轮状病毒不同基因型间交叉反应研究

发布时间：2018-07-28 18:15

【摘要】：轮状病毒(Rotavirus, RV)是世界范围内导致婴幼儿严重腹泻的主要原因。每年大约有60万左右的婴幼儿因轮状病毒的感染重症发病而死亡,其中80%以上发生在亚洲南部、非洲南部等经济及卫生条件相对落后的发展中国家和地区,轮状病毒造成的胃肠炎在世界各国都有不同程度的经济与疾病负担。目前尚无治疗轮状病毒感染的特效药物,使用疫苗是预防和控制轮状病毒感染,降低重症死亡率的最经济有效的手段。轮状病毒基因组由11条片段组成,分别编码6个结构蛋白(VP1-VP4与VP6-VP7)和6个非结构蛋白(NSP1-NSP6)。到目前为止,根据结构蛋白VP6(组抗原)可以分为A-H 8个不同的组,其中引起婴幼儿感染的主要是A组轮状病毒。另外,依据结构蛋白VP7(G型抗原或糖蛋白)和VP4(P型抗原或蛋白水解酶敏感蛋白)间的基因差异分为27个G基因型(G1-G27)和35个P基因型(P[1]-P[35])。疫苗研发的型别选择主要是依据流行型而定。已有3种轮状病毒减毒活疫苗用于预防轮状病毒感染,分别是葛兰素史克公司生产的单价人轮状病毒疫苗Rotarix(基因型为G1P[8]),默克公司生产的五价人-牛重组轮状病毒疫苗RotaTeq(基因型为G1,G2, G3, G4, G6和P[8])以及由兰州生物制品研究所研发的单价轮状病毒口服活疫苗罗特威(基因型为G10P[12])。现有的轮状病毒疫苗有单价和多价,这两种疫苗在制备工艺及质控方面都有所不同,多价疫苗相对困难。因此,单型能覆盖多型的尽可能用单价疫苗。而问题在于能否用单价疫苗获得多价疫苗的效果,从目前研究的结果表明,尚不能确定其否。尽管最终的效果取决于人体免疫后的资料,但人体资料的获得周期长,评价体系复杂。因此,建立实验室评价方法对考虑上述问题具有重要的指导意见和前瞻性。本课题研究旨在对单价和多价轮状病毒免疫原的免疫原性进行比较,评价单价免疫原引起的交叉保护作用与多价免疫原间的差异。本课题研究选取三种不同基因型的轮状病毒标准株：Wa(基因型为G1P[8]), SA11(基因型为G3P[1])和Gottfried(基因型为G4P[6]),这三株轮状病毒标准株单独作为单价轮状病毒免疫原,将这三株轮状病毒标准株按照不同的比例进行混合制备成多价轮状病毒免疫原。实验动物ICR小鼠随机分组为8个组,分别是3个单价轮状病毒免疫原组(Wa组,SA11组,Gottfried组),3个二价轮状病毒免疫原组(Wa+SA11组,Wa+Gottfried组,SA11+Gottfried组),1个三价轮状病毒免疫原组(Wa+SA11+Gottfried组)以及1个对照组(PBS组),每组按照相同的总剂量免疫轮状病毒免疫原。免疫后利用ELISA,中和试验以及IgA ELISA试剂盒等方法分别对各组血清样本中的轮状病毒特异性IgG水平,轮状病毒特异性中和抗体滴度以及血清IgA浓度进行检测与评价,从而对单价和多价轮状病毒免疫原的免疫原性进行比较分析。实验结果显示,单价轮状病毒免疫原免疫后,不同基因型之间能产生低于亲本株的血清交叉中和抗体；二价轮状病毒免疫原诱导的免疫反应要强于单价轮状病毒免疫原,但在二价轮状病毒免疫原的三种组合中,对Wa株及SA11株的免疫原性强于Gottfried株；三价轮状病毒免疫原诱导的免疫反应略高于单价轮状病毒免疫原,而略低于二价轮状病毒免疫原,其中免疫原性仍以Wa株最强,SA11次之,Gottfried较低。通过以上实验结果得出结论,多价轮状病毒免疫原比单价轮状病毒免疫原能够诱导产生更多的血清轮状病毒特异性抗体,产生的血清IgA抗体也高于单价轮状病毒免疫原,而且,多价轮状病毒免疫原比单价轮状病毒免疫原能够诱导交叉免疫反应的速度更快。鉴于以上实验结果,目前还不能考虑用单价轮状病毒疫苗代替多价轮状病毒疫苗。
[Abstract]:Rotavirus (RV) is the main cause of severe diarrhoea in infants worldwide. About 600 thousand of infants and young children die from the severe incidence of rotavirus infection every year. More than 80% of them occur in southern Asia, South Africa and other developing countries and regions with relatively backward economic and health conditions, rotavirus At present, there are no special drugs for the treatment of rotavirus infection, and the use of vaccines is the most economical and effective means to prevent and control rotavirus infection and reduce severe mortality. The rotavirus genome consists of 11 fragments, which encode 6 structural proteins (VP 1-VP4 and VP6-VP7) and 6 non structural proteins (NSP1-NSP6). Up to now, the structural protein VP6 (group antigen) can be divided into 8 different groups of A-H, among which, the main cause of infantile infection is group A rotavirus. In addition, the gene between structural protein VP7 (G type antigen or sugar egg white) and VP4 (P type antigen or protein hydrolase sensitive protein) The differences are divided into 27 G genotypes (G1-G27) and 35 P genotypes (P[1]-P[35]). The type selection of the vaccine development is mainly based on the epidemic type. There are 3 rotavirus attenuated live vaccines used in the prevention of rotavirus infection, respectively, the monovalent human rotavirus vaccine Rotarix (genotype G1P[8]) produced by GlaxoSmithKline Co, Merck Co. The production of the pentavalant bovine rotavirus vaccine RotaTeq (genotype G1, G2, G3, G4, G6 and P[8]) and the orally active rotavirus (genotype of G10P[12]) of the monovalent rotavirus developed by the Lanzhou Institute of biological products. The existing rotavirus vaccines have univalent