本文關(guān)鍵詞：白介素-7促進狂犬病病毒免疫記憶的作用機制研究　出處：《華中農(nóng)業(yè)大學》2017年博士論文　論文類型：學位論文

  更多相關(guān)文章： 狂犬病病毒 白介素-7 濾泡性T輔助細胞 生發(fā)中心B細胞 記憶性B細胞 漿細胞 免疫記憶 體液免疫

【摘要】：狂犬病(Rabies)作為最古老的傳染病之一,是由狂犬病病毒(Rabies Virus,RABV)引起的人獸共患中樞神經(jīng)系統(tǒng)傳染病,分布于世界大部分地區(qū)。我國每年死于狂犬病的人數(shù)居世界第二位及全國各類傳染病死亡人數(shù)的前五位,其中95%以上的病例是因直接或間接接觸患病或帶毒的動物引發(fā)的。目前,狂犬病發(fā)病后的臨床治療仍是一個世界性難題,其致死率高達100%,尚無有效的治療手段。數(shù)據(jù)顯示,70%以上犬的免疫覆蓋率可以有效控制人間狂犬病,因此,動物的免疫預防成為遏制人類狂犬病最為有效的措施。當前用于動物狂犬病免疫的疫苗主要為安全的滅活疫苗,但價格昂貴,并需多次免疫才能獲得良好的長期保護,阻礙了其在發(fā)展中國家的廣泛應用。因此,發(fā)展低毒、單劑量及長期有效的狂犬病疫苗對控制狂犬病具有重要意義。近來研究表明,過表達樹突狀細胞(Dendritic cells,DCs)刺激因子的重組狂犬病病毒可在淋巴結(jié)及外周血中招募并成熟DCs及B淋巴細胞,進而迅速產(chǎn)生顯著高水平的抗病毒中和抗體(Virus-neutralizing antibodies,VNA),但其有效持續(xù)時間短。當前,尚未有研究通過應用免疫記憶的作用機制來延長狂犬病疫苗誘導的體液免疫。本研究通過反向遺傳技術(shù),在弱毒疫苗株r LBNSE的基礎(chǔ)上過表達對免疫細胞的生長、存活及分化方面具有廣泛效應的白介素-7(Interleukin-7,IL-7),并拯救獲得重組狂犬病病毒(r LBNSE-IL7)。在確定r LBNSE-IL7的體外生長特性不因外源基因IL-7的插入而受到影響之后,通過ELISA檢測重組病毒感染細胞上清中IL-7的表達,發(fā)現(xiàn)只有rLBNSE-IL7以劑量依賴形式表達IL-7,并對細胞無明顯副作用。將r LBNSE-IL7在BSR細胞中連續(xù)傳代10次,各代次均保持IL-7的穩(wěn)定遺傳。為鑒定IL-7的表達對狂犬病病毒致病性的影響,將rLBNSE、rLBNSE-IL7以及陰性對照重組病毒rLBNSE-IL7(-)腦內(nèi)途徑感染免疫功能完全和不完全的小鼠。我們發(fā)現(xiàn)腦內(nèi)高表達的IL-7能夠限制病毒在腦內(nèi)的轉(zhuǎn)錄和復制,進一步減弱狂犬病病毒的致病力,而非IL-7的插入造成病毒基因組長度的增加所導致。為進一步分析IL-7對狂犬病病毒免疫原性的作用,將重組病毒后肢肌肉途徑免疫小鼠,進行依賴T細胞的與B細胞相互作用的相關(guān)免疫機制研究。與親本病毒r LBNSE相比,重組病毒表達的IL-7可以顯著增加淋巴結(jié)中濾泡性T輔助細胞(T follicular helper cells,Tfh cells)、生發(fā)中心B細胞(Germinal center cells,GC B cells)、長壽命記憶性B細胞(Memory B cells,Bmem)的數(shù)量和骨髓中長壽命漿細胞(Plasma cells,PCs)的比例。由單劑量免疫rLBNSE-IL7所增殖的長壽命PCs可產(chǎn)生更加強大的免疫效力,維持顯著高水平的VNA和相關(guān)抗體亞類長達360天,且能夠長期有效保護絕大多數(shù)小鼠抵抗強毒株攻擊。此外,IL-7所增加的長壽命Bmem能夠于再次共同免疫r LBNSE后,分化成抗體分泌細胞,迅速產(chǎn)生更高水平的VNA及相應抗體亞類。上述結(jié)果表明IL-7促進了Tfh細胞的增殖,并在其協(xié)助下增強了GCs反應,進一步生成與維持免疫記憶相關(guān)的長壽命Bmem和PCs,從而增強了機體針對狂犬病病毒的特異性初次免疫反應和再次免疫應答。綜上所述,本研究成功構(gòu)建了表達IL-7的重組狂犬病病毒r LBNSE-IL7,鑒定了外源基因IL-7在病毒基因組中的表達不影響病毒的體外生長特性;且腦內(nèi)高表達的IL-7可以降低狂犬病病毒的致病力;并通過促進GCs中的Tfh細胞與B細胞的相關(guān)免疫反應從而增殖長壽命Bmem和PCs以增強狂犬病病毒的免疫記憶,改善初次免疫保護性反應和再次免疫應答。此研究結(jié)果表明rLBNSE-IL7具有成為低毒、高效的動物狂犬病疫苗的潛能。
[Abstract]:Rabies virus (Rabies) infection as one of the oldest, is composed of rabies virus (Rabies Virus, RABV) zoonotic infectious disease caused by central nervous system, distributed in most parts of the world. China's annual number of deaths in the rabies ranked second in the world and all kinds of infectious diseases before the five, of which more than 95% the cases are caused by direct or indirect contact with diseased or poisonous animal caused. At present, the incidence of rabies after clinical treatment is still a difficult problem in the world, the mortality rate as high as 100%, there is no effective treatment. The data show that more than 70% of the coverage rate of immune dogs can effectively control rabies, therefore. Animal immune prevention is the most effective containment of human rabies. The measures currently used for animal rabies vaccines are inactivated vaccine is safe, but the price is expensive, and needs more time to be immune Have good long-term protection, has hindered its widespread use in the developing world. Therefore, the development of low toxicity, single dose and long-term effective rabies vaccine is important for rabies control. Recent research showed that over expression of dendritic cells (Dendritic, cells, DCs) stimulation of recombinant rabies virus factors in lymph nodes and peripheral blood recruit and mature DCs and B lymphocytes, and then quickly produced significantly higher levels of neutralizing antibody (Virus-neutralizing, antibodies, VNA), but