全人源炭疽保护性抗原中和抗体的研究

发布时间：2018-05-15 16:27

本文选题：全人源单抗 + 炭疽重组PA疫苗　；参考：《中国人民解放军军事医学科学院》2015年博士论文

【摘要】：炭疽芽胞杆菌(Bacillus anthracis)致死率极高,是人畜共患烈性传染病-炭疽病(Anthrax)的病原体。并且炭疽芽胞杆菌来源广泛、易于培养,被作为一种A类生物战剂进行管理,其防生和反恐一直以来都具有重要的军事意义和社会意义。炭疽的防控手段包括预防和治疗,二者分别主要依赖于炭疽疫苗和抗菌素,但是这两种药物均需在感染前或感染初期立即使用。而炭疽毒素中和抗体在宿主感染后,能快速有效中和体内产生的炭疽毒素,同时发挥补体依赖细胞毒性作用和抗体依赖细胞毒性作用,是目前最有前景的炭疽治疗药物。自从1975年杂交瘤技术出现后,多年来单抗治疗主要依赖于鼠源单抗。但是鼠源单抗半衰期短、毒副作用明显,大大制约了其临床应用。全人源单克隆抗体应用于人体时具有无免疫原性、半衰期长,国外大多数新开发项目现已转向全人源单抗的研发。目前全人源单抗制备的方法主要是通过人人杂交瘤技术、转基因鼠技术、抗体库技术和单细胞PCR技术,其中通过单细胞PCR技术可直接从人单个B细胞中扩增抗体基因,无需细胞融合培养和抗体多轮筛选的过程,能够在最短7天内筛选获得有效的全人源单抗,而其他几种抗体制备方法均需要数周甚至数月,建立这种快速高通量抗体筛选平台,将有助于提升我国新发突发传染病的应对能力;并且,单细胞PCR技术完整保留了抗体重链轻链在人体内的天然配对方式,因此可在获得治疗抗体的同时更加直观、深入地研究人体免疫机制。但是,由于单个细胞内基因拷贝数非常低,单细胞PCR技术的实现难度大,目前国内罕见成功通过该技术获得单抗的研究团队。目前大部分炭疽单抗仍然是鼠源单抗,少数研究者从大猩猩、转基因鼠和人体中筛选获得了炭疽单抗,而我国仍未见全人源的抗炭疽毒素单抗报道。鉴于炭疽毒素作用机制的复杂性,研发更多的针对不同中和表位的炭疽全人源单抗,能够有效避免炭疽毒素被人为突变带来的生物恐怖威胁,为炭疽的治疗提供更多选择。鉴于此,本文目的在于建立流式分选-单细胞PCR快速高通量的抗体筛选技术平台,并通过该技术平台从重组炭疽保护性抗原(protective antigen,PA)疫苗免疫的志愿者体内,筛选获得全人源PA中和抗体,并对单抗的亲和力、抗原结合表位、毒素中和机制、单抗联合用药效果等进行深入研究。首先对一名男性志愿者在第0天和28天两次免疫了重组PA疫苗,并在第0、28、35天采集志愿者血液样品。为确保能够在合适的时间点采集志愿者阳性的血液样品,从而提高后续PA抗体筛选的阳性率,本文通过ELISA和TNA的方法,对不同时间点血清中PA结合和中和抗体滴度进行测量。结果表明,免疫第35天血清中PA结合抗体和中和抗体滴度均明显升高。同时,通过ELISpot的方法,对不同时间点血液中抗体分泌细胞的数量变化进行检测。结果显示抗PA抗体阳性的抗体分泌细胞占总Ig G阳性的抗体分泌细胞的比例,从免疫后第28天的0.8%上升至免疫后第35天的23.8%,可见抗PA抗体阳性的抗体分泌细胞个数大幅升高。表明重组PA疫苗在志愿者体内有效激发了体液免疫反应,在免疫后第35天时体液免疫水平显著升高,为后续抗体的分选提供保证。为了实现单个B细胞的分选和抗体基因的单细胞PCR扩增,我们尝试了多种细胞分选和单细胞PCR的方法,在经历多次失败后,最终采用流式细胞分选抗体分泌细胞,以及多重引物组合的单细胞反转录-巢式两步PCR的方法,成功从人单个B细胞中扩增获得抗体基因。在单细胞PCR条件优化的过程中,我们发现引物设计、酶的种类和模板量对于扩增效率的影响较大,而退火温度对于扩增效果影响甚微。在成功实现研究中抗体分选的关键性技术-流式分选单个抗体分泌细胞和单细胞PCR技术后,采集了志愿者免疫后第35天的血液样品,通过密度梯度沉降法从血液样品中分离外周血单个核细胞(peripheral blood mononuclear cell,PBMC),在对一组抗体分泌细胞表面特异的白细胞分化抗原簇(cluster of differentiation,CD)分子进行荧光染色后,通过流式细胞分选仪从PBMC中分选获得2112个单个抗体分泌细胞克隆,通过单细胞PCR扩增单个细胞中的抗体基因。结果显示,529个单细胞克隆重链和轻链同时扩增成功的比率,即单细胞PCR的阳性率约为25%。在硬件条件满足的情况下,仅需1~2天即可完成以上单细胞的分选和抗体基因扩增的过程。如果采用传统的克隆方法,构建529对抗体基因的表达质粒,非常耗时费力。为了快速、高通量表达抗体基因,我们设计了可直接通过PCR融合启动子、前导序列、抗体全长基因、多聚A尾(poly(A)tail)的线性表达框。我们对线性表达框PCR条件进行优化,并使用一株对照抗体验证线性表达框所表达抗体的表达量和功能。结果显示,经线性表达框方法表达的抗体浓度可达0.4μg/m L,且良好保持了抗体中和效果,能够满足后续抗体筛选实验的要求,通过我们设计的线性表达框可在最短3天内表达供后续筛选用的抗体。在确保方法的可行性后,我们按照优化的重叠延伸PCR条件,构建了529对抗体重链和轻链线性表达框,并将配对的基因共转染至HEK 293T细胞中进行表达。继而,通过ELISA和TNA的方法对529株全人源单抗进行PA结合和中和活性的检测,并鉴定了单抗的特异结合的PA结构域。结果显示,我们成功通过建立的抗体筛选平台技术获得34株PA结合单抗,其中4株单抗(2A6、4A3、8H6、22F1)具有PA中和活性。中和单抗22F1与PA不结合,而是通过与PA的变构体PA63结合发挥作用。另外,研究发现PA结合单抗中有一种能够加速细胞死亡的毒素增强抗体(Toxin enhancing m Ab),占PA结合单抗的比例达55.9%之多。为了进一步探究PA中和抗体和毒素增强抗体的特性,构建4株PA中和抗体和1株毒素增强抗体8A7的表达质粒,转染并纯化抗体蛋白;并从以下几个方面对抗体的功能进行研究:1)通过BLI技术测定了5株重点研究的单抗的亲和力,结果显示除22F1外单抗亲和力均在纳摩尔级;2)测定4株中和单抗在体内和体外的中和活性,结果显示4株中和单抗中22F1单抗中和活性最强,其体外保护效果较上市PA单抗报道结果高出近一个数量级,而单抗2A6仅在体外具有保护活性;3)鉴定中和单抗的中和机制,结果表明8H6单抗对PA呈现抗体浓度依赖的酶切抑制作用,4A3单抗通过促进PA63的降解而抑制PA七聚体形成,4A3这种类似酶活作用的毒素中和机制尚未见报道,提示本研究可能发现了一株新中和机制的PA中和单抗。毒素增强单抗8A7能促进PA七聚体的形成,并与PA七聚体结合,提示其毒素增强作用的可能机制;4)分析单抗的联合用药效果,结果表明2A6和4A3、8H6和22F1表现出较弱的协同作用;2A6和8H6、2A6和22F1表现出无关作用;4A3和8H6、4A3和22F1表现出拮抗作用。而单抗联合用药协同效果最为显著的,是毒素增强单抗8A7与仅在体外具有中和活性的单抗2A6,二者联合用药能够在体内发挥高水平的毒素中和效果。结果提示我们,在人体内之所以大量产生毒素增强抗体的可能原因是,这种抗体在血液中能够辅助其他抗体发挥良好的机体保护效果。并且,8A7单抗针对的PA结构域3是目前普遍认为的PA非中和位点,根据本文所得结果推测针对PA结构域3表位的单抗,在人体内能够通过与其他抗体协同发挥重要的生物学功能,这种辅助性单抗在抗体筛选过程中容易被遗漏,但是其在“鸡尾酒”治疗中发挥的重要作用不容忽视。综上所述,本文建立了基于流式分选和单抗PCR技术的快速抗体筛选平台,能够为新发突发传染病抗体研究提供技术储备;筛选制备了多株不同中和机制的全人源PA中和单抗,其中包含新中和机制的单抗和高中和活性的单抗,是具有前景的炭疽治疗药物;同时,对抗体中和机制、表位、联合用药效果的研究,也为炭疽毒素作用机理研究、疫苗设计和“鸡尾酒”抗体药物设计提供有力依据;