and polyvalent, and these two vaccines have the preparation process and quality control. As a result, polyvalent vaccines are relatively difficult. Therefore, the single type can cover the multiple type of vaccine as much as possible. The problem is whether a single valent vaccine can be used to obtain the effect of a multivalent vaccine. The results from the present study show that it is not sure. Although the final effect depends on the data of the human body after the epidemic, the cycle of human data is long, The evaluation system is complex. Therefore, the establishment of laboratory evaluation method has important guidance and foresight to consider the above problems. The aim of this study is to compare the immunogenicity of the monovalent and polyvalent rotavirus immunogen, and to evaluate the difference between the cross protection and the multivalent immunogen caused by the monovalent immunogen. Three rotavirus standard strains of different genotypes: Wa (genotype G1P[8]), SA11 (genotype G3P[1]) and Gottfried (genotype G4P[6]), the three rotavirus standard strains were used individually as monovalent rotavirus immunogens, and the three rotavirus standard strains were mixed in different proportions to prepare polyvalent rotavirus free virus. The experimental animal ICR mice were randomly divided into 8 groups, which were 3 monovalent rotavirus immunogen groups (group Wa, group SA11, Gottfried), 3 two valence rotavirus immunogen groups (group Wa+SA11, Wa+Gottfried group, SA11+Gottfried group), 1 trivalent rotavirus immunogen group (Wa+SA11+Gottfried group) and 1 control groups (PBS group), each group was treated by the same group. The same total dose of the immune rotavirus immunogen was taken. After immunization, ELISA, neutralization test and IgA ELISA kit were used to detect and evaluate the specific IgG level of rotavirus, the titer of rotavirus specific neutralization antibody and the concentration of IgA in serum, so that the monovalent and polyvalent rotavirus was used. The immunogenicity of the immunogen was compared and analyzed. The results showed that after immunization of the monovalent rotavirus, different genotypes could produce serum cross neutralization antibody below the parent strain, and the immune response induced by the two rotavirus immunogen was stronger than the monovalent rotavirus immunogen, but three of the two valence rotavirus immunogen. In the combination, the immunogenicity of Wa strain and SA11 strain was stronger than that of the Gottfried strain; the immune response induced by the trivalent rotavirus immunogen was slightly higher than that of the monovalent rotavirus immunogen, but slightly lower than the two rotavirus immunogen, in which the immunogenicity was still the strongest in Wa, SA11 times and low in Gottfried. The immunogen of rotavirus immunogen can induce more serum rotavirus specific antibodies than the monovalent rotavirus immunogen, and the produced serum IgA antibody is also higher than the monovalent rotavirus immunogen. Moreover, the immunogen of the polyvalent rotavirus immunogen can induce the cross immune response faster than the monovalent rotavirus immunogen. It is not possible to replace the rotavirus vaccine with single valent rotavirus vaccine.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R725.1

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