新型结核病亚单位疫苗的制备和有效性评价及疫苗Ⅰ型过敏反应评价

发布时间：2018-06-22 15:44

本文选题：结核分枝杆菌 + 亚单位疫苗　；参考：《山东大学》2010年博士论文

【摘要】： 结核病(Tuberculosis, TB)是由结核分枝杆菌(Mycobacterium tuberculosis,Mtb)引起的一种古老而漫长的感染性疾病,由其导致的死亡率居全球第二位,给各国尤其是发展中国家带来了沉重的负担。据WHO统计,2007年全球新增结核病人937万,因结核病死亡人数达132万(不包括HIV阳性患者)。新增病例中亚洲和非洲分别占55%和31%,其中中国以130万新增病例位居第二位,结核病防控形势非常严峻。目前,卡介苗是唯一一种获得批准的广泛使用的结核病预防疫苗,对于儿童卡介苗具有良好的保护效果,但对于成人其保护效果不容乐观,对于抑制潜伏期结核的复发更是毫无作用。因此研制新的结核病疫苗迫在眉睫,未来的结核病疫苗不仅要对刚出生婴儿以及未感染结核菌的人群提供良好的预防作用,对于已经感染结核菌但尚未发病的已感染人群也要具有增强细胞免疫、加速清除结核菌的保护作用。本研究用分子生物学技术构建了两种克隆表达载体pET30a-Ag85b和pET30a-HspX,表达并纯化了结核分支杆菌H37Rv的保护性抗原Ag85b和休眠期高表达蛋白HspX,并与另外两种分泌性蛋白CFP-10和ESAT-6(已制备成融合蛋白CFP-10：ESAT-6)混合后,与铝佐剂和CpG DNA佐剂联合应用,制备成新型结核病亚单位疫苗。此种亚单位疫苗的制备为国内外首次报道。对此疫苗进行有效性评价,观察此疫苗的免疫原性和保护力；同时用OVA和Al(OH)3作为免疫原免疫豚鼠初步建立豚鼠Ⅰ型过敏反应模型和相关的定量评价指标，为今后疫苗的Ⅰ型过敏评价奠定基础。一、新型结核病亚单位疫苗的制备运用分子生物学技术成功构建了两种克隆表达载体pET30a-Ag85b和pET30a-HspX,并将两种载体分别转化入表达宿主菌大肠杆菌BL-21,通过IPTG诱导成功表达了两种蛋白。经验证,Ag85b为包涵体表达而HspX为上清表达；经过一系列前期处理,Ag85b上样样品经过Source30 Q阴离子交换柱层析进行一步纯化,HspX上样样品经过QHP阴离子交换柱层析,疏水层析,QHP阴离子交换柱层析进行三步纯化,分别得到了较纯的的蛋白样品。经过SDS-PAGE、蛋白质测序和WB等一系列实验,初步验证了蛋白分子量、纯度和等电点等基本理化性质以及蛋白的生物学活性，，为后期的有效性评价包括免疫原性评价和保护力评价奠定了基础。两种蛋白样品与事先制备好的另一种融合蛋白CFP-10：ESAT-6按照一定比例进行混合，之后按照顺序与铝佐剂和CpG佐剂进行混合制备成新型结核病亚单位疫苗。二、新型结核病亚单位疫苗的免疫原性评价将上述疫苗免疫小鼠对其免疫原性进行初步评价。对BALB／c小鼠进行颈背部皮下免疫,每周一次共免疫三次,末次免疫后一周,小鼠摘眼球放血处死,无菌取脾分离小鼠脾淋巴细胞；检测小鼠血清中针对三种蛋白的特异性IgG水平；体外用三种抗原分别刺激小鼠脾淋巴细胞后,通过淋巴细胞增殖实验和ELISPOT实验检测小鼠脾淋巴细胞的增殖能力和脾淋巴细胞中能分泌抗原特异性IFN-γ的细胞数量；分离小鼠腹腔巨噬细胞,体外用三种抗原刺激后,检测分泌IL-12的水平。结果表明,混合抗原与两种佐剂同时应用的组(Ag+Al+CpG)小鼠血清中特异性IgG水平、小鼠脾淋巴细胞的增殖能力、小鼠脾淋巴细胞能够分泌针对三种蛋白的特异性IFN-γ细胞数量以及巨噬细胞分泌IL-12水平均显著高于阴性对照组,与单纯混合抗原组相比也有显著升高。这表明新型疫苗在小鼠体内能同时引起较高的细胞免疫和体液免疫,同时也表明重组蛋白必须要与佐剂联合使用才能满足亚单位疫苗的要求,这也是为什么近几年进入临床评价的亚单位疫苗无一例外的采用了强大的佐剂系统。三、新型结核病亚单位疫苗的保护性评价用结核杆菌H37Rv通过腹股沟皮下注射的方式攻击豚鼠,造成结核菌感染后模型，然后用新型亚单位疫苗通过后腿肌肉注射方式进行免疫,每两周免疫一次,共免疫三次,末次免疫后两周,豚鼠腹腔注射戊巴比妥钠进行安乐死,取肝脏、脾脏和肺脏进行病理检查,计算病变评分；取脾脏研磨后接种改良罗氏培养基四周后计数菌落数计算脾菌负荷量。