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標題: 酵母菌表現豬瘟病毒醣蛋白E2次單位疫苗之效力分析
Efficacy of yeast expressed classical swine fever virus glycoprotein E2 subunit vaccine
作者: 林光展
Lin, Guang-Jan
Contributors: 黃千衿
Chienjin Huang
微生物暨公共衛生學研究所
關鍵字: 豬瘟病毒;酵母菌表現系統;E2重組蛋白;次單位疫苗
classical swine fever virus;Pichia pastoris expressed system;recombinant E2 protein;subunit marker vaccine
日期: 2012
Issue Date: 2013-11-19 12:51:19 (UTC+8)
Publisher: 微生物暨公共衛生學研究所
摘要: 豬瘟(classical swine fever; CSF)是由豬瘟病毒(classical swine fever virus; CSFV)所引起的豬隻重要傳染病,在世界各地造成嚴重的經濟損失。豬瘟病毒醣蛋白Erns與E2可誘發宿主產生中和抗體,本研究即利用酵母菌Pichia pastoris蛋白表現系統大量生產豬瘟病毒醣蛋白Erns與E2,並進一步進行豬隻免疫試驗以評估其做為豬瘟次單位疫苗之效力。酵母菌表現之豬瘟病毒醣蛋白Erns (yErns)與E2 (yE2)皆為N–linked醣基化蛋白且可形成同源雙體結構,進一步分別以yErns或yE2免疫6週齡大小的無特定病原(specified pathogen free; SPF)豬隻,並在初次免疫3週後進行補強免疫以評估其免疫原性。只有免疫yE2的豬隻在補強免疫後可產生高力價之中和抗體,以1× 105 TCID50豬瘟病毒進行攻毒試驗,免疫yErns的豬隻在攻毒1週後即出現嚴重豬瘟臨床症狀並需進行安樂死犧牲,而yE2免疫豬隻則無任何豬瘟症狀,進一步分析免疫豬隻血清抗Erns與E2抗體的變化,結果顯示yErns與yE2都具有正確的抗原性可於補強免疫後誘發免疫豬隻產生相對應的抗體反應,但只有yE2所誘發的抗體具有保護效力。為了解具疫苗效力的yE2做為豬瘟次單位疫苗時所需最適劑量,分別以200、300或400 μg的yE2免疫豬隻並分析其血清的中和抗體力價,免疫3種不同yE2劑量的豬隻其血清中均含有高力價的中和抗體,且在攻毒試驗後僅有免疫200 μg yE2的豬隻產生輕微發燒現象,其他yE2免疫豬隻則無任何症狀,而發燒的豬隻在短時間內也回復正常,故選定300 μg yE2總分泌蛋白為豬瘟次單位疫苗之最適劑量。且yE2免疫豬隻所引發的中和抗體亦具有中和3種不同基因型豬瘟病毒的能力。進一步將哨兵豬(sentinels)與攻毒3天後的yE2免疫豬隻共同圈養以進行同居感染試驗(cohabitation test),哨兵豬在試驗後並無豬瘟臨床症狀,證實yE2能有效防止病毒平行傳染。綜合以上結果顯示酵母菌Pichia pastoris蛋白表現系統可生產具有正確抗原性的yErns與yE2,而yE2可進一步做為豬次單位疫苗能有效誘發豬隻產生保護性免疫反應與防止病毒之平行傳染。
Classical swine fever (CSF) is an economically important swine disease worldwide and caused by the classical swine fever virus (CSFV). The viral envelope glycoprotein Erns and E2 are major targets for eliciting antibodies against CSFV in infected animals. The recombinant Erns and E2 proteins were constructed using the yeast Pichia pastoris expression system and evaluated for its vaccine efficacy. The yeast–expressed E2 (yE2) and Erns (yErns) were shown to have N-linked glycosylation and to form homodimer molecules. Six–week–old specified–pathogen–free (SPF) piglets were intramuscularly immunized with yE2 or yErns and twice at 3–week intervals. All yE2–immunized pigs could mount an anamnestic response after booster vaccination with neutralizing antibody titers. Vaccinated pigs were subjected to challenge infection with a dose of 1× 105 TCID50 (50% tissue culture infective dose) virulent CSFV strain. At 1 week post challenge infection, all of the yE2–immunized pigs were alive and without symptoms or signs of CSF. All of the yE2–immunized pigs were Erns antibody negative and had seroconverted against Erns post challenge infection. The yErns–immunized pigs seroconverted to CSFV–Erns–specific antibody after booster vaccination but no neutralizing antibody was detected and none survived after challenge infection, suggesting yErns and yE2 retain correct immunogenicity but only the yE2 is able to induce a protective immune response. All three doses of yE2 (200, 300, and 400 μg) could elicit high titers of neutralizing antibodies and protective responses after challenge. The yE2/200 group demonstrated a mild fever response but recovered soon, and none of the yE2/300 and yE2/400 pigs became febrile. The optimal dose of yE2 was recommended to be 300 μg of the total amount of secreted proteins. In addition, the yE2 vaccine could cross–protect from all three genotypes of viruses. Further, the yE2 vaccine efficacy in preventing virus horizontal transmission was evaluated by cohabitation of unimmunized sentinels 3 days after challenge infection. All the sentinel pigs were alive and had no clinical symptoms confirming yE2 vaccine could confer a protective immune response and prevent horizontal transmission of CSFV. The results elucidate yeast–expressed yErns and yE2 have correct immunogenicity, but only yE2 could induce a protective immune response against CSFV and prevent CSFV horizontal transmission, confirming yE2 was a classical swine fever E2 subunit vaccine candidate.
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