Ad5-HIVgp160疫苗药效学及IL15基因佐剂对其免疫效果增强作用

发布时间：2018-03-29 16:26

本文选题：艾滋病疫苗　切入点：佐剂　出处：《北京工业大学》2014年硕士论文

【摘要】：研制安全有效的艾滋病疫苗控制人类免疫缺陷病毒（HIV）传播是遏制艾滋病流行的最好方法。虽然已有几十种疫苗进行了临床试验，但是还没有一种疫苗对高危人群具有有效的保护作用。继续进行新型艾滋病疫苗研发刻不容缓，艾滋病治疗性疫苗能够在较长的时间内控制病毒载量、延缓艾滋病发病进程，为艾滋病疫苗研发的主要方向，，同时利用各种佐剂增强艾滋病疫苗的免疫效力也是目前研究中的新策略。本研究首先对本课题组前期构建的表达HIV-1gp160的腺病毒5型载体（Ad5-HIVgp160）疫苗进行临床前小鼠药效学研究。Ad5-HIVgp160疫苗以2×106VP/只、2×107VP/只、2×108VP/只、2×109VP/只、2×1010VP/只剂量分别免疫小鼠，采用ELISPOT法和ELISA法检测免疫小鼠体内特异性细胞免疫反应和体液免疫反应，结果各剂量组小鼠均可诱导出特异性细胞免疫反应和体液免疫反应，剂量为2×108VP/只时能够在小鼠体内激发出高水平的细胞免疫反应和体液免疫反应。之后，研究IL15基因佐剂协同poly(I:C)对表达HIV-1B亚型gp160DNA及腺病毒载体疫苗免疫增强作用，并探讨其作用机制。首先构建表达IL15的质粒pVR-IL15，将pVR-IL15联合pVR-HIVgp160初免Ad5-HIVgp160加强方式免疫小鼠，采用IFN-γ ELISPOT、ELISA和CCK-8等方法比较疫苗单独免疫组小鼠与疫苗加IL15佐剂组小鼠诱导的细胞免疫、体液免疫反应及淋巴细胞增殖反应的强度。结果显示疫苗加IL15佐剂组小鼠诱导的细胞免疫反应、体液免疫反应及淋巴细胞增殖反应比疫苗单独免疫组小鼠相比均有显著增强。pVR-IL15协同poly(I:C)联合pVR-HIVgp160初免Ad5-HIVgp160加强方式免疫小鼠，同样采用上述方法检测免疫小鼠体内特异性细胞免疫反应和淋巴细胞增殖反应，结果显示pVR-IL15协同poly(I:C)组小鼠特异性细胞免疫反应及淋巴细胞增殖反应均显著高于单独使用pVR-IL15组小鼠。进一步实验说明，pVR-IL15协同poly(I:C)组小鼠在免疫后3天即可诱导出高水平的特异性细胞免疫反应，高于其它各组小鼠，并且平稳保持至免疫后第8周。荧光定量PCR实验进一步显示，pVR-IL15协同poly(I:C)组小鼠淋巴细胞中Th1型细胞因子IFN-γ及抗凋亡因子Bcl-2的表达均高于其他各组小鼠，并且Bcl-2表达量与特异性细胞免疫反应及淋巴细胞增殖反应强度呈一定相关性。综上所述，Ad5-HIVgp160疫苗可以诱导机体产生特异性细胞免疫应答和体液免疫应答，有可能成为有效的艾滋病治疗性疫苗；IL15基因佐剂协同poly(I:C)可以增强HIV DNA疫苗初免/腺病毒疫苗加强免疫策略的免疫效果，延长免疫反应作用时间，为IL15佐剂协同poly(I:C)临床应用提供实验支持。
[Abstract]:The development of a safe and effective AIDS vaccine to control the spread of human immunodeficiency virus (HIV) is the best way to contain the AIDS epidemic. Although dozens of vaccines have been tested in clinical trials, However, none of the vaccines has an effective protective effect on high-risk groups. It is urgent to continue the development of new AIDS vaccines. AIDS therapeutic vaccines can control the load of the virus in a longer period of time and delay the onset of AIDS. The main direction of AIDS vaccine research and development, and the use of various adjuvants to enhance the immune effectiveness of AIDS vaccine is also a new strategy in current research. In this study, we first studied the preclinical pharmacodynamics of Ad5-HIVgp160 (Ad5-HIVgp160) vaccine constructed by our research group. Ad5-HIVgp160 vaccine was immunized with 2 脳 106VP/, 2 脳 107VP/, 2 脳 108VP/, 2 脳 109VP/ and 2 脳 1010VP/, respectively. The specific cellular and humoral immune responses of immunized mice were detected by ELISPOT and ELISA methods. The results showed that specific cellular and humoral immune responses could be induced in each dose group of mice. When the dose of 2 脳 108VP/ was 2 脳 108VP/ only, the high level of cellular and humoral immune response could be stimulated in mice. After that, the effects of IL15 gene adjuvant combined with polysid I: C on the expression of HIV-1B subtype gp160DNA and adenovirus vector vaccine were studied, and the mechanism was discussed. Firstly, the plasmid pVR-IL15 expressing IL15 was constructed, and the combination of pVR-IL15 and pVR-HIVgp160 was used to immunize mice with Ad5-HIVgp160. IFN- 纬 Elispot Elisa and CCK-8 were used to compare the cellular immunity induced by vaccine alone and IL15 adjuvant. The results showed that the immune response of mice induced by vaccine and IL15 adjuvant was induced by humoral immune reaction and lymphocyte proliferation. The humoral immune response and lymphocyte proliferation reaction were significantly enhanced compared with the mice immunized with vaccine alone. PVR-IL15 combined with pVR-HIVgp160 combined with Ad5-HIVgp160 was used to immunize mice. The above methods were also used to detect the specific cellular immune response and lymphocyte proliferation in immunized mice. The results showed that the specific cellular immune response and lymphocyte proliferation of mice in pVR-IL15 combined with polysid I: C group were significantly higher than those in mice treated with pVR-IL15 alone. Further experiments showed that the mice in the group of pVR-IL15 combined with polycyclic I: C) could induce high water content 3 days after immunization. The specific cellular immune response, The expression of Th1 type cytokine IFN- 纬 and anti-apoptotic factor (Bcl-2) in the lymphocytes of the mice treated with pVR-IL15 combined with polygonal I: C- (1) was higher than that of the other groups, and the expression of IFN- 纬 and anti-apoptotic factor Bcl-2 in the lymphocytes of the mice with pVR-IL15 combined with polymorphic I: C was higher than that of the other groups. The expression of Bcl-2 was correlated with the specific cellular immune response and lymphocyte proliferation. In conclusion, Ad5-HIVgp160 vaccine can induce specific cellular and humoral immune responses. It is possible to be an effective adjuvant of IL15 gene for AIDS treatment vaccine combined with poly (I: C). It can enhance the immune effect of HIV DNA vaccine primary immunization / adenovirus vaccine and prolong the time of immune response. To provide experimental support for clinical application of IL15 adjuvant in collaboration with polyacrylamide I: C.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R392

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