IL-15对HIV疫苗免疫效应的影响及HIV感染者TCR Vδ1 CDR3肽及其识别的抗原表位肽的筛选与鉴定

发布时间：2017-12-30 23:25

本文关键词：IL-15对HIV疫苗免疫效应的影响及HIV感染者TCR Vδ1 CDR3肽及其识别的抗原表位肽的筛选与鉴定　出处：《北京协和医学院》2010年博士论文　论文类型：学位论文

【摘要】：自从上个世纪80年代人类免疫缺陷病毒(human immunodeficiency virus, HIV)被发现以来,感染HIV所致的获得性免疫缺陷综合征,简称艾滋病(acquired immune deficiency syndrome, AIDS)在全球迅速传播,现已成为一种严重危害人类健康的疾病,对HIV/AIDS的预防、诊断、治疗也因此成为医学工作者的首要任务之一。研制安全有效的HIV疫苗是预防HIV感染的重要措施之一,研究者们一直致力于如何激发机体产生足够强度的细胞免疫和体液免疫并加强免疫记忆。本课题组前期研究显示,IL-15在抗肿瘤免疫中具有增强免疫反应的作用,且小鼠实验证实IL-15质粒能够增强HIV gag疫苗的免疫效应,同时能促进中枢记忆性T细胞分化及增强长期记忆性免疫应答。因此,本研究的第一部分将观察以痘苗病毒天坛株为载体的重组IL-15在小鼠体内对重组HIV gag痘苗病毒载体疫苗免疫效应的影响,以及在灵长类动物体内IL-15质粒对HIV疫苗免疫效应的影响,以进一步探索IL-15作为HIV疫苗佐剂的前景。在以痘苗病毒天坛株为载体的研究中,将IL-15基因编码片段插入至复制型痘苗病毒载体,体外检测了IL-15蛋白的表达及表达上清中IL-15的生物活性。将重组IL-15痘苗病毒与重组HIV gag痘苗病毒共同免疫小鼠后,酶联免疫斑点法(enzyme-linked immunospot assay, ELI SPOT)测定结果显示,在痘苗病毒的高剂量组和低剂量组,IL-15共免疫均未能增加小鼠体内抗HIV gag抗原特异性IFN-γ分泌的CD8+T淋巴细胞频率,反而使其降低；同时IL-15对HIV特异性体液免疫也无增强作用。在灵长类动物研究中,本研究以IL-15重组质粒与HIV多价的DNA疫苗共同免疫恒河猴,然后用重组HIV痘苗病毒疫苗进行加强免疫。结果显示,IL-15重组质粒与HIV DNA疫苗共同免疫后,恒河猴体内的HIV特异性的T细胞反应与HIV疫苗单独免疫无明显差异,体液免疫在两组间也无明显差异。在HIV痘苗病毒疫苗加强免疫后,IL-15重组质粒共免疫的恒河猴的HIV特异性T细胞反应比HIV疫苗单独免疫组稍强,而且前者的抗HIV env蛋白抗体滴度明显高于后者。流式细胞术检测显示,在几乎所有的检测时间点,IL-15重组质粒共免疫组的动物外周血中记忆性CD8+T细胞的比例明显高于HIV疫苗单独免疫组,提示IL-15能够诱导更多的效应性T细胞向记忆性T细胞转化。综上所述,重组IL-15天坛株痘苗病毒对HIV gag痘苗病毒疫苗无免疫增强作用,反而减弱其免疫效应,故IL-15可能不适合用于天坛株痘苗病毒载体疫苗体系；在灵长类动物实验中,IL-15能够增加恒河猴外周血中记忆性T细胞的比例,同时还可以增强长期的体液免疫反应,提示IL-15可以增强HIV疫苗的长期免疫反应。本文的第二部分为HIV感染者TCR Vδ1 CDR3肽及其识别的抗原表位肽的筛选与鉴定,研究的对象是HIV感染者体内的γδT细胞。γδT细胞作为连接固有性免疫和特异性免疫的桥梁,虽然其在外周血T细胞中所占的比例仅为1-10%,但是在抗感染过程中发挥着重要的作用。业已证明,在HIV感染者外周血中γδT细胞在绝对数量上有4-5倍的扩增,且主要是Vδ1亚型,而Vγ9/δ2γδT细胞却处于免疫无能状态。这种改变的具体机制目前仍不清楚。本实验室主要从事γδT细胞研究,证实了TCRδ链的CDR3区是γδT细胞与抗原结合的关键部位,本研究从以上理论出发,进行了如下实验：首先分离9例HIV感染者外周血单个核细胞,经反转录聚合酶链式反应(reverse transcription-polymerase chain reaction, RT-PCR)扩增γδTCR Vδ1区基因,将扩增片段重组到pGEM-T载体上进行测序,结果获得了两条优势的CDR3肽：CALGVTTALIQWGFVYTDKLIF (HP1)和CALGEPPIYFNWGIRVTDKPIF (HP2)。然后利用得到的两条优势的CDR3肽(HP1和HP2)为探针,筛选噬菌体随机十二肽库。通过非特异性洗脱和特异性洗脱两种方法,我们得到了3条优势十二肽：WHWQWTPWSIQP、WHWNAWNWSSQQ和WHWSWIQNAAPN,这3条十二肽的共有序列为WHW。合成以上3条十二肽,进行初步的生物学分析：通过酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)和流式细胞术(flow cytometry, FCM),在分子和细胞水平验证十二肽与探针和γδTCR的结合；利用固相扩增的方法,通过流式细胞术和二甲基偶氮唑蓝(3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide, MTT)法验证合成的十二肽对γδT细胞群体的促增殖作用。以上结果显示,本研究得到了HIV感染者外周血中TCRδ1链的优势CDR3肽的序列,并利用优势的CDR3δ肽通过筛选噬菌体随机十二肽库得到了Vδ1 T细胞识别的候选抗原表位肽,为研究HIV病程中Vδ1 T细胞的作用提供了新思路。
[Abstract]:Since the last century in 80s, human immunodeficiency virus (human immunodeficiency, virus, HIV) has been found infected by HIV acquired immunodeficiency syndrome, AIDS (referred to as acquired immune deficiency syndrome, AIDS) spread rapidly around the world, has become a serious hazard to human health disease, prevention, diagnosis of HIV/AIDS, treatment it becomes one of the most important tasks of medical workers. The development of safe and effective HIV vaccine is one of the important measures for prevention of HIV infection, the