI antibody production) against NDV HN proteinexpressed by a recombinant rFPV/HN although chIL-12 also enhanced cell mediatedimmunity (CMI). Thus, different regulatory mechanisms exist among differentantigens by the same cytokine chIL-12. This research proposal is to prepare variousvaccine combinations consisting of proteins VP2 or HN with chIL-12 or oil adjuvant.The prepared vaccines are used to co-culture with splenic cell or to vaccinate chickens.The expression of several genes related to immune responses is assessed to insight theregulatory mechanisms of chicken immune responses against VP2 or HN protein bychIL-12. The time table for this proposal includes 3 years. In the first year, proteinsVP2, HN and chIL-12 will be purified from the recombinant virus-infected DF-1 cellsand their biological functions are analyzed. Meanwhile, the gene coded for a uniquemarker CD83, for chicken dendritic cells (DC) is cloned and CD83 is expressed. Thepurified CD83 will be further used to immunized BALB/c mice to prepare polyclonaland monoclonal antibody. In the second year, the DC, macrophage and lymphocyteare isolated from spleens and cultured. The cultured cells will be treated with variousvaccine combinations as described in the first year, followed by detecting several geneexpression related to immune responses against VP2 or HN protein by real timeRT-PCR. Also, phagocytosis capacity of the treated cells is assessed by theirendocytosis of Dextran-FITC. Finally, to assess if the numbers of chickenIgG-secreting cells increase, the cells treated with vaccine combinations are examinedby the ELISPOT techniques. The vaccine combinations are also used to vaccinatechickens. Spleens are removed and DC and splenic lymphocytes are prepared for theanalysis of gene expression related to immune responses against VP2 and HN and theincrease of numbers of chicken IgG-secreting cells by the ELISPOT, as described forin vitro study. The results obtained from those in cultured cells (in vitro) and inchickens (in vivo) are assessed and expected to figure out the mechanisms of immuneresponses against VP2 and HN proteins regulated by chIL-12. These findings mayprovide useful information for the development of more effective vaccines againstIBD and ND

Investigation on the Virulence Factor of Avian Reovirus

本計劃擬利用反向遺傳學系統 (reverse genetics system ) 技術探討家禽里奧病毒 (ARV) 腳底接種後，S1133毒株引起關節炎或2408毒株引起急性死亡之毒力因子。擬分三年進行。第一年著重於ARV S1133 及2408 各10段RNA基因體片段之全長cDNA製造並嵌入pcDNA 3.1 plasmid中，構築上游含T7 啟動子序列及下游含D 型肝炎病毒終止密碼序列之pT7-ARVcDNA-HDVR 之重組質體，其中10 源自S1133 毒株；另10 種則源自2408毒株。另外構築T7聚合酶重組質體pT7-pol. 於重組質體鑑定後，利用共同轉染( cotransfection )及基因 reassortment 原理，製備包括9 段源自S1133毒株RNA 及1 段源自2408 毒株之重組病毒10 株；以相同方法製備包括9段源自2408 毒株RNA 和1 段源自S1133 毒株之重組ARV 10 株。接著以SPF 雞接種分析試驗，鑑定ARV 哪一段RNA 基因體交換後，會導致病原顯著改變，以確定哪一段基因體 (簡稱 X片段)和病徵有關。接著構築分別來自S1133及2408毒株X片段之chimeric X cDNA片段。並依上述相同方法構築成重組質體pT7-chimeric X cDNA-HDVR 。再和pT7-ARVcDNA-HDVR以外的9種重組其體及pT7-pol共同轉染以製造chimeric重組ARV。再接種SPF雞隻，分析chimeric 重組ARV 病原性的改變，從而得知ARV X片段RNA哪些區域和ARV導致病徵相同。最後再利用點變異技術，將上述和毒力因子相關區域內的毒株間差異的胺基酸進行點變異，進而以相同方法製造出點變異病毒株，再接種SPF雞隻分析ARV X片段RNA 哪些胺基酸和病毒毒力相關，從而鑑定ARV 致病毒力因子。Avian reovirus (ARV) S1133 has been reported as an etiological agent. Recently, Wefound that ARV 2408 could cause high mortality in infected birds with coagulativenecrosis lesions in liver, at 2-3 days postinoculation. Thus, this research proposal isto analyze the virulence factor involved in arthritis or acute death caused by ARV,using a reverse genetics system. The time table for this proposal includes 3years. Inthe first year, cDNA of 10 segments of each of ARV S1133 or 2408 genomic RNAwill be synthesized and cloned into pcDNA3.1 plasmid to generate a series ofrecombinant plasmids (RP) (pT7-ARV cDNA-HDVR), which includes T7 promoterupstream of ARV cDNA and hepatitis D virus ribozyme ( HDRV ) sequence. A totalof 20 RP will be constructed, 10 RP from each of 10 genome segments of S1133 and10 RP from each of 10 genome segments of 2408. In addition, a RP (pT7-pol) whichincludes T7 polymerase will also be constructed in this study. Nine RPs of ARVS1133, one RP of 2408, and T7-pol will be used to cotransfect into DF-1 cell line togenerate 10 recombinant viruses (RV). The genomic RNA segments of each theseRV includes 9 RNA from S1133 and one segment RNA from 2408. Following thesame procedures, 10 RV will be generate. The genome of each these RV include 9RNA segments from 2408 and one RNA from S1133. These RV will be used toinfect SPF birds to determine which viral RNA segment ( called X RNA segment )involves in virulence. In the second year, to further determine if region(s) of X RNAsegment involve(s) in virulence factor(S), chimeric X cDNA fragments derived fromS1133 and 2408 X RNA segment will be constructed to generate chimeric RP(pT7-chimeric X cDNA-HDVR). Nine RPs of S1133, one RP of chimeric X cDNA,and pT7-pol will be used to cotransfect cells to generate chimeric RV. The sameprocedures will be used to generate chimeric RV using 9 RP of 2408 forcotransfection. The region of X RNA segment which involves virus virulent factorwill be assessed in birds infected with the chimeric RV. Finally, to generated pointmutation RV to determine if amino acid(s) in the region of X RNA segmentinvolve(s) in virulence factor of ARV, point mutation of amino acid in the region ofX RNA segment will be changed to generate point mutant RV. These RV will beused to inoculate birds for the evaluation of ARV virulence. The amino acid-basedvirulence factors will be finally determined