手足口病（EV71和CVA16）病毒樣顆粒疫苗免疫原性及保護(hù)性研究
發(fā)布時(shí)間：2018-08-11 18:49
【摘要】：手足口病(Hand,Foot and Mouth Disease,HFMD)是一種全球范圍內(nèi)的傳染性疾病,流行區(qū)域主要集中在發(fā)展中國(guó)家(特別是亞太地區(qū))。中國(guó)流行病學(xué)調(diào)查結(jié)果顯示:手足口病位居丙類(lèi)傳染病之首,其主要致病病原體是Enterovirus 71(EV71)型和Coxsackievirus A16(CVA16)型,兩種病毒的流行率在80%以上。目前臨床上還沒(méi)有有效的藥物治療手足口病,疫苗的研發(fā)與接種是控制手足口病流行的重要手段。根據(jù)中國(guó)大陸臨床試驗(yàn)結(jié)果證實(shí),EV71滅活疫苗具有良好的保護(hù)效果,可以預(yù)防EV71所引起手足口病的保護(hù)率在90%以上,預(yù)防EV71相關(guān)疾病的保護(hù)率在80%以上,其誘導(dǎo)的中和抗體具有良好的廣譜交叉活性,但其血清不能中和CVA16以及其它引起手足口病的病毒,所以包含EV71和CVA16的聯(lián)合疫苗的研制顯得尤其迫切。目前CVA16滅活疫苗仍處于臨床前研究階段,限制了二者聯(lián)合疫苗在臨床上的應(yīng)用。EV71和CVA16分別只有一個(gè)血清型,但每種病毒含有多個(gè)基因亞型,而各基因亞型之間的中和抗體交叉活性有所不同。因此篩選出中和抗體覆蓋率高、免疫原性好的疫苗株,制備成聯(lián)合疫苗預(yù)防由EV71和CVA16引起的手足口病是疫苗研發(fā)的首選。目前研究只檢測(cè)了單價(jià)疫苗的廣譜性中和抗體交叉活性,而關(guān)于聯(lián)合疫苗的中和抗體交叉活性是否有差別尚未有任何報(bào)道,聯(lián)合疫苗同時(shí)接種時(shí)兩種疫苗之間是否產(chǎn)生免疫抑制和免疫干擾,聯(lián)合疫苗接種時(shí)是否可以預(yù)防兩種病毒的入侵等方面亦沒(méi)有詳細(xì)、全面的研究。HFMD疫苗的研究形式以滅活疫苗和病毒樣顆粒(VLPs)疫苗為主,隨著基因工程技術(shù)的不斷發(fā)展VLPs疫苗以其獨(dú)有的天然優(yōu)勢(shì)而被廣泛應(yīng)用。本論文針對(duì)以上問(wèn)題進(jìn)行更為深入、具體、全面的研究,提供真實(shí)、可靠的動(dòng)物試驗(yàn)數(shù)據(jù),期望解決目前聯(lián)合疫苗存在的問(wèn)題,開(kāi)發(fā)出有效、安全、交叉活性好的EV71 VLPs和CVA16 VLPs聯(lián)合疫苗。另一方面,疫苗的抗原劑量效應(yīng)關(guān)系(ED_(50))直接反映出疫苗的效力及最低免疫劑量,對(duì)疫苗的臨床前評(píng)價(jià)和疫苗的臨床免疫劑量具有指導(dǎo)意義。EV71滅活疫苗在臨床前評(píng)價(jià)了抗原的ED_(50),但對(duì)于EV71 VLPs疫苗的ED_(50)還沒(méi)有詳細(xì)、可靠的研究數(shù)據(jù)。本論文就這一問(wèn)題也進(jìn)行了研究:應(yīng)用層析純化之后的EV71 VLPs作為免疫原,選擇不同免疫劑量接種小鼠,不僅對(duì)EV71 VLPs抗原的ED_(50)進(jìn)行了測(cè)定,而且測(cè)定了其抗體的ED_(50)�？乖涂贵wED_(50)的測(cè)定對(duì)EV71 VLPs疫苗的后續(xù)研究具有重要的指導(dǎo)意義。第一部分:單價(jià)疫苗和聯(lián)合疫苗免疫原性及保護(hù)性研究一、本課題根據(jù)中國(guó)大陸流行病學(xué)調(diào)查,選用EV71流行株C4a(EU703814.1)及CVA16流行株B1a(AF177911.1),應(yīng)用昆蟲(chóng)細(xì)胞桿狀病毒表達(dá)系統(tǒng)生產(chǎn)EV71病毒樣顆粒(VLPs)、CVA16病毒樣顆粒(VLPs),陽(yáng)性對(duì)照疫苗為EV71滅活疫苗和CVA16滅活疫苗,分別制備出單價(jià)疫苗和聯(lián)合疫苗。二、依托本實(shí)驗(yàn)室的假病毒檢測(cè)平臺(tái),評(píng)價(jià)了單價(jià)疫苗和聯(lián)合疫苗的免疫原性,結(jié)果顯示不論是單價(jià)疫苗還是聯(lián)合疫苗都可以誘導(dǎo)出很強(qiáng)的中和抗體滴度;VLPs單價(jià)疫苗與滅活單價(jià)疫苗相比中和抗體滴度無(wú)顯著性差異;聯(lián)合疫苗同時(shí)接種時(shí)與單價(jià)疫苗相比中和抗體滴度亦沒(méi)有顯著性差異;聯(lián)合免疫后兩種疫苗之間不存在免疫抑制和免疫干擾;在此首次測(cè)定了聯(lián)合疫苗的廣譜性中和抗體。三、保護(hù)力試驗(yàn)是評(píng)價(jià)疫苗有效性的一個(gè)重要指標(biāo)。本研究選用1日齡ICR乳鼠,分別建立出EV71乳鼠動(dòng)物模型和CVA16乳鼠動(dòng)物模型,并評(píng)價(jià)了單價(jià)疫苗和聯(lián)合疫苗的保護(hù)力水平。結(jié)果顯示無(wú)論是單價(jià)疫苗還是聯(lián)合疫苗對(duì)新生乳鼠都可以提供很好的保護(hù)力水平,聯(lián)合疫苗可以預(yù)防兩種病毒的侵入。第二部分:EV71 VLPs抗原和抗體ED_(50)的研究本部分研究與中國(guó)食品藥品檢定研究院(NIFDC)合作,評(píng)價(jià)層析純化之后的EV71 VLPs抗原和抗體的ED_(50)。EV71 VLPs不同劑量分別與鋁佐劑吸附后免疫接種小鼠,中和抗體呈現(xiàn)出明顯的劑量效應(yīng)關(guān)系。通過(guò)乳鼠保護(hù)力實(shí)驗(yàn)結(jié)果顯示根據(jù)不同免疫劑量新生乳鼠腦內(nèi)攻毒后的存活率,計(jì)算出EV71 VLPs抗原ED_(50)為0.20μg/dose,根據(jù)攻毒時(shí)間點(diǎn)不同免疫劑量組的中和抗體滴度,計(jì)算出在此劑量下誘導(dǎo)出的中和抗體滴度大約為50。應(yīng)用兩種乳鼠保護(hù)力評(píng)定方式分別初步確定了抗體的ED_(50)大約為54和24。綜上所述,本研究利用昆蟲(chóng)細(xì)胞桿狀病毒系統(tǒng)表達(dá)出EV71 VLPs、CVA16VLPs,并應(yīng)用假病毒中和抗體檢測(cè)體系評(píng)價(jià)單價(jià)疫苗和聯(lián)合疫苗的中和抗體滴度趨勢(shì);首次應(yīng)用了所有基因亞型的假病毒評(píng)價(jià)了單價(jià)疫苗和聯(lián)合疫苗的廣譜性中和抗體滴度,證明聯(lián)合疫苗中和抗體滴度與單價(jià)疫苗相比在相同基因亞型的假病毒中無(wú)顯著性差異;并且聯(lián)合疫苗同時(shí)接種不存在免疫抑制及免疫干擾。應(yīng)用乳鼠動(dòng)物模型體內(nèi)評(píng)價(jià)了單價(jià)疫苗及聯(lián)合疫苗的有效性,兩種保護(hù)力評(píng)價(jià)方式結(jié)果證明聯(lián)合疫苗可以同時(shí)預(yù)防兩種病毒的攻擊。廣譜性中和抗體測(cè)定及乳鼠保護(hù)力的評(píng)價(jià)為聯(lián)合疫苗的后續(xù)研究和臨床應(yīng)用提供了真實(shí)、可靠的數(shù)據(jù)。另外,本研究還首次研究了層析純化后的EV71 VLPs抗原和抗體的ED_(50),為EV71 VLPs疫苗的進(jìn)一步研究提供了試驗(yàn)基礎(chǔ)。