[Abstract]:Bacillus anthracis (Bacillus anthracis) is highly lethal and is the pathogen of human zoonotic strong infectious disease, anthrax (Anthrax). And Bacillus anthracis is widely used as a kind of biological warfare agent to manage, and has important military and social significance. The prevention and anti terrorism of anthrax has been of great importance in the prevention and treatment of anthrax. Control means include prevention and treatment, the two are mainly dependent on anthrax vaccine and antibiotics, but both of these two drugs need to be used immediately before or in the early stage of infection. The anthrax neutralization antibody and antibody can quickly and effectively neutralize the anthrax toxin produced in the body after the infection of the host, and play the complement dependent cytotoxic effect and the antibody. The toxic effects of LYC are the most promising anthrax treatment drugs. Since the emergence of hybridoma technology in 1975, McAbs have been mainly dependent on rat McAbs for many years. But the half life of the mAb is short and the toxic and side effects are obvious, which greatly restrict its clinical application. Most of the new development projects in foreign countries have now turned to the R & D of all human McAbs. At present, the methods of preparing all human monoclonal antibodies are mainly through human hybridoma technology, transgenic mouse technology, antibody library technology and single cell PCR technology, in which single cell PCR technology can directly amplify the antibody genes from individual B cells. The process of cell fusion and antibody multi wheel screening can screen effective total human monoclonal antibodies in the shortest 7 days, and several other antibody preparation methods need several weeks or even months. The establishment of this rapid and high flux antibody screening platform will help to improve the ability to respond to new onset infectious diseases in China; and single cell PCR technology. The natural pairing of antibody heavy chain light chain in human body is preserved, so it can be more intuitionistic and in-depth study on human immune mechanism while obtaining therapeutic antibodies. However, the realization of single cell PCR technology is very difficult because of the very low gene copy number within single cell. The research team. Most of the anthrax monoclonal antibodies are still murine McAbs. A few researchers have screened the anthrax McAbs from gorillas, transgenic mice and human bodies. However, there is no full human anti anthrax monoclonal antibody reported in our country. In view of the complexity of the mechanism of anthrax toxin, more anthrax against different neutralization epitopes has been developed. Source McAbs can effectively avoid the bioterrorism threat of anthrax toxin caused by human mutation, and provide more options for the treatment of anthrax. In view of this, the aim of this paper is to establish a rapid and high flux antibody screening technology platform for flow separation single cell PCR, and the technology platform from the recombinant anthrax protective antigen (protective antigen, PA). All human PA neutralization antibodies were screened in vaccinated volunteers, and the affinity of mAb, antigen binding epitopes, toxin neutralization mechanism, and the effect of McAb combined use were studied. First, a male volunteer was immunized with recombinant PA vaccine at zeroth and 28 days, and the blood samples were collected on day 0,28,35. In order to ensure that the positive blood samples can be collected at the appropriate time point to improve the positive rate of subsequent PA antibody screening, the PA binding and neutralizing antibody titer in serum at different time points were measured by ELISA and TNA. The results showed that the titer of PA binding antibody and neutralizing antibody in the serum of thirty-fifth days were all immunized. At the same time, the number of antibody secreting cells in blood at different time points was detected by ELISpot method. The results showed that the proportion of antibody secreting cells with positive anti PA antibody secreting cells accounted for the total Ig G positive antibody secreting cells, rising from 0.8% days after immunization to thirty-fifth days after