本文關(guān)鍵詞：表達(dá)免疫刺激因子的重組狂犬疫苗對狂犬病的暴露前及暴露后預(yù)防研究　出處：《吉林大學(xué)》2010年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 狂犬病 狂犬病毒 免疫刺激因子 暴露前 暴露后

【摘要】： 狂犬病(Rabies)是由RNA病毒引起、通過患狂犬病動物咬、抓傷傳播的嚴(yán)重威脅人類健康的人獸共患傳染病。每年,全世界大約有55000人死于狂犬病感染,且多于1000萬的人接受狂犬病暴露后免疫治療。大多數(shù)人類狂犬病主要發(fā)生在亞洲、非洲等發(fā)展中國家。在發(fā)達(dá)國家,由于對寵物的常規(guī)免疫,已基本消除了人類狂犬病。當(dāng)前用于人狂犬病免疫的疫苗主要為滅活疫苗,此種疫苗具有安全、有效等優(yōu)點(diǎn),但狂犬病暴露人群通常需要在較長時間(14天)內(nèi)進(jìn)行多次免疫注射(至少4次)。此外,此種疫苗價格昂貴,進(jìn)一步阻止了其在發(fā)展中國家的使用。弱毒疫苗或重組活疫苗也被批準(zhǔn)用于動物,尤其是野生動物的免疫。這些疫苗已在歐洲及北美洲很大范圍內(nèi)消除了野生動物的狂犬病,但通常會引起副反應(yīng),且對犬及臭鼬沒有良好的保護(hù)效果。因此,狂犬病的控制需要更加廉價、有效的疫苗,尤其是在廣大發(fā)展中國家。目前,狂犬病仍沒有有效的治療手段,一旦出現(xiàn)臨床癥狀,幾乎所有的方法都無能無力。截止今天,全世界共有9人在出現(xiàn)狂犬病癥狀后依然存活,然而他們中的大多數(shù)或產(chǎn)生嚴(yán)重的神經(jīng)系統(tǒng)后遺癥或死于持續(xù)的疾病。因此,探討新的狂犬病治療方法對于控制狂犬病具有重要意義。 近來研究表明,先天性免疫應(yīng)答的激活,尤其是趨化因子及干擾素,是狂犬病致弱的重要分子機(jī)制。為進(jìn)一步研究趨化因子在狂犬病感染中的作用,通過反向遺傳技術(shù),將巨噬細(xì)胞炎性蛋白(Macrophage inflammatory protein 1α, MIP-1α)克隆入HEP-Flury致弱狂犬病毒株,拯救獲得重組病毒并命名為rHEP-MIP1α。rHEP-MIP1α與母本病毒在NA細(xì)胞的生長特性相似,這表明重組病毒的體外增殖不受外源基因表達(dá)的影響。ELISA檢測病毒感染細(xì)胞后MIP-1α的表達(dá),發(fā)現(xiàn)rHEP-MIP1α以劑量依賴形式表達(dá)MIP-1α。將重組病毒腦內(nèi)注射小鼠,檢測MIP-1α表達(dá)對重組病毒致病性的影響。與母本病毒相比,rHEP-MIP1α可在中樞神經(jīng)系統(tǒng)(Central nervous system, CNS)誘導(dǎo)短暫的趨化因子表達(dá)及炎性細(xì)胞浸潤,且其對小鼠的致病性降低。結(jié)果說明rHEP-MIP1α可通過誘導(dǎo)短暫的CNS先天性免疫應(yīng)答進(jìn)一步降低病毒的致病性。 為進(jìn)一步研究rHEP-MIP1α的免疫原性,將重組病毒肌肉免疫小鼠后,以快速熒光灶抑制試驗(yàn)(RFFIT)測定病毒誘導(dǎo)機(jī)體產(chǎn)生的中和抗體水平。與母本病毒相比,rHEP-MIP1α肌肉注射后可誘導(dǎo)機(jī)體產(chǎn)生顯著高的狂犬病中和抗體。以CVS-24強(qiáng)毒進(jìn)行攻擊后,顯著多的rHEP-MIP1α免疫小鼠可抵抗強(qiáng)病毒感染。通過熒光定量PCR分析免疫局部的病毒增殖、趨化因子表達(dá)及先天性免疫應(yīng)答分子聚集,結(jié)果發(fā)現(xiàn)不同病毒在免疫局部的增殖相似,這說明rHEP-MIP1α誘導(dǎo)的適應(yīng)性免疫應(yīng)答與病毒在免疫局部的增殖無關(guān)。與母本病毒相比,rHEP-MIP1α可在免疫局部誘導(dǎo)表達(dá)更高水平的MIP-1α、IL-4、CD19及CD11c。此外,流式細(xì)胞術(shù)分析發(fā)現(xiàn)rHEP-MIP1α可在淋巴結(jié)及外周血聚集并激活更多的樹突狀細(xì)胞(Dendritic cells, DCs)及B淋巴細(xì)胞。這些數(shù)據(jù)說明,rHEP-MIP1α可在免疫局部表達(dá)高水平的MIP-1α,進(jìn)而在淋巴結(jié)及外周血聚集并激活更多的DCs及B細(xì)胞,從而產(chǎn)生高水平的中和抗體。因此,DCs的聚集及激活在增強(qiáng)病毒的保護(hù)性免疫應(yīng)答過程中起重要作用。