researchers have been working on how to stimulate the body to produce cellular immunity and humoral immunity of sufficient strength and strengthen immune memory. Our previous studies showed that IL-15 could enhance the immune response in tumor immunity, and the mice experiment proved that the IL-15 plasmid can enhance the immune effect of HIV gag vaccine, also can promote the central memory T Long term memory cell differentiation and enhance immune response. Therefore, the first part of this study will observe the effect of IL-15 in recombinant vaccinia virus Tiantan strain as carrier in vivo vaccine immune effect of recombinant HIV gag vaccinia virus vector, as well as in the primate animal IL-15 Piasmid effect on HIV vaccine immune effect, to further exploration of IL-15 as adjuvant HIV vaccine in the future.
In the research on the Tiantan strain of vaccinia virus vector, IL-15 gene encoding fragment inserted into replicating vaccinia virus vector, and the supernatant was detected in vitro biological activity of IL-15 protein expression of IL-15 HIV gag. The recombinant vaccinia virus IL-15 recombinant vaccinia virus and common immune mice after ELISPOT (enzyme-linked immunospot assay, ELI SPOT) the results showed that the high dose group and low dose group in vaccinia virus, CD8+T lymphocyte IL-15 was able to increase the frequency of immune mice against HIV gag antigen specific IFN- secretion rate, but decreased; while IL-15 on HIV specific humoral immunity without enhancement.
In primate animal studies, this research is based on the IL-15 plasmid and HIV multivalent DNA vaccine of Ganges RIver common monkey, and then use the HIV recombinant vaccinia virus vaccine for immunization. The results showed that the recombinant plasmid of IL-15 and HIV DNA vaccine immunization, no significant difference between the Ganges RIver macaques HIV specific T cell responses and solo HIV vaccine, humoral immunity in the two groups have no significant difference. To strengthen the immunity in HIV vaccinia virus vaccine, HIV specific T cell responses of IL-15 recombinant plasmid were immune to the Ganges RIver monkey than HIV Vaccine Immunized slightly, and anti HIV env antibody titer was significantly higher than that of the latter. The former shows flow FCM detection, detection in almost all the time points, the proportion of memory CD8+T cells in the peripheral blood of IL-15 recombinant plasmid co immunization group was significantly higher than that of the animal immunized HIV vaccine, suggesting that IL-15 can induce More effector T cells convert to memory T cells.
In summary, the recombinant IL-15 vaccinia virus Tiantan strain without HIV gag to enhance the immune function of vaccinia virus vaccine, it weakens the immune effect, so IL-15 might not be suitable for Tiantan vaccinia virus vector vaccine system; in primate animal experiments, IL-15 can increase the memory T cells in the peripheral blood of Ganges RIver monkey proportion, also can enhance the humoral immune response to long-term, suggesting that IL-15 can enhance the long-term immune response to HIV vaccine.