the effective duration is short. At present, there has not been a mechanism through the application of immune memory to extend the humoral immunity induced by rabies vaccine. This study by reverse genetic technique, overexpression of immune cell growth based on attenuated vaccine strain R LBNSE, has a wide effect on survival and differentiation of interleukin -7 (Interleukin-7, IL-7), and. Save the recombinant rabies virus (R LBNSE-IL7) r LBNSE-IL7. After determining the in vitro growth characteristics due to the insertion of exogenous gene IL-7 affected, recombinant virus ELISA infection by detecting the expression of IL-7 in the supernatant, found that only rLBNSE-IL7 expression of IL-7 in a dose dependent form, and no obvious side effects on R LBNSE-IL7 cells. In BSR cells were passaged 10 times and each generation are to maintain the stability of the IL-7 genetic expression. To identify the effect of IL-7 on the pathogenicity of rabies virus, rLBNSE, rLBNSE-IL7 and negative control recombinant virus rLBNSE-IL7 (-) complete and incomplete the immune function of mice infected brain. We found high expression in the brain the IL-7 transcription and replication in the brain to limit the virus, further attenuated rabies virus pathogenicity, rather than the increase of IL-7 due to the insertion of the viral genome length caused by in. Further analysis of the effect of IL-7 on the immunogenicity of rabies virus, the recombinant virus immunized mice by way of hindlimb muscles, dependent T cells and B cells related to immune mechanism of the interaction of LBNSE and R. Compared with the parental virus, the recombinant virus IL-7 can significantly increase the expression of T in lymph node follicular helper cells (T follicular helper cells, Tfh cells), germinal center B cell (Germinal center cells GC, B cells), long-lived memory B cells (Memory B cells, Bmem) and the number of long-lived plasma cells in the bone marrow (Plasma, cells, PCs). The proportion of long-lived PCs proliferation by single dose immunization can be rLBNSE-IL7 have stronger immunity, maintain significantly higher levels of VNA and related antibody subclasses for up to 360 days, and can effectively protect the most mice against virulent attacks. In addition, long-life Bmem added to the IL-7 to another LBNSE R after immunization, differentiate into antibody secreting cells, quickly produce higher levels of VNA and the corresponding subunits. These results indicated that IL-7 promoted the proliferation of Tfh cells and enhanced GCs reaction in its assistance, further generation associated with maintaining immunological memory long life Bmem and PCs, so as to enhance the the specificity of the primary immune response to rabies virus and secondary immune response. In summary, this study successfully constructed IL-7 expressing recombinant rabies virus R LBNSE-IL7, identified the expression of IL-7 gene in the viral genome does not affect the virus in vitro growth characteristics; and in the brain of the high expression of IL-7 can reduce the rabies virus force; and through the immune response in GCs Tfh cells and promote B cells proliferation and long life Bmem and PCs to enhance the rabies virus immune memory, improve primary immune protection The results of this study suggest that rLBNSE-IL7 has the potential to become a low toxic and efficient animal rabies vaccine.

【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S852.65

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