immunization, and the anti PA antibody positive was found. The number of antibody secreting cells increased significantly. It showed that the recombinant PA vaccine effectively stimulated the humoral immune response in the volunteers. The level of humoral immunity increased significantly at thirty-fifth days after immunization, which provided a guarantee for the separation of subsequent antibodies. In order to realize the separation of single B cells and the single cell PCR amplification of the antibody base, we tried a variety of details. The method of cell sorting and single cell PCR, after many failures, was finally used to separate the antibody secreting cells by flow cytometry and the single cell reverse transcription two step PCR with multiple primers combination. The antibody gene was successfully amplified from individual B cells. The primer design was found in the optimization of single cell PCR conditions. The type of enzyme and the amount of template have great influence on the amplification efficiency, and the annealing temperature has little effect on the amplification effect. After the successful implementation of the antibody separation of single antibody secreting cells and single cell PCR technology, the blood samples were collected thirty-fifth days after the volunteers' immunization, and the density gradient sedimentation method was used. After separating the peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC) from the blood samples, after the fluorescent staining of a group of specific leukocyte differentiation antigen clusters (cluster of differentiation, CD) molecules on a group of antibody secreting cells, 2112 single antibody secreting cells were selected from PBMC through a flow cytometry. Cloning, the antibody genes in single cells were amplified by single cell PCR. The results showed that the ratio of 529 single cell clone heavy chain and light chain amplification successfully, that is, the positive rate of single cell PCR is about 25%. when the hardware conditions are satisfied, only 1~2 days can be used to complete the separation of single cell and the process of amplification of antibody genes. It is very time-consuming and time-consuming to construct 529 pairs of antibody gene expression plasmids with the traditional cloning method. In order to express the antibody genes rapidly and high throughput, we designed the linear expression frame that can directly through the PCR fusion promoter, the preamble sequence, the full length gene of the antibody and the poly A tail (poly (A) tail). We optimize the linear expression frame PCR condition, and we optimize the linear expression frame of the linear expression frame. A control antibody was used to verify the expression and function of the antibody expressed in the linear expression frame. The results show that the antibody concentration in the linear expression frame method can reach 0.4 g/m L, and it keeps the antibody neutralizing effect well, and can meet the requirements of the follow-up antibody screening experiment. The linear expression frame designed by us can be used in the shortest day for 3 days. After ensuring the feasibility of the method, we constructed 529 pairs of antibody heavy chain and light chain linear expression frame according to the optimized overlap extension PCR conditions, and co transfected the paired genes into HEK 293T cells for expression. Then, 529 all human monoclonal antibodies were combined by ELISA and TNA. The specific binding PA domain of the monoclonal antibody was identified and identified. The results showed that we successfully obtained 34 PA binding monoclonal antibodies by establishing the antibody screening platform technique, of which 4 monoclonal antibodies (2A6,4A3,8H6,22F1) had PA neutralization activity. The neutralizing monoclonal antibody 22F1 