[Abstract]:Hand, Foot and Mouth Disease (HFMD) is a worldwide infectious disease, and the epidemic areas are mainly concentrated in developing countries (especially in the Asia-Pacific region). The results of epidemiological survey in China show that HFMD is the most common type of infectious diseases, and the main pathogens are Enterovirus 71 (EV71) and Coxsackievirus. At present, there is no effective drug to treat HFMD. The development and vaccination of vaccine is an important means to control the epidemic of HFMD. According to the results of clinical trials in mainland China, the inactivated vaccine of EV71 has a good protective effect and can prevent the HFMD caused by EV71. The protective rate against EV71-related diseases is above 90%, and the protective rate against EV71-related diseases is above 80%. The induced neutralizing antibodies have good broad-spectrum cross-reactivity, but their serum can not neutralize CVA 16 and other viruses causing HFMD. Therefore, the development of a combined vaccine containing EV71 and CVA 16 is particularly urgent. EV71 and CV16 have only one serotype, but each virus contains multiple genotypes, and the neutralizing antibody cross-reactivity among the genotypes is different. Therefore, the vaccine strains with high coverage and good immunogenicity were screened and prepared to be linked. Preventing hand-foot-mouth disease (HFMD) caused by EV71 and CV16 with a combined vaccine is the first choice for vaccine development. Current studies have only examined the broad-spectrum neutralizing antibody cross-activity of monovalent vaccines, but there is no report on the difference in neutralizing antibody cross-activity between the combined vaccines. Whether there is immunity between the two vaccines when the combined vaccines are vaccinated simultaneously has not been reported. There is no detailed and comprehensive study on inhibition and immune interference, and whether combined vaccination can prevent the invasion of two viruses. HFMD vaccines mainly consist of inactivated vaccines and virus-like particles (VLPs) vaccines. With the development of genetic engineering technology, VLPs vaccines have been widely used for their unique natural advantages. Aiming at the above problems, this paper carries out a more in-depth, specific and comprehensive study to provide real and reliable animal test data, expecting to solve the existing problems of the combined vaccine and develop an effective, safe and cross-active EV71 VLPs and CVA16 VLPs combined vaccine. On the other hand, the antigen dose-response relationship (ED_ (50)) of the vaccine directly reflects. EV71 inactivated vaccine has been evaluated for ED_ (50) of antigen before clinic, but there is no detailed and reliable data for ED_ (50) of EV71 VLPs vaccine. This paper also studies this problem: application chromatography purity. The ED_ (50) of EV71 VLPs antigen and the ED_ (50) of its antibody were determined. The determination of antigen and antibody ED_ (50) is of great significance for the follow-up study of EV71 VLPs vaccine. Part I: Monovalent vaccine and combined vaccine immunization. First, according to the epidemiological investigation in mainland China, we selected EV71 strain C4a (EU703814.1) and CVA16 strain B1a (AF177911.1) to produce EV71 virus-like particles (VLPs), CVA16 virus-like particles (VLPs) by using insect cell baculovirus expression system. The positive control