本文關(guān)鍵詞：IL-15對HIV疫苗免疫效應(yīng)的影響及HIV感染者TCR Vδ1 CDR3肽及其識別的抗原表位肽的篩選與鑒定　出處：《北京協(xié)和醫(yī)學(xué)院》2010年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： HIV IL-15 記憶性T細(xì)胞 γδT細(xì)胞 CDR3肽

【摘要】：自從上個世紀(jì)80年代人類免疫缺陷病毒(human immunodeficiency virus, HIV)被發(fā)現(xiàn)以來,感染HIV所致的獲得性免疫缺陷綜合征,簡稱艾滋病(acquired immune deficiency syndrome, AIDS)在全球迅速傳播,現(xiàn)已成為一種嚴(yán)重危害人類健康的疾病,對HIV/AIDS的預(yù)防、診斷、治療也因此成為醫(yī)學(xué)工作者的首要任務(wù)之一。研制安全有效的HIV疫苗是預(yù)防HIV感染的重要措施之一,研究者們一直致力于如何激發(fā)機(jī)體產(chǎn)生足夠強(qiáng)度的細(xì)胞免疫和體液免疫并加強(qiáng)免疫記憶。本課題組前期研究顯示,IL-15在抗腫瘤免疫中具有增強(qiáng)免疫反應(yīng)的作用,且小鼠實驗證實IL-15質(zhì)粒能夠增強(qiáng)HIV gag疫苗的免疫效應(yīng),同時能促進(jìn)中樞記憶性T細(xì)胞分化及增強(qiáng)長期記憶性免疫應(yīng)答。因此,本研究的第一部分將觀察以痘苗病毒天壇株為載體的重組IL-15在小鼠體內(nèi)對重組HIV gag痘苗病毒載體疫苗免疫效應(yīng)的影響,以及在靈長類動物體內(nèi)IL-15質(zhì)粒對HIV疫苗免疫效應(yīng)的影響,以進(jìn)一步探索IL-15作為HIV疫苗佐劑的前景。 在以痘苗病毒天壇株為載體的研究中,將IL-15基因編碼片段插入至復(fù)制型痘苗病毒載體,體外檢測了IL-15蛋白的表達(dá)及表達(dá)上清中IL-15的生物活性。將重組IL-15痘苗病毒與重組HIV gag痘苗病毒共同免疫小鼠后,酶聯(lián)免疫斑點法(enzyme-linked immunospot assay, ELI SPOT)測定結(jié)果顯示,在痘苗病毒的高劑量組和低劑量組,IL-15共免疫均未能增加小鼠體內(nèi)抗HIV gag抗原特異性IFN-γ分泌的CD8+T淋巴細(xì)胞頻率,反而使其降低；同時IL-15對HIV特異性體液免疫也無增強(qiáng)作用。 在靈長類動物研究中,本研究以IL-15重組質(zhì)粒與HIV多價的DNA疫苗共同免疫恒河猴,然后用重組HIV痘苗病毒疫苗進(jìn)行加強(qiáng)免疫。結(jié)果顯示,IL-15重組質(zhì)粒與HIV DNA疫苗共同免疫后,恒河猴體內(nèi)的HIV特異性的T細(xì)胞反應(yīng)與HIV疫苗單獨免疫無明顯差異,體液免疫在兩組間也無明顯差異。在HIV痘苗病毒疫苗加強(qiáng)免疫后,IL-15重組質(zhì)粒共免疫的恒河猴的HIV特異性T細(xì)胞反應(yīng)比HIV疫苗單獨免疫組稍強(qiáng),而且前者的抗HIV env蛋白抗體滴度明顯高于后者。流式細(xì)胞術(shù)檢測顯示,在幾乎所有的檢測時間點,IL-15重組質(zhì)粒共免疫組的動物外周血中記憶性CD8+T細(xì)胞的比例明顯高于HIV疫苗單獨免疫組,提示IL-15能夠誘導(dǎo)更多的效應(yīng)性T細(xì)胞向記憶性T細(xì)胞轉(zhuǎn)化。 綜上所述,重組IL-15天壇株痘苗病毒對HIV gag痘苗病毒疫苗無免疫增強(qiáng)作用,反而減弱其免疫效應(yīng),故IL-15可能不適合用于天壇株痘苗病毒載體疫苗體系；在靈長類動物實驗中,IL-15能夠增加恒河猴外周血中記憶性T細(xì)胞的比例,同時還可以增強(qiáng)長期的體液免疫反應(yīng),提示IL-15可以增強(qiáng)HIV疫苗的長期免疫反應(yīng)。 本文的第二部分為HIV感染者TCR Vδ1 CDR3肽及其識別的抗原表位肽的篩選與鑒定,研究的對象是HIV感染者體內(nèi)的γδT細(xì)胞。γδT細(xì)胞作為連接固有性免疫和特異性免疫的橋梁,雖然其在外周血T細(xì)胞中所占的比例僅為1-10%,但是在抗感染過程中發(fā)揮著重要的作用。業(yè)已證明,在HIV感染者外周血中γδT細(xì)胞在絕對數(shù)量上有4-5倍的擴(kuò)增,且主要是Vδ1亞型,而Vγ9/δ2γδT細(xì)胞卻處于免疫無能狀態(tài)。這種改變的具體機(jī)制目前仍不清楚。本實驗室主要從事γδT細(xì)胞研究,證實了TCRδ鏈的CDR3區(qū)是γδT細(xì)胞與抗原結(jié)合的關(guān)鍵部位,本研究從以上理論出發(fā),進(jìn)行了如下實驗： 首先分離9例HIV感染者外周血單個核細(xì)胞,經(jīng)反轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(reverse transcription-polymerase chain reaction, RT-PCR)擴(kuò)增γδTCR Vδ1區(qū)基因,將擴(kuò)增片段重組到pGEM-T載體上進(jìn)行測序,結(jié)果獲得了兩條優(yōu)勢的CDR3肽：CALGVTTALIQWGFVYTDKLIF (HP1)和CALGEPPIYFNWGIRVTDKPIF (HP2)。 然后利用得到的兩條優(yōu)勢的CDR3肽(HP1和HP2)為探針,篩選噬菌體隨機(jī)十二肽庫。通過非特異性洗脫和特異性洗脫兩種方法,我們得到了3條優(yōu)勢十二肽：WHWQWTPWSIQP、WHWNAWNWSSQQ和WHWSWIQNAAPN,這3條十二肽的共有序列為WHW。合成以上3條十二肽,進(jìn)行初步的生物學(xué)分析：通過酶聯(lián)免疫吸附法(enzyme-linked immunosorbent assay, ELISA)和流式細(xì)胞術(shù)(flow cytometry, FCM),在分子和細(xì)胞水平驗證十二肽與探針和γδTCR的結(jié)合；利用固相擴(kuò)增的方法,通過流式細(xì)胞術(shù)和二甲基偶氮唑藍(lán)(3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide, MTT)法驗證合成的十二肽對γδT細(xì)胞群體的促增殖作用。 以上結(jié)果顯示,本研究得到了HIV感染者外周血中TCRδ1鏈的優(yōu)勢CDR3肽的序列,并利用優(yōu)勢的CDR3δ肽通過篩選噬菌體隨機(jī)十二肽庫得到了Vδ1 T細(xì)胞識別的候選抗原表位肽,為研究HIV病程中Vδ1 T細(xì)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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R392

【共引文獻(xiàn)】

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