脾菌负荷量和脏器病变评分显示其保护效果有限,虽然好于生理盐水对照组和单纯抗原组,但尚未有统计学差异。导致保护效果有限的原因可能与疫苗制备工艺尚不成熟,各组分配比以及佐剂混合顺序不同有关,此外,结核菌感染后模型的不完善也可能是原因之一,腹股沟皮下注射H37Rv建立豚鼠感染后模型的方法有待进一步优化。本研究中的效力学实验并未设置BCG阳性对照组原因在于BCG对于结核菌感染后人群并无保护效果,而本实验效力学评价使用的豚鼠模型为感染后模型。四、疫苗Ⅰ型过敏反应阳性模型的建立疫苗的安全性评价是疫苗临床前评价的重要内容,为了给疫苗免疫毒性中Ⅰ型过敏反应的评价提供一个阳性模型,同时建立Ⅰ型过敏反应定量评价指标,本研究以OVA和Al(OH)3作为免疫原免疫豚鼠,初次免疫采取背部皮下注射的方式,然后通过后腿肌肉注射的方式进行三次加强免疫；末次免疫后一周,对模型组豚鼠用OVA通过后腿静脉注射的方式进行激发,观察并记录豚鼠的Ⅰ型过敏反应症状；对激发后的豚鼠采取支气管肺泡灌洗液,离心后收集上清检测Ⅰ型过敏反应介导介质组胺和白三烯水平,对未激发的模型组豚鼠直接采取腹腔灌洗液分离腹腔肥大细胞,体外经OVA激发后检测上清中组胺和白三烯水平。结果表明,模型组豚鼠在经OVA激发后出现明显的Ⅰ型过敏反应症状如挠耳、挠脸、摆头、竖毛、咳嗽、喷嚏、呼吸困难、步态不稳、痉挛等,对支气管肺泡灌洗液中组胺和白三烯水平进行检测发现两者水平均有显著升高；未激发的豚鼠的腹腔肥大细胞体外经OVA刺激后分泌组胺和白三烯水平也显著升高。这些结果表明我们成功的建立了豚鼠I型过敏反应模型,并且建立了几种定量评价I型过敏反应的指标,今后对于疫苗的I型过敏反应评价,我们除了观察I型过敏反应症状等定性指标外,还可以对支气管肺泡灌洗液中组胺和白三烯水平以及腹腔肥大细胞所分泌组胺和白三烯水平进行定量检测。五、结论本研究首次将结核菌保护性抗原Ag85b、潜伏期蛋白HspX和另外两种早期分泌蛋白CFP10：ESAT6混合,并与两种佐剂CpG DNA和Al(OH)3联用,制备成一种新型结核病亚单位疫苗。对这种新型疫苗进行免疫原性和结核菌感染后的保护力评价,发现具有较强的免疫原性但对于结核菌感染后豚鼠保护力有限,我们计划经过后续对蛋白表达形式的优化和疫苗配比的改进以期获得更好的保护效果,从而为结核菌已感染人群提供一种理想的治疗性疫苗。同时本疫苗含有结核菌早期分泌蛋白成分也使其可能成为针对未感染人群的一种预防性疫苗或者BCG的加强疫苗。初步建立了一种豚鼠Ⅰ型过敏反应模型,建立了BALF上清中和腹腔肥大细胞体外刺激培养液中组胺和白三烯的检测方法,建立的Ⅰ型过敏反应模型和组胺、白三烯等定量检测指标可为疫苗的Ⅰ型过敏反应评价提供新的方法。
[Abstract]:Tuberculosis (TB) is an ancient and long, long, infectious disease caused by Mycobacterium tuberculosis (Mtb). The death rate is the second largest in the world, bringing a heavy burden to all countries, especially the developing countries. According to WHO statistics, the total number of new TB patients worldwide in 2007 was killed by tuberculosis. The number of dead people was 1 million 320 thousand (excluding HIV positive). Among the new cases, Asia and Africa accounted for 55% and 31% respectively, of which 1 million 300 thousand of the new cases in China ranked second, and the situation of tuberculosis prevention and control was very severe.
