本文選題：狂犬病病毒 + 馴化　； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2012年博士論文

【摘要】：在我國，每年被犬咬傷的人數(shù)多達1千萬以上，并因此導(dǎo)致至少2000～3000人死于狂犬病。國產(chǎn)滅活疫苗發(fā)展緩慢是我國人狂犬病持續(xù)多發(fā)的原因之一。我國犬的狂犬病疫苗產(chǎn)品供應(yīng)多年來一直依賴少量進口滅活疫苗和國產(chǎn)弱毒活疫苗。目前，進口疫苗供應(yīng)量只能占總需求量的1～3%，弱毒疫苗由于安全性問題在發(fā)達國家早已不再用于家畜動物免疫，少數(shù)幾家國產(chǎn)滅活疫苗產(chǎn)品雖然在近幾年已完成注冊，但由于效力和成本等各種原因遲遲未能上市或剛剛啟動生產(chǎn)，而且產(chǎn)能有限，其數(shù)量和質(zhì)量遠不能保證有效免疫的高覆蓋率。因此，提高細胞培養(yǎng)狂犬病病毒的產(chǎn)毒滴度和研制高效疫苗佐劑，是我國動物狂犬病滅活疫苗研究的兩個主要方向，也是解決疫苗效力和生產(chǎn)成本問題的重要途徑。 針對上述問題，本研究開展了以下試驗： 1.我國狂犬病病毒流行毒株的細胞馴化。試驗內(nèi)容：①毒株的細胞培養(yǎng)。對分離自我國江西地區(qū)的一株鼬獾狂犬病病毒JX08-45株在BHK-21細胞上進行連續(xù)傳代和培養(yǎng)滴度（TCID50）測定。②病毒細胞適應(yīng)株的致病性檢測。以腦內(nèi)和肌肉注射途徑，分別在犬和小鼠進行病毒致病性檢測。③全基因組測序分析。對病毒適應(yīng)細胞培養(yǎng)前后的全基因組核苷酸和氨基酸變化進行分析，并與其他疫苗株和流行株進行比對，從而確定影響病毒細胞適性的關(guān)鍵氨基酸位點。④免疫原性檢測。通過小鼠免疫試驗，對該細胞適應(yīng)株和其他疫苗株的滅活后免疫原性進行比較，評價本毒株的免疫原性。 2.狂犬病疫苗佐劑的研制。試驗內(nèi)容：①新型佐劑和免疫增強劑的篩選。進行一系列佐劑的制備，利用小鼠免疫試驗和狂犬病中和抗體滴度測定，對佐劑的免疫增強作用進行檢測，篩選出適用于狂犬病滅活疫苗的高效佐劑。②佐劑的作用機制研究。通過淋巴細胞增殖試驗、血液Th/Tc細胞亞群鑒定和細胞因子檢測，對佐劑誘導(dǎo)的免疫類型及增強抗體反應(yīng)的機制進行研究。③佐劑的安全性評價。通過超劑量接種，檢測小鼠的生長發(fā)育和生育能力變化，以及內(nèi)臟組織的病理學(xué)變化。 3.高效佐劑狂犬病疫苗的研制。試驗內(nèi)容：①疫苗的制備。通過在小鼠和犬的免疫效果評價，確定病毒的最佳培養(yǎng)工藝和佐劑/抗原的最佳配比，并進行三批實驗室產(chǎn)品的制備。②疫苗的安全性評價。通過在犬的超劑量免疫，檢測疫苗對犬內(nèi)臟組織、體溫、行為和飲食的影響。③與國內(nèi)同類疫苗產(chǎn)品免疫效果的比較。通過在犬的免疫試驗，對國內(nèi)使用的進口疫苗和本疫苗誘導(dǎo)的體液免疫水平進行檢測和比較。④疫苗效力檢測方法的建立。利用狂犬病中和抗體標(biāo)準(zhǔn)檢測方法（FAVN法），建立適用于本疫苗效力檢驗的小鼠血清學(xué)疫苗效力測定方法，并對其檢測的準(zhǔn)確性和可重復(fù)性進行驗證。 通過以上試驗，獲得以下結(jié)果： 1.細胞馴化結(jié)果顯示，病毒在BHK-21傳至120代時增殖滴度能達到108.0TCID50/mL，并將其重新命名為JX08-45CC株。JX08-45CC株腦內(nèi)接種可使小鼠100%致死，外周攻毒僅能使部分小鼠死亡，但外周接種對犬不致病。JX08-45CC適應(yīng)細胞后基因組中共發(fā)生了17個核苷酸位點的突變和7個氨基酸突變，其中6個氨基酸突變點在G蛋白，但并未改變其中和抗原表位和主要的毒力相關(guān)位點。6個G蛋白氨基酸突變點中，有4個位點與其他疫苗株序列一致，其中2個位點與流行株序列不同。JX08-45CC株滅活后的免疫原性與國內(nèi)使用的疫苗株相比，在相同病毒滴度下不低于現(xiàn)有疫苗株。 2.免疫佐劑的制備研究結(jié)果顯示，以水包油型佐劑和黃芪多糖為佐劑的疫苗，能誘導(dǎo)產(chǎn)生顯著高于普通氫氧化鋁佐劑疫苗的狂犬病中和抗體，而且多糖能加速疫苗誘導(dǎo)的體液免疫應(yīng)答。佐劑接種對小鼠發(fā)育和生育能力無明顯影響，對小鼠主要內(nèi)臟未造成損傷。水包油型佐劑和黃芪多糖均可刺激淋巴細胞明顯增殖，能誘導(dǎo)血液中Th1/Tc1細胞亞群的極化和多種細胞因子（IL-1β、IL-5、IL-6、MCP-1、GM-CSF和IFN-α）水平上調(diào)，，主要通過誘導(dǎo)細胞免疫增強機體的抗體反應(yīng)水平。 3.高效佐劑疫苗研究結(jié)果顯示，病毒的轉(zhuǎn)瓶培養(yǎng)條件：5%同步接種，37℃培養(yǎng)4h，34℃培養(yǎng)7～8天；并確定疫苗配制體積比（佐劑:病毒液）為1:4，黃芪多糖在疫苗的工作濃度為10mg/mL。根據(jù)以上確定的疫苗配制方案，完成了3批實驗室狂犬病滅活疫苗（JX08-45CC株）制備（批號：201001Vac、201002Vac和201003Vac），疫苗效力分別為3.15IU/mL、3.10IU/mL和3.05IU/mL。本疫苗對犬生長發(fā)育和主要臟器均無明顯影響。本疫苗在犬誘導(dǎo)的抗體水平總體高于目前國內(nèi)使用的進口疫苗，對攻毒犬具有完全保護能力。根據(jù)疫苗生產(chǎn)需要，建立了一種快速、簡便的半定量檢測疫苗效力的小鼠血清學(xué)方法，具有周期短、成本低、重復(fù)性好的特點，適用于生產(chǎn)中疫苗效力檢驗。 