EIAV减毒疫苗诱导体液免疫反应时相及膜抗原改造研究
发布时间：2018-10-23 18:04
【摘要】： 马传染性贫血病毒(EIAV)是与HIV同科同属的马属动物慢病毒,其病毒形态、基因组结构,病毒编码蛋白的结构和功能及抗原特性等方面与HIV极为相似。中国EIAV减毒活疫苗是目前世界上唯一大规模应用的慢病毒疫苗。因此深入探索该疫苗的免疫保护机制将为HIV疫苗的研制提供有益的借鉴。 本课题利用研究室前期工作中构建的EIAV感染性分子克隆pLGFD3制备减毒疫苗pLGFD3-V,免疫动物后,定期采集血样本,通过ELISA方法检测各个时点血清中针对EIAV膜蛋白(Env)Gp90,和基质蛋白p26结合抗体的滴度;同时,采用细胞蚀斑染色方法检测中和抗体的滴度,分析EIAV减毒疫苗诱导的体液免疫反应的成熟时相。结果显示,EIAV减毒疫苗免疫马后,针对Env Gp90和Gag p26蛋白均产生了特异性的体液免疫反应,Gp90结合抗体在2个半月左右达峰值(1:320,1:320),随后尽管有轻微的波动,但一直维持在较高的水平直至免疫后5至6个月,随后开始轻微回落,在免疫后32个月时仍能检测到稳定存在。而p26结合抗体则存续时间较短,在免疫后1-2个月达峰值(1:320,1:40),随后呈明显回落,在4个月时回落至检测水平以下。EIAV减毒疫苗诱导的特异性中和抗体的水平同样在2个月左右达峰值(约1:2800—1:3000),在整个观测期内持续稳定存在。上述结果证实,EIAV减毒疫苗免疫后,能诱导产生特异性的Gp90,p26结合抗体以及中和抗体,而且GpgO特异性结合抗体和中和抗体可持续稳定存在。 为了增强减毒疫苗诱导中和抗体的免疫原性,我们尝试对env基因V3,V4区以及连接片层上糖基化位点进行了缺失型改造,探索上述糖基化位点的缺失对体液免疫反应的影响。首先构建了上述Env区糖基化位点缺失的的DNA疫苗(pDRVI—none)和痘苗载体疫苗(rTTV—none),以减毒疫苗膜蛋白的DNA疫苗(pDRVI—env)及痘苗载体活疫苗(rTTV—env),以及空白的DNA疫苗/痘苗病毒载体(pDRVI—sv1.0/rTTV—7521)为对照,采用DNA初免/痘苗加强的策略,分别免疫小鼠。分组如下(1)pDRVI—env/rTTV—env,(2)pDRVI—none/rTTV—none,(3)pDRVI—sv1.0/rTTV—7521和(4)生理盐水。免疫2个月后,采用与上述相同的实验方法,检测各组基因工程疫苗诱导的针对gp90的结合抗体和中和抗体水平。结果显示,和减毒疫苗膜蛋白未经改造组相比,糖基化位点的缺失对膜蛋白诱导结合抗体的能力没有形成显著的差异,但降低了膜蛋白诱导中和抗体的频率和中和滴度。说明减毒疫苗膜蛋白在V3,V4区所分别保留的199位,217位糖基化位点对于有效中和抗体的诱导是必须的。今后应该探索新的策略,增强疫苗诱导体液免疫反应的免疫原性。
[Abstract]:Equine infectious anemia virus (EIAV) is a kind of equine lentivirus belonging to the same family as HIV. Its morphology, genome structure, structure and function of virus coding protein and antigenic characteristics of the virus are very similar to HIV. Chinese EIAV attenuated live vaccine is the only large-scale application of lentivirus vaccine in the world. Therefore, further exploring the immune protection mechanism of the vaccine will provide useful reference for the development of HIV vaccine. In this study, the attenuated vaccine pLGFD3-V, immunized animals were prepared by cloning pLGFD3 from EIAV infectious molecules constructed in the previous work of the laboratory, and blood samples were collected regularly. The titer of EIAV membrane protein (Env) Gp90, and matrix protein p26 binding antibody was detected by ELISA method, and the neutralization antibody titer was detected by cell plaque staining. The mature phase of humoral immune response induced by EIAV attenuated vaccine was analyzed. The results showed that EIAV attenuated vaccine produced a specific humoral immune response against both Env Gp90 and Gag p26 protein, and the Gp90 binding antibody reached its peak in about two and a half months (1: 320min: 1: 320), although there was a slight fluctuation. But it remained at a high level until 5 to 6 months after immunization, followed by a slight drop, and a steady presence was detected 32 months after immunization. On the other hand, the survival time of p26 binding antibody was shorter, reaching its peak at 1-2 months after immunization (1: 320 or 1: 40), and then decreased significantly. The level of specific neutralizing antibody induced by EIAV attenuated vaccine also reached its peak at about 2 months (about 1: 2800-1: 3000) and remained stable throughout the observation period. The above results confirmed that EIAV attenuated vaccine could induce the production of specific Gp90,p26 binding antibody and neutralizing antibody, and the existence of GpgO specific binding antibody and neutralizing antibody could be sustained and stable. In order to enhance the immunogenicity of neutralizing antibody induced by attenuated vaccine, we attempted to modify the deletion type of the V3V4 region of env gene and the glycosylation site on the connecting lamellar, and to explore the effect of the above glycosylation sites on humoral immune response. First of all, DNA vaccine (pDRVI-none) and vaccinia vector vaccine (rTTV-none) with missing glycosylation sites in Env region were constructed for attenuated DNA vaccine (pDRVI-env) and vaccinia vector live vaccine (rTTV-env), as well as blank DNA vaccine / vaccinia vaccine. Seedling virus vector (pDRVI-sv1.0/rTTV-7521) was used as control. Mice were immunized with DNA / vaccinia. The groups were as follows: (1) pDRVI-env/rTTV-env, (2) pDRVI-none/rTTV-none, (3) pDRVI-sv1.0/rTTV-7521 and (4) normal saline. After 2 months of immunization, the levels of binding and neutralizing antibodies against gp90 induced by each group of genetically engineered vaccines were detected by the same experimental method as those mentioned above. The results showed that the absence of glycosylation sites had no significant difference in the ability of membrane protein to induce antibody, but decreased the frequency and titer of membrane protein induced neutralizing antibody. The results showed that the 199 and 217 glycosylation sites of attenuated vaccine membrane proteins in V3 / V4 region were necessary for the induction of effective neutralizing antibodies. New strategies should be explored to enhance the immunogenicity of humoral immune response induced by vaccine.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