At present, Bacillus Calmette Guerin is the only widely used vaccine for tuberculosis prevention, which has good protective effect on BCG in children, but it is not optimistic for adults and has no effect on inhibiting the recurrence of latent tuberculosis. Therefore, the development of a new tuberculosis vaccine is imminent and the future tuberculosis epidemic is imminent. The vaccine should not only provide a good preventive effect on the newborn babies and those who have not infected the Mycobacterium tuberculosis, but also have the enhancement of cellular immunity to the infected people who have been infected but have not yet been infected, and accelerate the protection of the tuberculosis.
In this study, two cloned expression vectors, pET30a-Ag85b and pET30a-HspX, were constructed with molecular biology techniques, expressing and purifying the protective antigen Ag85b of Mycobacterium tuberculosis H37Rv and the hibernation high expression protein HspX, and mixed with two other secretory proteins, CFP-10 and ESAT-6 (prepared fusion protein CFP-10:ESAT-6). In combination with CpG DNA adjuvant, a new subunit vaccine for tuberculosis was prepared. The preparation of this subunit vaccine was first reported at home and abroad. The effectiveness of the vaccine was evaluated, the immunogenicity and protective ability of the vaccine were observed. At the same time, OVA and Al (OH) 3 were used as immunogenic immunogenic guinea pigs to establish guinea pig type I anaphylaxis model. And related quantitative evaluation indicators will lay the foundation for future type I allergy evaluation of vaccines.
Preparation of a new subunit vaccine for tuberculosis
Two kinds of cloning expression vectors pET30a-Ag85b and pET30a-HspX were successfully constructed by molecular biology technology, and two vectors were transformed into Escherichia coli BL-21 respectively, and two proteins were successfully expressed through IPTG. It was proved that Ag85b was expressed as inclusion body and HspX was expressed in the supernatant; after a series of preprocessing, Ag85b Samples were purified by Source30 Q anion exchange column chromatography. Samples of HspX samples were purified by QHP anion exchange column chromatography, hydrophobicity chromatography and QHP anion exchange column chromatography. A series of pure protein samples were obtained respectively. After a series of experiments, such as SDS-PAGE, protein sequencing and WB, the protein molecules were preliminarily verified. The basic physicochemical properties of the quantity, purity and isoelectric point, as well as the biological activity of the protein, lay the foundation for the evaluation of the later effectiveness, including the evaluation of immunogenicity and the evaluation of protective ability.
The two protein samples were mixed in a certain proportion with a pre prepared fusion protein CFP-10:ESAT-6, then mixed with aluminum and CpG adjuvant in order to prepare a new subunit vaccine for tuberculosis.
Two. Evaluation of the immunogenicity of a new subunit vaccine against tuberculosis.
The immunogenicity of the mice immunized with the above vaccine was preliminarily evaluated. The BALB / c mice were immunized with the back of the neck. The mice were immunized three times a week. One week after the last immunization, the mice were killed and the spleen lymphocytes were isolated from the spleen, and the specific IgG level of the three proteins in the mice serum was detected. After three antigens were used to stimulate the spleen lymphocytes of mice, the proliferation of lymphocyte and the number of antigen specific IFN- gamma cells in splenic lymphocytes were detected by lymphocyte proliferation test and ELISPOT test, and the level of IL-12 was detected after three kinds of antigen stimulation in vitro of peritoneal macrophages in mice. The results showed that the specific IgG level in the serum of the mixed antigen and the two adjuvants (Ag+Al+CpG), the proliferation of mouse spleen lymphocyte, the number of specific IFN- gamma cells secreted by the spleen lymphocyte and the secretion of IL-12 in the macrophage were significantly higher than that of the negative control group. This shows that the new vaccine can cause higher cellular immunity and humoral immunity in mice, and that the recombinant protein must be combined with the adjuvant in order to meet the requirements of subunit vaccine, which is why the subunit vaccine entered into clinical evaluation in recent years is no exception. A powerful adjuvant system is used.