was not combined with PA, but it played a role by combining with the PA's allosteric PA63. In addition, the study found that there is a toxin enhanced antibody (Toxin enhancing m Ab) that can accelerate cell death in PA binding monoclonal antibody, which accounts for more than 55.9% of the proportion of PA binding monoclonal antibodies. In order to further explore the characteristics of PA neutralization antibody and toxin enhanced antibody, 4 expression plasmids of PA neutralizing antibody and 1 toxin enhanced antibody 8A7 are constructed and transfected and purified. The antibody function was studied in the following aspects: 1) the affinity of 5 key monoclonal antibodies was measured by BLI technology. The results showed that the affinity of McAbs except 22F1 was at nanmore level; 2) neutralization activity in vivo and in vivo was measured in 4 neutralization McAbs in vivo and in vitro, and the results showed that the neutralization activity of 22F1 monoclonal antibodies in 4 neutralization McAbs. In vitro, its protective effect is nearly one order of magnitude higher than that of the PA monoclonal antibody reported in the market, while the monoclonal antibody 2A6 only has protective activity in vitro; 3) the neutralization mechanism of neutralization mAb is identified. The results show that the 8H6 monoclonal antibody has the inhibitory effect on the concentration dependent enzyme of the antibody to PA, and the 4A3 single antibody inhibits the formation of PA seven polymer by promoting the degradation of PA63, 4A 3 the toxin neutralization mechanism similar to the enzyme activity has not yet been reported, suggesting that a new neutralization mechanism of PA neutralization McAb may be discovered in this study. Toxin enhanced monoclonal antibody 8A7 can promote the formation of PA seven polymer and combine with PA seven polymer, suggesting the possible mechanism of the enhancement of the toxin; 4) analysis of the effect of the combination of McAbs and the results of 2A 6 and 4A3,8H6 and 22F1 showed a weak synergistic effect; 2A6 and 8H6,2A6 and 22F1 showed irrelevant effects; 4A3 and 8H6,4A3 and 22F1 showed antagonism. The synergistic effect of mAb combined drugs was the most significant, and the toxin enhanced the monoclonal antibody 8A7 and the neutralization activity of the monoclonal antibody only in vitro, and the combination of the two was able to play high water in the body. The results suggest that the possible cause of a large number of toxin enhanced antibodies in the human body is that the antibody can assist other antibodies in the blood to protect the body's protective effect. And the PA domain 3 of the 8A7 monoclonal antibody is a commonly considered PA non neutralization site, according to the results obtained in this article. It is presumed that the monoclonal antibody against the 3 epitopes of the PA domain can play an important biological function in coordination with other antibodies in the human body. This auxiliary monoclonal antibody is easily omitted during the antibody screening process, but its important role in the "cocktail" treatment can not be ignored. The rapid antibody screening platform for anti PCR technology can provide technical reserve for the study of new onset infectious disease antibodies; screening and preparing all human PA neutralization McAbs with different neutralization mechanisms, including new neutralization mAb, high school and active mAb, is a promising anthrax treatment drug; meanwhile, antibody neutralization mechanism, The study of epitope and combination effect also provides a strong basis for the research of the mechanism of anthrax toxin, the design of vaccine and the design of cocktail antibody drugs.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R517.2

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