灭活轮状病毒与灭活甲肝病毒联合疫苗的制备及免疫学效果评价

发布时间：2018-01-17 23:02

本文关键词：灭活轮状病毒与灭活甲肝病毒联合疫苗的制备及免疫学效果评价　出处：《昆明医学院》2011年硕士论文　论文类型：学位论文

【摘要】：使用联合疫苗早在上世纪八十年代末,世界卫生组织(WHO)在制定儿童疫苗免疫计划(CVI)就提出能否用20-30年时间研制出更多的一次免疫预防多种疾病的疫苗。因此,研制联合疫苗不仅是目前也是未来疫苗研发的方向,联合疫苗不仅解决受接种者的经济负担,也减轻了精神负担。目前已有一些联合疫苗用于我国的免疫规划,包括百白破,麻腮风,甲乙肝疫苗等,但至今我国还没有以轮状病毒为基础的联合疫苗。人轮状病毒(human rotavirus, HRV)是世界范围内引起5岁以内婴幼儿严重腹泻最主要的病原体,HRV感染是造成婴幼儿腹泻死亡的主要原因。至今每年约有60万婴儿死于RV感染引起的腹泻。目前的减毒RV活疫苗在预防RV感染中发挥了一定作用,但疫苗引起的副反应如外源因子污染及可能发生毒力返祖的问题,尚未完全得到解决。世界卫生组织(world health organization, WHO)正积极推动不同形式轮状病毒疫苗的研究与开发。灭活RV也是WHO提倡研制的RV疫苗之一。为开展联合疫苗的研制,我们已完成了实验室规模灭活RV疫苗的制备,为联合疫苗的研制提供了条件。病毒性肝炎是由多种肝炎病毒引起的以肝脏损害为主的全身性急性或持续性传染病,在世界各地均有发病流行。在肝炎患者中30-50%属于甲型肝炎。甲肝的流行,其发生与社会经济的发达程度,环境卫生水平密切相关。发达国家感染率达到50-1000/10万人,发展中国家为100-300/10万人。我国大部分地区属于甲肝高流行区。由于甲肝疫苗的使用,我国甲肝的流行与发病率已大幅下降。其中灭活甲肝疫苗发挥了重要的作用。如果能将两种或两种以上的有效疫苗配制成联合疫苗将减轻受种者的经济和精神负担。基于这一考虑,本研究认为IRV和IHAV疫苗理化性质相同,免疫对象相同,免疫程序基本一致。因此,进行了灭活轮状病毒与灭活甲肝病毒联合疫苗的制备及免疫学效果评价。实验第一部分：联合疫苗的制备。实验方法：规模化培养的P[2]G3轮状病毒收获液,经离心、微滤、超滤和层析等步骤纯化,获得结构完整,呈典型的轮状病毒特征,具有感染性,基因组未发生突变的病毒液,加入终浓度0.00925%甲醛进行灭活,先在56℃加热30min,然后在37℃持续孵育至72h,经盲传三代证明没有残余毒力,病毒被完全灭活。经抗原定量检测,再将灭活的P[2]G3轮状病毒与氢氧化铝佐剂吸附,与成品甲肝试剂混合制备成联合疫苗。实验第二部分：免疫学效果评价。实验方法：将小鼠分为空白组,对照组,实验组。然后在小鼠上进行联合疫苗的免疫效果评价。联合疫苗经肌肉注射,每次免疫间隔两周,共免疫3次。自第1次免疫后两周检测小鼠血清中抗RV特异性抗体和粪便中的分泌性抗体,分析免疫后CD4+T淋巴细胞和CD8+T淋巴细胞的变化情况。实验结果：初次免疫后第2周,小鼠全部抗RV抗体阳转；二次免疫后第4周时,检测到抗HAV抗体；在第3次免疫后第6周时,抗体水平到达高峰小鼠血清抗RV酶标抗体滴度达80865.76±1953.97,抗RV分泌性抗体滴度为4.54±0.58,抗RV中和抗体效价为10240,抗甲肝抗体效价为28.50876±6.53。细胞免疫反应CD4+T达到14.52±3.83,CD8+T达到3.9±0.72。经t检验,P0.05。以上实验结果表明,灭活轮状病毒与灭活甲肝病毒联合疫苗可诱导小鼠产生特异性抗RV体液免疫反应和抗HAV体液免疫反应,也能产生明显的细胞免疫反应。这项研究为研制IRV和IHAV联合疫苗提供了一些科学依据。
[Abstract]:The use of combined vaccine as early as in the last century at the end of 80s, WHO (WHO) in the development of vaccine immunization for children plan (CVI) proposed to 20-30 years in developing a vaccine immune prevention of various diseases more. Therefore, development of combined vaccine is not only the present also is the future direction of vaccine development, not only solved by combined vaccine with the economic burden, but also reduce the mental burden. There are some combined vaccine for China's immunization program, including DPT, MMR, a hepatitis B vaccine, but so far our country has not combined vaccine with rotavirus based.
Human rotavirus (human rotavirus HRV) is less than 5 year of severe diarrhea in infants and young children the main pathogen worldwide, HRV infection is the main cause of infantile diarrhea death. Every year about 600 thousand babies died of diarrhea caused by RV infection. The attenuated live vaccine RV play a role in the prevention of RV the infection, but the side effects caused by exogenous factors such as vaccine contamination and possible virulence reversion problems have not been completely solved. The WHO (World Health Organization, WHO) is actively promoting the research and development of different forms of rotavirus vaccine. Inactivated RV vaccine is one of the RV WHO to promote the development. For the development of joint the vaccine, we have completed the laboratory scale preparation of inactivated RV vaccine, provided the conditions for the development of vaccine.