Three, the protective evaluation of a new subunit vaccine against tuberculosis.
The guinea pig was attacked by subcutaneous injection of Mycobacterium tuberculosis H37Rv, resulting in the model of Mycobacterium tuberculosis after infection, and then immunized with a new subunit vaccine through the hind leg muscle injection, immunized once every two weeks, three times, and two weeks after the last immunization, and pentobarbital was injected into the abdominal cavity of guinea pigs for euthanasia, liver and spleen. The pathological examination was carried out with the lungs to calculate the lesion score, and the spleen load was calculated after the inoculation of the spleen after lapping the improved Roche culture medium. The spleen load and organ lesion score showed that the protective effect was limited, although it was better than the normal saline control group and the former group of Dan Chunkang, but there was no statistical difference. The cause of the limited fruit may be not mature with the vaccine preparation process, the distribution ratio of each group and the mixing order of the adjuvant are different. In addition, the imperfect model of the Mycobacterium tuberculosis may be one of the reasons. The method of subcutaneous injection of H37Rv in the groin to establish the model of the guinea pig after infection needs to be further optimized. The reason for the setting of BCG positive control group was that BCG had no protective effect on the population after tuberculosis infection, and the guinea pig model used in the effectiveness evaluation of this experiment was the post infection model.
Four, the establishment of an allergic reaction model for vaccine type I
The safety evaluation of the vaccine is an important part of the pre clinical evaluation of the vaccine. In order to provide a positive model for the evaluation of type I anaphylaxis in vaccine immuno toxicity, and to establish a quantitative evaluation index of type I anaphylaxis, this study takes OVA and Al (OH) 3 as the immunogenicity of the immunogen mouse, the first immunization by subcutaneous injection of the back. After the last week, the guinea pigs in the model group were stimulated by the injection of OVA through the hind leg vein to observe and record the symptoms of type I anaphylaxis in the guinea pig. The bronchoalveolar lavage fluid of the guinea pigs after the stimulation was taken and the supernatant was collected to detect type I allergy after the centrifuge was centrifuged after the last immunization. The reaction mediates the level of histamine and leukotriene, and the abdominal mast cells are isolated from the non excited model group of guinea pigs directly by intraperitoneal lavage solution, and the levels of histamine and leukotrienes in the supernatant are detected by OVA in vitro.
The results showed that the guinea pigs in the model group had obvious symptoms of type I anaphylaxis, such as ear, flex, head, hair, coughing, sneezing, dyspnea, gait instability, spasm and so on. The levels of histamine and leukotrienes in bronchoalveolar lavage fluid were significantly increased, and the abdominal cavity of unstimulated Guinea pigs was found. The secretory histamine and leukotriene levels of the mast cells also increased significantly after OVA stimulation in vitro. These results suggest that we have successfully established the guinea pig I anaphylaxis model, and established several indicators for quantitative evaluation of the I anaphylaxis. In the future, we evaluate the I anaphylaxis of the vaccine, in addition to observing the symptoms of the I allergic reaction. In addition, the levels of histamine and leukotrienes in bronchoalveolar lavage fluid and the levels of histamine and leukotrienes secreted by mast cells in the abdominal cavity can also be quantified.
Five. Conclusion
For the first time, a new type of subunit vaccine for tuberculosis was prepared by mixing the protective antigen Ag85b, latent protein HspX and two other early secretory protein CFP10:ESAT6 and combined with two kinds of adjuvant, CpG DNA and Al (OH) 3.
To evaluate the immunogenicity of the new vaccine and the protective effect of Mycobacterium tuberculosis infection, it is found that the vaccine has a strong immunogenicity but limited protection force for the guinea pig after tuberculosis infection. We plan to improve the protein expression and improve the ratio of the vaccine in order to obtain better protective effect, so that the Mycobacterium tuberculosis has been felt. The infected population provides an ideal therapeutic vaccine. The vaccine contains early secretory protein components from Mycobacterium tuberculosis and may also make it a preventive vaccine against uninfected people or a strengthened vaccine for BCG.
A guinea pig type I anaphylaxis model was established. The detection methods of histamine and leukotriene in the culture medium of BALF supernatant and peritoneal mast cells in vitro were established. The model of type I anaphylaxis and histamine, leukotrienes and other quantitative indicators could provide a new method for the evaluation of type I anaphylaxis.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

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