通過以上結(jié)果，得出以下結(jié)論： 1.獲得一株適應(yīng)BHK-21細胞高滴度培養(yǎng)的狂犬病病毒JX08-45CC株，其致病性和基因組序列變化等背景清楚，免疫原性強，具備疫苗候選株的基本條件。 2.制備了兩種用于狂犬病滅活疫苗的佐劑，闡明了佐劑通過誘導(dǎo)細胞免疫增強體液免疫的基本作用機制，并證明其免疫增強作用顯著、安全性良好，制備工藝簡單，適用于獸用狂犬病疫苗生產(chǎn)。 3.利用馴化毒株和兩種佐劑研制了狂犬病滅活疫苗（JX08-45CC株），其制備工藝簡單、效力檢驗方法簡便、生產(chǎn)成本低廉、安全性良好、免疫效力較高，符合我國犬狂犬病免疫的現(xiàn)實需求。 論文創(chuàng)新點： 1.為我國獸用疫苗生產(chǎn)獲得了第一個具有自主知識產(chǎn)權(quán)、培養(yǎng)滴度高、免疫原性好的狂犬病疫苗候選株。 2.研制出用于狂犬病滅活疫苗的新型佐劑，闡明了其基本作用機制，并證明具有顯著免疫增強作用，適用于狂犬病滅活疫苗生產(chǎn)。 3.利用自主馴化的毒株和制備的佐劑，研制出狂犬病滅活疫苗（JX08-45CC株），免疫效果總體好于目前國內(nèi)市售疫苗。
[Abstract]:In China, the number of people bitten by dogs is more than 10 million in our country each year, and at least 2000~3000 people die of rabies. The slow development of domestic inactivated vaccine is one of the reasons for the continuous occurrence of human rabies in China. Before, the supply of imported vaccines could only account for 1 to 3% of the total demand, and the vaccine was no longer used for livestock and animal immunity in developed countries because of the safety problem in the developed countries. A few domestic inactivated vaccine products have been registered in recent years. It is limited, its quantity and quality can not guarantee the high coverage of effective immunization. Therefore, it is the two main direction for the study of rabies inactivated vaccine in China, and it is also an important way to solve the problem of vaccine effectiveness and production cost.
In view of the above problems, the following experiments were carried out in this study.
1. the cell acclimatization of the rabies virus strain of our country. Test contents: (1) the cell culture of the virus strain. The continuous generation and culture titer (TCID50) of a ferret badger rabies virus JX08-45 strain from the isolated Jiangxi region of China were measured on the BHK-21 cells. Detection of viral pathogenicity in dogs and mice. (3) whole genome sequencing analysis. Analysis of all genome nucleotide and amino acid changes before and after the virus adaptive cell culture, and comparison with other vaccine strains and epidemic strains to determine the key amino acid loci that affect the suitability of the virus cells. (4) immunogenicity detection. The immunogenicity of the cell adapted strain and other vaccine strains was compared by immunization test in mice, and the immunogenicity of the strain was evaluated.
