探討登革病毒外套膜蛋白質上不同抗體之抗原辨識位及其中和能力、專一性與病毒成熟度關係

Study of the epitopes on dengue virus envelop protein recognized by different antibodies and the relationship to neutralization potency, specificity and maturation status

林宏恩(Hong-En Lin)

指導教授 ： 王維恭

國立臺灣大學/醫學院/微生物學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.00154

摘要

四種血清型之登革病毒是目前人類蟲媒病毒最主要的致病原，目前尚未有正式許可的登革疫苗上市。登革病毒的外套膜蛋白質是抗體中和病毒的主要目標，也是疫苗發展的主要對象。本研究的長期目標是經由對登革病毒中和性抗體在外套膜蛋白質上抗原辨識位的瞭解，提供有助於登革疫苗研發的重要資訊。本研究的目的是探討不同登革病毒中和性抗體在外套膜蛋白質上抗原辨識位，包括其保守程度與抗體專一性之關係以及不同抗體之抗原辨識位與登革病毒成熟度及中和能力之關係。
本研究之第一目標為建立快速標定抗原辨識位的方法，我們利用一組包括外套膜蛋白質上67個外露胺基酸之丙胺酸突變點，快速地找尋單株抗體以及血清中多株抗體的主要抗原辨識位，再以類病毒顆粒捕捉酵素連結免疫分析法確認。分析12株小鼠單株抗體，有三株具有新發現的抗原辨識位，其組成胺基酸落在外套膜蛋白質第II區域中央交界面上，此外有三株的抗原辨識位是由第Ш區域和第II區域側邊上胺基酸共同組成，此種跨區域的抗原辨識位，能被本研究中使用的方法找到。我們以同樣方法找出登革病人血清中多株抗體的主要抗原辨識位，由第II區域之融合環及其周圍胺基酸組成。
本研究的第二標為根據外套膜蛋白質胺基酸在黃質病毒血清群間保守程度決定各個抗原辨識位組成胺基酸之保守指標，進而計算出各抗原辨識位之保守分數。我們發現抗原辨識位的保守分數與其抗體專一性有極好的相關性，利用不同抗體之抗原辨識位之保守分數可以解釋不同抗體之專一性。
本研究的第三個目標是建立產生完全成熟的登革類病毒顆粒方法，病分析登革類病毒顆粒成熟與否對不同單株抗體之結合能力之影響。我們發現大多數GR類單株抗體對未成熟病毒顆粒的結合力較強，強過於對完全成熟之類病毒顆粒，顯示這類抗體可能主要由未成熟病毒顆粒所誘發。
綜合以上，本研究建立了一個快速找尋登革病毒外套膜蛋白質上抗原辨識位的方法，包括數個跨區域的抗原辨識位。本研究分析發展出的抗原辨識位之保守分數可以解釋不同抗體之專一性。此外我們發現登革類病毒顆粒成熟與否會影響辨識不同抗原辨識位之不同類單株抗體之結合能力。這些對未來登革疫苗之研發提供重要資訊。

關鍵字

登革病毒 ； 外套膜蛋白質 ； 單株抗體 ； 抗原辨識位

並列摘要

The four serotypes of dengue virus (DENV) are the leading cause of arboviral diseases in humans; currently here is no licensed dengue vaccine. The envelope (E) protein of DENV is the major target of neutralizing antibodies and vaccine development. The long-term goal of this study is to provide important information for the development of dengue vaccine. The objective of this study is to investigate the epitopes on dengue virus envelop protein recognized by different antibodies and the relationship of epitopes to specificity, maturation status of particles and neutralization potency of different antibodies.
The first specific aim is to establish a high-throughput method of epitope mapping. We used a panel of 67 alanine mutants of surface exposed E residues to rapidly identify the epitopes of anti-E monoclonal antibodies (mAbs) and the predominant epitope of anti-E antibodies in polyclonal sera, followed by verification with capture-ELISA using virus-like particles (VLPs). Of the 12 mouse anti-E mAbs, three recognized a novel epitope in the E protein domain II central interface, and three recognized an epitope involving domain III and lateral ridge of domain II. This interdomain epitope can be recognized by this method. Using the same approach, we found the predominant epitope of anti-E antibodies in polyclonal sera of dengue patients consisted of the fusion loop of domain II and surrounding residues.
The second aim is to determine the conservation index of each epitope residue based on the degree of conservation among different serocomplexes of flaviviruses and calculate the conservation scores of each epitope. We found a good correlation between the conservation scores of epitope and the specificity of antibodies. Conservation scores of epitope can explain the specificity of different antibodies.
The third aim is to establish a method to produce mature DENV VLPs and study the effect of maturation status of VLPs to the binding of different anti-E mAbs. We found most group-reactive (GR) anti-E mAbs bound immature VLPs better than mature VLPs, suggesting that these antibodies are primarily induced by immature particles.
In summary, we establish a high-throughput method to identify the epitopes on DENV E protein, including several interdomain epitopes. The conservation scores of epitopes analyzed in this study can explain the specificity of different antibodies. Moreover, we found the maturation status of VLPs affected the binding of different categories of anti-E mAbs recognizing different epitopes. These provide important information for the future development of dengue vaccine.

並列關鍵字

dengue virus ； envelope protein ； monoclonal antibody ； epitope

參考文獻

WHO, Dengue and severe dengue hemorrhagic fever fact sheet no 117 Nov 2012

WHO, 2009. Dengue hemorrhagic fever, diagnosis, treatment, prevention and control .

Wu MH, Hwang KP, Tsai JJ, Wu TS, Huang YC, King, CC, 2005. Epidemiology of dengue fever/ dengue hemorrhagic fever in Taiwan, 2001-2003. Taiwan J Pub Health 24: 452-459.

Allison, S. L., Y. J. Tao, G. O'Riordain, C. W. Mandl, S. C. Harrison and F. X. Heinz (2003). "Two distinct size classes of immature and mature subviral particles from tick-borne encephalitis virus." J Virol 77: 11357-11366.

Ashburn, P. M., C. F. Craig and U. S. A. B. f. t. S. o. T. Diseases (2004). "Experimental investigations regarding the etiology of dengue fever. 1907." J Infect Dis 189: 1747-1783; discussion 1744-1746.