vaccine was EV71 inactivated vaccine and CVA16 inactivated vaccine. The results showed that both monovalent and combined vaccines could induce strong neutralizing antibody titers, and VLPs monovalent vaccines could neutralize antibody titers compared with inactivated monovalent vaccines. There was no significant difference in neutralizing antibody titer between the combined vaccine and the monovalent vaccine; there was no immunosuppression and immune interference between the two vaccines after combined immunization; the broad-spectrum neutralizing antibody of the combined vaccine was determined for the first time. Indicators. In this study, one-day-old ICR suckling mice were selected to establish EV71 suckling mice model and CVA16 suckling mice model, and the protective level of monovalent vaccine and combined vaccine was evaluated. The results showed that both monovalent vaccine and combined vaccine could provide a good protective level for neonatal suckling mice, and combined vaccine could prevent two kinds of vaccine. Invasion of the virus. Part II: Study on the antigen and antibody ED_ (50) of EV71 VLPs. This part, in cooperation with the China Institute of Food and Drug Control (NIFDC), evaluates the ED_ (50) of the purified EV71 VLPs antigen and antibody. Different doses of EV71 VLPs were immunized with aluminum adjuvant respectively, and the neutralizing antibody showed a significant dose. According to the protective effect of suckling mice, according to the survival rate of neonatal mice after intracerebral injection of different immune doses, the ED_ (50) of EV71 VLPs antigen was calculated to be 0.20 ug/dose. According to the titers of neutralizing antibodies at different immune doses at different time points, the titers of neutralizing antibodies induced at this dose were calculated to be about 50. In summary, the expression of EV71 VLPs and CVA116 VLPs by baculovirus system in insect cells and the neutralizing antibody titer trend of monovalent vaccine and combined vaccine were evaluated by using pseudovirus neutralizing antibody detection system. The broad-spectrum neutralizing antibody titers of the monovalent vaccine and the combined vaccine were evaluated by the pseudovirus genotype, which showed that there was no significant difference between the neutralizing antibody titers of the combined vaccine and the monovalent vaccine in the same genotype of pseudovirus, and there was no immunosuppression and immune interference in the combined vaccine. The validity of monovalent vaccine and combined vaccine was evaluated internally. The results showed that the combined vaccine could prevent the attack of both viruses simultaneously. The determination of broad-spectrum neutralizing antibody and the evaluation of suckling mice'protective power provided real and reliable data for the follow-up study and clinical application of the combined vaccine. The ED_ (50) of the purified antigen and antibody of EV71 VLPs was studied, which provided experimental basis for further study of EV71 VLPs vaccine.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R392
本文編號(hào)：2177899