2. development of the adjuvant of rabies vaccine. Test contents: (1) screening of a new adjuvant and immune enhancer. Preparation of a series of adjuvant, using mice immune test and test of antibody titer of rabies neutralization antibody, detecting the immune enhancement of the adjuvant, screening out the effective adjuvant for inactivated canine inactivated vaccine. Mechanism study. Through lymphocyte proliferation test, blood Th/Tc cell subgroup identification and cytokine detection, study on the immune type induced by adjuvant and the mechanism of enhancing antibody response. 3. Safety evaluation of adjuvant. Through super dose inoculation, the growth and fertility changes in mice and the pathology of visceral tissue are detected. Change.
3. the preparation of a highly effective adjuvant rabies vaccine. Test content: (1) preparation of the vaccine. By evaluating the immune effect of mice and dogs, the optimum culture technology of the virus and the optimum ratio of adjuvant / antigen were determined, and the preparation of three batch of laboratory products. The effects of visceral tissue, body temperature, behavior and diet. (3) comparison with the immunization effect of domestic similar vaccine products. Test and compare the level of humoral immunity induced by imported and present vaccines in China by the immunization test in dogs. (4) establishment of the method for testing the efficacy of vaccines. FAVN method) to establish a method for determining the potency of the serological vaccine suitable for the efficacy test of the vaccine, and to verify the accuracy and repeatability of the test.
Through the above experiments, the following results are obtained:
The results of 1. cell acclimation showed that the virus could reach 108.0TCID50/mL when the virus spread from BHK-21 to 120 generation, and renamed it to JX08-45CC strain.JX08-45CC strain, which could kill 100% of the mice. The peripheral inoculation only could cause some mice to die, but the peripheral inoculation of the canine after the non pathogenic.JX08-45CC adaptive cell genome occurred 17. A mutation at the nucleotide site and a 7 amino acid mutation, of which 6 amino acids mutated in G protein, but did not change the amino acid mutation points of the antigen epitopes and the major virulence related loci.6 G protein amino acids, 4 loci were consistent with the other vaccine strains, of which 2 sites were inactivated by different.JX08-45CC strains from the epidemic strain. Compared with the domestic vaccine strains, the immunogenicity is lower than that of the existing vaccine strains under the same virus titer.