本文编号：2290055
[Abstract]:Equine infectious anemia virus (EIAV) is a kind of equine lentivirus belonging to the same family as HIV. Its morphology, genome structure, structure and function of virus coding protein and antigenic characteristics of the virus are very similar to HIV. Chinese EIAV attenuated live vaccine is the only large-scale application of lentivirus vaccine in the world. Therefore, further exploring the immune protection mechanism of the vaccine will provide useful reference for the development of HIV vaccine. In this study, the attenuated vaccine pLGFD3-V, immunized animals were prepared by cloning pLGFD3 from EIAV infectious molecules constructed in the previous work of the laboratory, and blood samples were collected regularly. The titer of EIAV membrane protein (Env) Gp90, and matrix protein p26 binding antibody was detected by ELISA method, and the neutralization antibody titer was detected by cell plaque staining. The mature phase of humoral immune response induced by EIAV attenuated vaccine was analyzed. The results showed that EIAV attenuated vaccine produced a specific humoral immune response against both Env Gp90 and Gag p26 protein, and the Gp90 binding antibody reached its peak in about two and a half months (1: 320min: 1: 320), although there was a slight fluctuation. But it remained at a high level until 5 to 6 months after immunization, followed by a slight drop, and a steady presence was detected 32 months after immunization. On the other hand, the survival time of p26 binding antibody was shorter, reaching its peak at 1-2 months after immunization (1: 320 or 1: 40), and then decreased significantly. The level of specific neutralizing antibody induced by EIAV attenuated vaccine also reached its peak at about 2 months (about 1: 2800-1: 3000) and remained stable throughout the observation period. The above results confirmed that EIAV attenuated vaccine could induce the production of specific Gp90,p26 binding antibody and neutralizing antibody, and the existence of GpgO specific binding antibody and neutralizing antibody could be sustained and stable. In order to enhance the immunogenicity of neutralizing antibody induced by attenuated vaccine, we attempted to modify the deletion type of the V3V4 region of env gene and the glycosylation site on the connecting lamellar, and to explore the effect of the above glycosylation sites on humoral immune response. First of all, DNA vaccine (pDRVI-none) and vaccinia vector vaccine (rTTV-none) with missing glycosylation sites in Env region were constructed for attenuated DNA vaccine (pDRVI-env) and vaccinia vector live vaccine (rTTV-env), as well as blank DNA vaccine / vaccinia vaccine. Seedling virus vector (pDRVI-sv1.0/rTTV-7521) was used as control. Mice were immunized with DNA / vaccinia. The groups were as follows: (1) pDRVI-env/rTTV-env, (2) pDRVI-none/rTTV-none, (3) pDRVI-sv1.0/rTTV-7521 and (4) normal saline. After 2 months of immunization, the levels of binding and neutralizing antibodies against gp90 induced by each group of genetically engineered vaccines were detected by the same experimental method as those mentioned above. The results showed that the absence of glycosylation sites had no significant difference in the ability of membrane protein to induce antibody, but decreased the frequency and titer of membrane protein induced neutralizing antibody. The results showed that the 199 and 217 glycosylation sites of attenuated vaccine membrane proteins in V3 / V4 region were necessary for the induction of effective neutralizing antibodies. New strategies should be explored to enhance the immunogenicity of humoral immune response induced by vaccine.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
【参考文献】
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1 张晓燕;李红梅;梁华;沈_";马燕;相文华;沈荣显;邵一鸣;;EIAV减毒疫苗诱导的特异性细胞免疫应答[J];细胞与分子免疫学杂志;2006年01期
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