表达HIV基因的DNA、Ad5及DC疫苗在小鼠体内的免疫原性研究

发布时间：2018-09-15 06:35

【摘要】：人类免疫缺陷病毒(HIV)感染人类所导致的获得性免疫缺陷综合征(Acquired Immunodeficiency Syndrome,即AIDS)已经在全球范围内流行了30余年,但是迄今为止尚未找到能够彻底清除病毒感染并治愈AIDS的方法。本课题组主要致力于治疗性艾滋病疫苗(Therapeutic HIV vaccine)的研究,导师曾毅院士在国内外首次提出HIV多载体疫苗序贯重复免疫的策略,并在小鼠及恒河猴实验中证实该策略能诱导较强而且持久的HIV特异性细胞免疫反应,提示该策略有很好的应用前景。另外,基于树突状细胞(Dendritic Cells,DCs)的HIV疫苗在临床前和临床研究中都显示可有效控制病毒载量,我们拟在多载体疫苗序贯重复免疫的策略中增加这种DCs疫苗的应用。本研究目的是在前期研究的基础上,构建新的表达不同亚型基因候选艾滋病疫苗并进行免疫原性评价;同时也对负载Ad5-HIVgag/env的DC疫苗进行免疫原性评价为后期进一步进行包括上述各种疫苗在内的多载体疫苗联合免疫方案的优化奠定基础。本研究主要包括三部分,首先对表达HIV-1 CRF01_AE亚型gp145和gp160基因的DNA疫苗在小鼠体内的免疫原性进行了比较,实验结果显示,表达gp145和gp160基因的DNA疫苗都能够诱导抗原特异性免疫反应,反应峰值分别出现在免疫后3w和4w,表达gp145基因的DNA疫苗免疫后诱导的HIV特异性细胞免疫反应更快达到峰值,pVR-AE gp145在小鼠体内的免疫原性明显优于pVR-AE gp160,在后续研究中将选择pVR-AE gp145疫苗与其他载体疫苗进行联合免疫的进一步评价。其次,构建了表达HIV-1 B/C亚型gag基因的重组腺病毒疫苗(rAd5-HIV B/C gag),并对其在小鼠体内诱导的免疫反应进行了初步研究。ELISPOT结果显示,rAd5-HIV B/C gag免疫2周后,在小鼠内检测到了高水平的Gag特异性细胞免疫应答。此外,还在小鼠体内评价了负载Ad5-HIV gag/env的DCs疫苗的免疫原性。首先用贴壁法分离纯化BALB/c小鼠DCs,并通过细胞因子诱导DC细胞成熟后,采用流式细胞术鉴定DC纯度。利用重组腺病毒Ad5-HIV gag/env感染DC细胞,制备DC疫苗,并用所制备的DC疫苗单独或联合免疫小鼠,在免疫后的不同时间点,用ELISPOT方法检测小鼠体内HIV特异性细胞免疫应答强度。结果显示,Ad5-HIV gag/env负载的树突状细胞疫苗可以在小鼠体内诱导较强的HIV特异性细胞免疫反应。上述的DNA疫苗,Ad5疫苗和负载Ad5-HIVgag/env的DC疫苗都有很好的免疫原性,为进一步评价这些疫苗在小鼠体内序贯、重复免疫的效果奠定基础。
[Abstract]:Acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV) infection has been prevalent in the world for more than 30 years, but no method has been found to completely eliminate the virus infection and cure AIDS. Our research group is mainly devoted to the research of therapeutic AIDS vaccine (Therapeutic HIV vaccine). Academician Zeng Yi proposed the strategy of sequential repeated immunization of HIV multi-carrier vaccine for the first time at home and abroad. The results of experiments in mice and rhesus monkey showed that the strategy could induce strong and persistent HIV specific cellular immune response, which indicated that the strategy had a good application prospect. In addition, HIV vaccine based on dendritic cells (Dendritic Cells,DCs) has been shown to be effective in controlling viral load in both clinical and clinical studies. We intend to increase the application of this DCs vaccine in the strategy of sequential repeated immunization of multi-vector vaccines. The aim of this study was to construct a new candidate AIDS vaccine expressing different subtypes of genes and evaluate its immunogenicity on the basis of previous studies. At the same time, the immunogenicity evaluation of the DC vaccine loaded with Ad5-HIVgag/env laid the foundation for further optimization of the multi-vector vaccine combined immunization scheme, including the above-mentioned vaccines. This study mainly consists of three parts. Firstly, the immunogenicity of the DNA vaccine expressing HIV-1 CRF01_AE subtype gp145 and gp160 gene in mice was compared. Both DNA vaccines expressing gp145 and gp160 genes can induce antigen-specific immune responses. The peak value of the response occurred at 3w and 4ws after immunization, respectively. The immunogenicity of HIV specific cellular immune response induced by DNA vaccine expressing gp145 gene was faster than that of pVR-AE gp160, in mice after immunization. The immunogenicity of pVR-AE gp145 in mice was significantly better than that of pVR-AE gp160, in the follow-up study. PVR-AE gp145 vaccine and other vector vaccines will be selected for further evaluation of combined immunization. Secondly, the recombinant adenovirus vaccine (rAd5-HIV B / C gag),) expressing HIV-1 B / C subtype gag gene was constructed and the immune response induced by rAd5-HIV B / C gag), in mice was studied. The results showed that rAd5-HIV B / C gag was immunized for 2 weeks. A high level of Gag specific cellular immune response was detected in mice. In addition, immunogenicity of DCs vaccine loaded with Ad5-HIV gag/env was evaluated in mice. The DCs, of BALB/c mice was isolated and purified by adherent method. After the DC cells matured by cytokines, the purity of DC was identified by flow cytometry. The recombinant adenovirus Ad5-HIV gag/env was used to infect DC cells to prepare DC vaccine. Mice were immunized with the DC vaccine alone or in combination. The specific cellular immune response of HIV in mice was detected by ELISPOT method at different time points after immunization. The results showed that the dendritic cell vaccine loaded with Ad5-HIV gag/env could induce a strong HIV specific cellular immune response in mice. The above DNA vaccine, Ad5 vaccine, and DC vaccine loaded with Ad5-HIVgag/env have good immunogenicity, which lays a foundation for further evaluation of the sequential and repeated immunization effect of these vaccines in mice.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R392

【相似文献】