2. the results of the preparation of the immuno adjuvant showed that the vaccine with the oil and astragalus polysaccharide as adjuvant could induce the rabies neutralization antibody which was significantly higher than the ordinary aluminum hydroxide adjuvant vaccine, and the polysaccharide could accelerate the humoral immune response induced by the vaccine. The adjuvant inoculation had no obvious effect on the development and fertility of mice, and the effect of the adjuvant inoculation was small. The main viscera of rats did not cause injury. The water bag oil adjuvant and astragalus polysaccharides could stimulate the proliferation of lymphocytes. It could induce the polarization of Th1/Tc1 cell subsets in the blood and the up regulation of various cytokines (IL-1 beta, IL-5, IL-6, MCP-1, GM-CSF and IFN- alpha), mainly by inducing cell immunity to enhance the level of antibody response.
3. the results of the efficient adjuvant vaccine study showed that the culture conditions of the virus were: 5% synchronous inoculation, 37 C for 4h, 34 C for 7~8 days; and the vaccine preparation volume ratio (adjuvant: virus liquid) was 1:4, and the concentration of Astragalus Polysaccharide in the vaccine was 10mg/mL. based on the vaccine preparation scheme above, and the laboratory rabies inactivation was completed. The vaccine (JX08-45CC strain) was prepared (batch number: 201001Vac, 201002Vac and 201003Vac). The vaccine efficacy was 3.15IU/mL, 3.10IU/mL and 3.05IU/mL., respectively. The vaccine had no obvious effect on the growth and development of the dog and the main organs. The antibody level induced in the dog was higher than the imported vaccine in the country. According to the needs of vaccine production, a quick, simple and semi quantitative serological method for detecting the potency of the vaccine is established. It has the characteristics of short cycle, low cost and good reproducibility, which is suitable for the test of vaccine efficacy in production.
Through the above results, the following conclusions are drawn.
1. a strain of rabies virus (JX08-45CC strain) adapted to the high titer of BHK-21 cells was obtained. The pathogenicity and genome sequence changes were clear, the immunogenicity was strong, and the basic conditions of the vaccine candidate strains were obtained.
2. two kinds of adjuvant used for rabies inactivated vaccine were prepared, and the basic mechanism of adjuvant by inducing cell immunity to enhance humoral immunity was clarified, and it was proved that the immune enhancement was remarkable, the safety was good, the preparation process was simple, and it was suitable for the production of animal rabies vaccine.
3. the rabies inactivated vaccine (JX08-45CC strain) was prepared by the acclimated strain and two adjuvant. The preparation process was simple, the method of efficacy test was simple, the production cost was low, the safety was good, and the immune effect was high, which accords with the actual demand of rabies immunity in our country.
Innovation points of the paper:
1. the first rabies vaccine candidate with independent intellectual property rights, high titer and good immunogenicity was obtained for veterinary vaccine production in China.
2. a new adjuvant used for rabies inactivated vaccine was developed, and its basic mechanism was clarified. It proved that it had significant immune enhancement and was suitable for the production of rabies inactivated vaccine.
3. the rabies inactivated vaccine (JX08-45CC strain) was developed by using self domesticated strains and prepared adjuvants. The immunization effect is generally better than the domestic vaccines currently sold.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R392

【參考文獻】

相關(guān)期刊論文 前1條

1 張守峰;曹亮;張菲;李海濤;李清竹;扈榮良;;狂犬病毒核蛋白單抗和熒光抗體中和試驗的建立與應(yīng)用[J];中華檢驗醫(yī)學(xué)雜志;2006年06期

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