The second part is the screening and identification of a peptide antigen in HIV infected TCR V 8 1 CDR3 peptide and its recognition, the research object is HIV infection in vivo gamma delta T cells. T cells as a bridge between innate immunity and specific immunity, although its share in peripheral blood the proportion of T cells is only 1-10%, but the anti infection plays an important role in the process. It has been proved that the HIV infection in peripheral blood T cells in absolute numbers was 4-5 times, and is mainly the V delta 1 subtypes, and V gamma gamma delta T delta 2 9/ cells are in the immune anergy. The mechanism of these changes remains unclear. The laboratory is mainly engaged in the research of gamma delta T cells, confirmed the TCR CDR3 delta chain is a key part of combination of gamma delta T cells and antigen, this study from the above theory, experiments are conducted as follows:
9 cases of HIV infection in peripheral blood mononuclear cells separated first by reverse transcription polymerase chain reaction (reverse transcription-polymerase chain reaction, RT-PCR) amplified V delta gamma delta TCR 1 gene, the amplified fragment was recombined into pGEM-T vector and sequenced, the results obtained two advantages: CDR3 peptide (HP1) and CALGVTTALIQWGFVYTDKLIF CALGEPPIYFNWGIRVTDKPIF (HP2).
Then the CDR3 peptide by two to obtain the advantage (HP1 and HP2) as a probe, screening the phage random twelve peptide library. Through non-specific elution and specific elution of two methods, we obtain 3 advantages: WHWQWTPWSIQP WHWNAWNWSSQQ, twelve peptide and WHWSWIQNAAPN, the 3 of twelve peptides with the consensus sequence of WHW. synthesis more than 3 of twelve peptides, preliminary biological analysis: by enzyme-linked immunosorbent assay (enzyme-linked immunosorbent, assay, ELISA) and flow cytometry (flow cytometry, FCM), with validation of the twelve peptides with probe and gamma delta TCR at the molecular and cellular levels; using the method of solid-phase amplification by flow cytometry. Operation and two methyl thiazolyl tetrazolium (3- (4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide, MTT) method to verify the proliferation of twelve peptide synthesis of gamma delta T cell populations.
The above results indicate that this research has got the advantages of CDR3 sequence peptide HIV infection in peripheral blood of 1 TCR delta chain, CDR3 delta peptide and the use of the advantages of the V delta 1 T cell recognition candidate epitope peptide by phage random twelve peptide library, provides a new idea for the study in the course of HIV V 8 1 T cells.

【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】：2010
【分类号】：R392

【共引文献】