我国狂犬病流行毒株驯化及高效佐剂疫苗研究

发布时间：2018-05-24 22:33

本文选题：狂犬病病毒 + 驯化　；参考：《中国人民解放军军事医学科学院》2012年博士论文

【摘要】：在我国，每年被犬咬伤的人数多达1千万以上，并因此导致至少2000～3000人死于狂犬病。国产灭活疫苗发展缓慢是我国人狂犬病持续多发的原因之一。我国犬的狂犬病疫苗产品供应多年来一直依赖少量进口灭活疫苗和国产弱毒活疫苗。目前，进口疫苗供应量只能占总需求量的1～3%，弱毒疫苗由于安全性问题在发达国家早已不再用于家畜动物免疫，少数几家国产灭活疫苗产品虽然在近几年已完成注册，但由于效力和成本等各种原因迟迟未能上市或刚刚启动生产，而且产能有限，其数量和质量远不能保证有效免疫的高覆盖率。因此，提高细胞培养狂犬病病毒的产毒滴度和研制高效疫苗佐剂，是我国动物狂犬病灭活疫苗研究的两个主要方向，也是解决疫苗效力和生产成本问题的重要途径。针对上述问题，本研究开展了以下试验： 1.我国狂犬病病毒流行毒株的细胞驯化。试验内容：①毒株的细胞培养。对分离自我国江西地区的一株鼬獾狂犬病病毒JX08-45株在BHK-21细胞上进行连续传代和培养滴度（TCID50）测定。②病毒细胞适应株的致病性检测。以脑内和肌肉注射途径，分别在犬和小鼠进行病毒致病性检测。③全基因组测序分析。对病毒适应细胞培养前后的全基因组核苷酸和氨基酸变化进行分析，并与其他疫苗株和流行株进行比对，从而确定影响病毒细胞适性的关键氨基酸位点。④免疫原性检测。通过小鼠免疫试验，对该细胞适应株和其他疫苗株的灭活后免疫原性进行比较，评价本毒株的免疫原性。 2.狂犬病疫苗佐剂的研制。试验内容：①新型佐剂和免疫增强剂的筛选。进行一系列佐剂的制备，利用小鼠免疫试验和狂犬病中和抗体滴度测定，对佐剂的免疫增强作用进行检测，筛选出适用于狂犬病灭活疫苗的高效佐剂。②佐剂的作用机制研究。通过淋巴细胞增殖试验、血液Th/Tc细胞亚群鉴定和细胞因子检测，对佐剂诱导的免疫类型及增强抗体反应的机制进行研究。③佐剂的安全性评价。通过超剂量接种，检测小鼠的生长发育和生育能力变化，以及内脏组织的病理学变化。 3.高效佐剂狂犬病疫苗的研制。试验内容：①疫苗的制备。通过在小鼠和犬的免疫效果评价，确定病毒的最佳培养工艺和佐剂/抗原的最佳配比，并进行三批实验室产品的制备。②疫苗的安全性评价。通过在犬的超剂量免疫，检测疫苗对犬内脏组织、体温、行为和饮食的影响。③与国内同类疫苗产品免疫效果的比较。通过在犬的免疫试验，对国内使用的进口疫苗和本疫苗诱导的体液免疫水平进行检测和比较。④疫苗效力检测方法的建立。利用狂犬病中和抗体标准检测方法（FAVN法），建立适用于本疫苗效力检验的小鼠血清学疫苗效力测定方法，并对其检测的准确性和可重复性进行验证。通过以上试验，获得以下结果： 1.细胞驯化结果显示，病毒在BHK-21传至120代时增殖滴度能达到108.0TCID50/mL，并将其重新命名为JX08-45CC株。JX08-45CC株脑内接种可使小鼠100%致死，外周攻毒仅能使部分小鼠死亡，但外周接种对犬不致病。JX08-45CC适应细胞后基因组中共发生了17个核苷酸位点的突变和7个氨基酸突变，其中6个氨基酸突变点在G蛋白，但并未改变其中和抗原表位和主要的毒力相关位点。6个G蛋白氨基酸突变点中，有4个位点与其他疫苗株序列一致，其中2个位点与流行株序列不同。JX08-45CC株灭活后的免疫原性与国内使用的疫苗株相比，在相同病毒滴度下不低于现有疫苗株。 2.免疫佐剂的制备研究结果显示，以水包油型佐剂和黄芪多糖为佐剂的疫苗，能诱导产生显著高于普通氢氧化铝佐剂疫苗的狂犬病中和抗体，而且多糖能加速疫苗诱导的体液免疫应答。佐剂接种对小鼠发育和生育能力无明显影响，对小鼠主要内脏未造成损伤。水包油型佐剂和黄芪多糖均可刺激淋巴细胞明显增殖，能诱导血液中Th1/Tc1细胞亚群的极化和多种细胞因子（IL-1β、IL-5、IL-6、MCP-1、GM-CSF和IFN-α）水平上调，，主要通过诱导细胞免疫增强机体的抗体反应水平。 3.高效佐剂疫苗研究结果显示，病毒的转瓶培养条件：5%同步接种，37℃培养4h，34℃培养7～8天；并确定疫苗配制体积比（佐剂:病毒液）为1:4，黄芪多糖在疫苗的工作浓度为10mg/mL。根据以上确定的疫苗配制方案，完成了3批实验室狂犬病灭活疫苗（JX08-45CC株）制备（批号：201001Vac、201002Vac和201003Vac），疫苗效力分别为3.15IU/mL、3.10IU/mL和3.05IU/mL。本疫苗对犬生长发育和主要脏器均无明显影响。本疫苗在犬诱导的抗体水平总体高于目前国内使用的进口疫苗，对攻毒犬具有完全保护能力。根据疫苗生产需要，建立了一种快速、简便的半定量检测疫苗效力的小鼠血清学方法，具有周期短、成本低、重复性好的特点，适用于生产中疫苗效力检验。通过以上结果，得出以下结论： 1.获得一株适应BHK-21细胞高滴度培养的狂犬病病毒JX08-45CC株，其致病性和基因组序列变化等背景清楚，免疫原性强，具备疫苗候选株的基本条件。 2.制备了两种用于狂犬病灭活疫苗的佐剂，阐明了佐剂通过诱导细胞免疫增强体液免疫的基本作用机制，并证明其免疫增强作用显著、安全性良好，制备工艺简单，适用于兽用狂犬病疫苗生产。 3.利用驯化毒株和两种佐剂研制了狂犬病灭活疫苗（JX08-45CC株），其制备工艺简单、效力检验方法简便、生产成本低廉、安全性良好、免疫效力较高，符合我国犬狂犬病免疫的现实需求。论文创新点： 1.为我国兽用疫苗生产获得了第一个具有自主知识产权、培养滴度高、免疫原性好的狂犬病疫苗候选株。 2.研制出用于狂犬病灭活疫苗的新型佐剂，阐明了其基本作用机制，并证明具有显著免疫增强作用，适用于狂犬病灭活疫苗生产。 3.利用自主驯化的毒株和制备的佐剂，研制出狂犬病灭活疫苗（JX08-45CC株），免疫效果总体好于目前国内市售疫苗。
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

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