進(jìn)一步分析了表達(dá)粒細(xì)胞-巨噬細(xì)胞集落刺激因子(Granulocyte-macrophage colony-stimulating factor, GMCSF )、巨噬細(xì)胞來源趨化因子( Macrophage derived chemokines, MDC)及MIP-1α等DCs刺激分子的重組病毒的免疫原性。結(jié)果表明,重組病毒表達(dá)這些分子后可顯著增強(qiáng)DCs的聚集及激活,進(jìn)而增強(qiáng)病毒的保護(hù)性免疫應(yīng)答。 為研究表達(dá)免疫刺激因子的重組狂犬病毒對狂犬病的暴露后預(yù)防作用,在小鼠肌肉感染高致病性狂犬病街毒株后不同時間,用不同重組病毒以腦內(nèi)(i.c.)、肌注(i.m.)、皮下(i.d.)及滴鼻(i.n.)等途徑進(jìn)行治療。結(jié)果發(fā)現(xiàn),即使在街毒感染后5天開始治療,表達(dá)MIP-1α、GM-CSF、MDC及IP-10的重組病毒仍具有明顯的保護(hù)作用。盡管紫外線滅活的重組病毒誘導(dǎo)機(jī)體產(chǎn)生了較高的中和抗體,但對街毒感染沒有保護(hù)作用。測定熒光素鈉(Sodium fluorescein, NaF)由血液循環(huán)進(jìn)入CNS的量,發(fā)現(xiàn)表達(dá)免疫刺激因子的重組病毒可顯著提高大腦及小腦的血腦屏障(Blood brain barrier, BBB)通透性。通過熒光定量PCR、多重ELISA(Multiplex ELISA)及流式細(xì)胞術(shù)分析CNS的趨化因子、細(xì)胞因子表達(dá)及炎性細(xì)胞浸潤。結(jié)果表明,與紫外線滅活病毒相比,表達(dá)免疫刺激因子的重組病毒腦內(nèi)注射后在CNS及外周血引起更高水平的趨化因子及細(xì)胞因子表達(dá),炎性細(xì)胞浸潤。更為重要的是,可增強(qiáng)血腦屏障通透性的趨化因子MCP-1(Chemoattractant protein 1, MCP-1)顯著增加了滅活病毒的保護(hù)效果。這些數(shù)據(jù)說明,表達(dá)免疫刺激因子的重組病毒可通過在CNS誘導(dǎo)炎性因子表達(dá),增強(qiáng)炎性細(xì)胞浸潤及提高血腦屏障的通透性,允許更多的炎性細(xì)胞或免疫效應(yīng)因子進(jìn)入CNS,進(jìn)而加速病毒的清除及阻止狂犬病感染的發(fā)生。 本研究成功構(gòu)建了表達(dá)免疫刺激因子的重組狂犬病毒,證實(shí)了外源基因在病毒基因組的表達(dá)對病毒的體外生長沒有影響,且重組病毒可通過誘導(dǎo)短暫的先天性免疫應(yīng)答分子表達(dá)進(jìn)一步降低病毒的致病性,DCs的聚集及激活在增強(qiáng)病毒的保護(hù)性免疫應(yīng)答中具有重要作用,篩選獲得的重組疫苗不僅可用于狂犬病的暴露前及暴露后預(yù)防免疫,而且還可用于狂犬病的臨床治療。此研究結(jié)果為研究狂犬病的致病機(jī)理及篩選新型、低毒、高效狂犬病疫苗提供了重要理論依據(jù),并為狂犬病的治療提供了新的手段及工具。
[Abstract]:Rabies virus (Rabies) is caused by the RNA virus, rabies through animal bites, scratches the spread of a serious threat to human health zoonotic disease. Every year, there are about 55000 people died of rabies infection all over the world, and more than 10 million people received post exposure rabies immune therapy. Most human rabies occurred mainly in Asia, Africa etc. developing countries in developed countries, due to routine immunization of pets, has been basically eliminated. The current human rabies rabies vaccines used for human mainly for the inactivated vaccine, the vaccine is safe, effective and other advantages, but the population exposed to rabies usually in the long time (14 days) in the number of immunization (at least 4 time). In addition, the vaccine is expensive, further to