[Abstract]:Hand, Foot and Mouth Disease (HFMD) is a worldwide infectious disease, and the epidemic areas are mainly concentrated in developing countries (especially in the Asia-Pacific region). The results of epidemiological survey in China show that HFMD is the most common type of infectious diseases, and the main pathogens are Enterovirus 71 (EV71) and Coxsackievirus. At present, there is no effective drug to treat HFMD. The development and vaccination of vaccine is an important means to control the epidemic of HFMD. According to the results of clinical trials in mainland China, the inactivated vaccine of EV71 has a good protective effect and can prevent the HFMD caused by EV71. The protective rate against EV71-related diseases is above 90%, and the protective rate against EV71-related diseases is above 80%. The induced neutralizing antibodies have good broad-spectrum cross-reactivity, but their serum can not neutralize CVA 16 and other viruses causing HFMD. Therefore, the development of a combined vaccine containing EV71 and CVA 16 is particularly urgent. EV71 and CV16 have only one serotype, but each virus contains multiple genotypes, and the neutralizing antibody cross-reactivity among the genotypes is different. Therefore, the vaccine strains with high coverage and good immunogenicity were screened and prepared to be linked. Preventing hand-foot-mouth disease (HFMD) caused by EV71 and CV16 with a combined vaccine is the first choice for vaccine development. Current studies have only examined the broad-spectrum neutralizing antibody cross-activity of monovalent vaccines, but there is no report on the difference in neutralizing antibody cross-activity between the combined vaccines. Whether there is immunity between the two vaccines when the combined vaccines are vaccinated simultaneously has not been reported. There is no detailed and comprehensive study on inhibition and immune interference, and whether combined vaccination can prevent the invasion of two viruses. HFMD vaccines mainly consist of inactivated vaccines and virus-like particles (VLPs) vaccines. With the development of genetic engineering technology, VLPs vaccines have been widely used for their unique natural advantages. Aiming at the above problems, this paper carries out a more in-depth, specific and comprehensive study to provide real and reliable animal test data, expecting to solve the existing problems of the combined vaccine and develop an effective, safe and cross-active EV71 VLPs and CVA16 VLPs combined vaccine. On the other hand, the antigen dose-response relationship (ED_ (50)) of the vaccine directly reflects. EV71 inactivated vaccine has been evaluated for ED_ (50) of antigen before clinic, but there is no detailed and reliable data for ED_ (50) of EV71 VLPs vaccine. This paper also studies this problem: application chromatography purity. The ED_ (50) of EV71 VLPs antigen and the ED_ (50) of its antibody were determined. The determination of antigen and antibody ED_ (50) is of great significance for the follow-up study of EV71 VLPs vaccine. Part I: Monovalent vaccine and combined vaccine immunization. First, according to the epidemiological investigation in mainland China, we selected EV71 strain C4a (EU703814.1) and CVA16 strain B1a (AF177911.1) to produce EV71 virus-like particles (VLPs), CVA16 virus-like particles (VLPs) by using insect cell baculovirus expression system. The positive control vaccine was EV71 inactivated vaccine and CVA16 inactivated vaccine. The results showed that both monovalent and combined vaccines could induce strong neutralizing antibody titers, and VLPs monovalent vaccines could neutralize antibody titers compared with inactivated