Viral hepatitis is caused by a variety of viral hepatitis with liver damage mainly systemic acute or persistent infectious disease incidence in the popular all over the world. In patients with hepatitis 30-50% hepatitis. Hepatitis A epidemic belongs to, the development degree of social economy, is closely related to the level of environmental sanitation. The infection rate of developed countries 50-1000/10 million, 100-300/10 million people in developing countries. Most areas of our country belong to a high endemic area. Due to the use of hepatitis A vaccine, epidemic and the incidence of hepatitis A in China have dropped significantly. The inactivated hepatitis A vaccine has played an important role.
If two or more than two kinds of effective vaccine prepared combined vaccine will reduce the sort of economic and mental burden. Based on this consideration, this study shows that the IRV IHAV vaccine and physicochemical properties of the same object are the same, the immune, immune procedure is basically the same. Therefore, the preparation and evaluation of immune effect of inactivated live rotavirus and inactivated hepatitis A virus vaccine.
The first part of the experiment: the preparation of the combined vaccine.
Methods: P[2]G3 rotavirus harvest scale culture liquid by centrifugation, microfiltration, ultrafiltration and chromatography purification step, obtain the complete structure, is the typical characteristics of rotavirus, infectious virus genome, without liquid mutations, with a final concentration of 0.00925% formaldehyde inactivated, first at 56 C 30min heating at 37 DEG C, and then continue to 72h after incubation, three blind passages prove that there is no residual virulence, virus was completely inactivated. The quantitative detection of antigen, then inactivated rotavirus P[2]G3 and aluminum hydroxide adjuvant adsorption, and the product prepared by mixed reagent of hepatitis A vaccine.
The second part of the experiment: the evaluation of immunological effect.
Method: the experimental mice were divided into blank group, control group and experimental group. Then the evaluation of immune effect of vaccine in mice. The combined vaccine by intramuscular injection, each time interval of two weeks were immune, immune 3 times. Since the first two weeks after immunization were detected in serum anti RV antibodies and feces the secretion of antibodies, the changes of CD4+T lymphocyte and CD8+T lymphocyte immune analysis.
Results: second weeks after the first immunization, all mice anti RV antibody positive; two times at fourth weeks after immunization, anti HAV antibodies were detected in third; sixth weeks after immunization, the antibody level reached the peak of serum anti RV enzyme antibody titer was 80865.76 + 1953.97, RV antibody titers of anti secretory 4.54 + 0.58, neutralizing antibody titers of anti RV 10240, anti hav antibody titer of 6.53. cell immune reaction of CD4+T 28.50876 + to 14.52 + 3.83, CD8+T reaches 3.9 + 0.72. by t test, more than P0.05.. The experimental results show that the inactivated rotavirus and inactivated hepatitis A virus vaccine can induce specific anti RV anti HAV humoral immune response and humoral immune response, can also produce cellular immune reaction obviously. This study provides some scientific basis for the development of IRV and IHAV combined vaccine.

【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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