prevent its use in developing countries. The attenuated vaccine or recombinant live vaccine has been approved for animal, especially wild Animal immune system. These vaccines have been eliminated in the wild animal rabies in Europe and North America in a large range, but often cause side effects, and there is no good protective effect on canine and skunk rabies control. Therefore, the need for more cheap and effective vaccine, especially in developing countries. At present, rabies is still not the effective treatment method, once the clinical symptoms, almost all methods are powerless. As of today, 9 people throughout the world are still alive in rabies, but most of them or cause serious neurological sequelae or died of persistent disease. Therefore, to explore the methods of treatment for rabies is new the important significance of rabies control.
Recent studies have shown that activation of the innate immune response, particularly chemokines and interferons, is an important mechanism of rabies virus attenuation. Chemokines in rabies infection. For further research, by reverse genetic technique, the macrophage inflammatory protein (Macrophage inflammatory protein 1 alpha, alpha MIP-1) was cloned into HEP-Flury attenuated rabies virus strains, save the recombinant virus named rHEP-MIP1 alpha.RHEP-MIP1 alpha and female parent virus were similar in the growth characteristics of NA cells, suggesting that the detection of.ELISA virus infected cells MIP-1 expression on proliferation of recombinant virus in vitro by exogenous gene expression, rHEP-MIP1 expression of MIP-1 alpha in dose-dependent manners. The recombinant virus was injected into the brain of mice, to detect MIP-1 expression effects on pathogenicity of the recombinant virus. Compared with the parent virus, rHEP-MIP1 alpha in the central nervous system (Cen Tral nervous system (CNS) induced transient chemokine expression and inflammatory cell infiltration, and reduced its pathogenicity to mice. Results showed that rHEP-MIP1 alpha could further reduce the pathogenicity of virus by inducing transient CNS immune response.