monovalent vaccines. There was no significant difference in neutralizing antibody titer between the combined vaccine and the monovalent vaccine; there was no immunosuppression and immune interference between the two vaccines after combined immunization; the broad-spectrum neutralizing antibody of the combined vaccine was determined for the first time. Indicators. In this study, one-day-old ICR suckling mice were selected to establish EV71 suckling mice model and CVA16 suckling mice model, and the protective level of monovalent vaccine and combined vaccine was evaluated. The results showed that both monovalent vaccine and combined vaccine could provide a good protective level for neonatal suckling mice, and combined vaccine could prevent two kinds of vaccine. Invasion of the virus. Part II: Study on the antigen and antibody ED_ (50) of EV71 VLPs. This part, in cooperation with the China Institute of Food and Drug Control (NIFDC), evaluates the ED_ (50) of the purified EV71 VLPs antigen and antibody. Different doses of EV71 VLPs were immunized with aluminum adjuvant respectively, and the neutralizing antibody showed a significant dose. According to the protective effect of suckling mice, according to the survival rate of neonatal mice after intracerebral injection of different immune doses, the ED_ (50) of EV71 VLPs antigen was calculated to be 0.20 ug/dose. According to the titers of neutralizing antibodies at different immune doses at different time points, the titers of neutralizing antibodies induced at this dose were calculated to be about 50. In summary, the expression of EV71 VLPs and CVA116 VLPs by baculovirus system in insect cells and the neutralizing antibody titer trend of monovalent vaccine and combined vaccine were evaluated by using pseudovirus neutralizing antibody detection system. The broad-spectrum neutralizing antibody titers of the monovalent vaccine and the combined vaccine were evaluated by the pseudovirus genotype, which showed that there was no significant difference between the neutralizing antibody titers of the combined vaccine and the monovalent vaccine in the same genotype of pseudovirus, and there was no immunosuppression and immune interference in the combined vaccine. The validity of monovalent vaccine and combined vaccine was evaluated internally. The results showed that the combined vaccine could prevent the attack of both viruses simultaneously. The determination of broad-spectrum neutralizing antibody and the evaluation of suckling mice'protective power provided real and reliable data for the follow-up study and clinical application of the combined vaccine. The ED_ (50) of the purified antigen and antibody of EV71 VLPs was studied, which provided experimental basis for further study of EV71 VLPs vaccine.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前5條
1 高帆;姚昕;毛群穎;梁爭(zhēng)論;;新型腸道病毒71型全病毒滅活疫苗質(zhì)量控制研究[J];中國(guó)病毒病雜志;2015年02期
2 Kaw Bing Chua;Abdul Rasid Kasri;;Hand Foot and Mouth Disease Due to Enterovirus 71 in Malaysia[J];Virologica Sinica;2011年04期
3 葛菁萍;高冬妮;陳娜;樓莊偉;平文祥;;桿狀病毒——高效蛋白表達(dá)載體[J];中國(guó)預(yù)防獸醫(yī)學(xué)報(bào);2009年10期
4 楊朝輝;尹少甫;張宗久;許文波;;柯薩奇病毒A組16型[J];中國(guó)疫苗和免疫;2009年01期
5 ;Expression,purification and characterization of enterovirus-71 virus-like particles[J];World Journal of Gastroenterology;2006年06期
，本文編號(hào)：2177899
本文鏈接：http://sikaile.net/yixuelunwen/jichuyixue/2177899.html
最近更新
教材專(zhuān)著