For further study of immunogenicity of rHEP-MIP1 alpha, the recombinant virus muscle of mice immunized with the rapid fluorescent focus inhibition test (RFFIT) neutralizing antibody level determination of virus induced. Compared with the parent virus, rHEP-MIP1 alpha after intramuscular injection can induce significantly higher rabies neutralizing antibody attack to CVS-24. After inoculation, rHEP-MIP1 alpha mice significantly more resistant to strong virus infection. By fluorescence quantitative PCR analysis of virus proliferation of local immune, chemokine expression and innate immune response of molecular aggregation, found with the virus in the local immune proliferation is similar, which shows that the adaptive immune response and virus induced by rHEP-MIP1 in the independent the proliferation of local immune. Compared with the parent virus, alpha rHEP-MIP1 can induce local higher expression levels of MIP-1, CD19 and CD11c. in the immune IL-4, in addition, flow cytometry Analysis found that rHEP-MIP1 alpha in lymph nodes and peripheral blood aggregation and activation of dendritic cells (Dendritic more cells, DCs) and B lymphocytes. These data suggest that rHEP-MIP1 alpha can be local expression of high levels of MIP-1 in the immune system, and in the lymph node and peripheral blood aggregation and activating DCs and B cells. In order to produce high levels of neutralizing antibodies. Therefore, recruitment and activation of DCs play an important role in the enhancement of virus protective immune response. Further analysis of the expression of granulocyte macrophage colony stimulating factor (Granulocyte-macrophage colony-stimulating, factor, GMCSF), macrophage derived chemokine (Macrophage derived, chemokines, MDC) immunogenicity DCs and MIP-1 alpha stimulation recombinant virus molecule. The results showed that the recombinant virus expression of these molecules can significantly enhance aggregation and induced DCs activities, thus enhancing the virus Protective immune response.
Immune stimulating factor recombinant rabies virus for prevention of rabies exposure on expression in muscle of mice infected with highly pathogenic strains of rabies street after different time, with different recombinant virus in the brain (i.c.), (i.m.), subcutaneous injection (i.d.) and intranasal (i.n.) and other means of treatment. Found that, even in the street virus infection 5 days after the start of treatment, the expression of MIP-1 alpha, GM-CSF, recombinant virus MDC and IP-10 still has obvious protective effect. Although recombinant virus UV inactivated induce neutralizing antibodies is higher, but no protective effect on street virus infection. The determination of fluorescein sodium (Sodium fluorescein. NaF) from the circulation into the amount of CNS, found that the expression of the recombinant virus immune stimulating factor can significantly improve the blood brain barrier in brain and cerebellum (Blood brain, barrier, BBB). The permeability by fluorescence quantitative PCR, multiplex ELISA (Multiplex ELISA) and flow cytometry analysis of CNS chemokines, cytokines and inflammatory cell infiltration. The results showed that compared with UV inactivated virus, expression of recombinant virus immune stimulating factor in brain after injection of CNS in peripheral blood and induced higher levels of chemokines and cytokines, inflammation infiltration of inflammatory cells. More importantly, can enhance the permeability of blood brain barrier chemokine MCP-1 (Chemoattractant protein 1, MCP-1) significantly increased the protective effect of inactivated virus. These data suggest that expression of recombinant virus immune stimulating factors can be induced by inflammatory cytokine expression in CNS enhanced inflammatory cells infiltration and increase the permeability of the blood-brain barrier, allowing inflammatory cells or immune effectors more into CNS, thereby accelerating the clearance and prevents the occurrence of rabies virus.
This study successfully constructed recombinant rabies virus expressing immunostimulatory factors, confirmed the expression of exogenous gene in the viral genome of the virus in vitro growth was not affected, and the recombinant virus can induce transient innate immune response molecule expression further reduced the pathogenicity of the virus, recruitment and activation of DCs play an important role in the enhancement of protection immune responses to the virus, the screening of recombinant vaccine can be used not only for rabies pre exposure and post exposure immunization, but also can be used for clinical treatment of rabies. The results of this research and study the pathogenic mechanism of rabies and screening model, low toxicity, provides an important basis for effective rabies vaccine, and provides new means and tools for the treatment of rabies.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王磊;馮娜;李天松;劉玉秀;高玉偉;王鐵成;楊松濤;夏咸柱;;犬瘟熱病毒重組囊膜糖蛋白(H/F)全長質(zhì)粒的構(gòu)建[J];中國動物檢疫;2011年06期

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本文編號：1373454