2型登革病毒特异性单克隆抗体保护作用的实验研究

发布时间：2018-06-19 15:27

本文选题：登革病毒 + 单克隆抗体　；参考：《第三军医大学》2008年博士论文

【摘要】： 登革病毒(Dengue Virus,DENV)是黄病毒属、有包膜的单股正链RNA病毒,由于包膜蛋白的抗原性不同,又分为四种血清型,即DENV1～4。DENV主要以埃及伊蚊和白纹伊蚊为媒介而广泛流行于热带和亚热带地区。每年,DENV会导致数百万人感染,引起登革热(dengue classical fever,DF)和登革出血热/休克综合症(dengue hemorrhagic fever/dengue shock syndrome, DHF/DSS)。DF是自限性发热性疾病;而DHF/DSS则是威胁患者生命的重症,其主要特征是血管通透性显著增加,导致血浆渗漏。每年有大约50万DHF/DSS患者需要及时入院治疗,否则病死率高达50 %。然而,DHF/DSS的发病机制,以及DENV与宿主细胞之间相互作用的机制尚不清楚。虽然在过去几十年中,研究者从多方式、多角度入手研制DENV的疫苗,但至今仍没有预防和治疗DENV感染的疫苗和药物。无论是减毒活疫苗、多价疫苗,还是核酸疫苗、蛋白成分疫苗都遇到DENV流行株抗原变异、抗体增强效应(antibody dependent enhancement,ADE)等障碍而进展缓慢。基于近年来DHF/DSS患者数量明显增加和上述研究现状,被动免疫或者说是治疗性单克隆抗体(Monoclonal antibodies,mAb)再次引起了研究者的关注。早在1907年人体抗血清就被用来治疗麻疹病毒感染的患者。20世纪40年代起,从高免疫的血清中提取的免疫球蛋白来预防和治疗病毒感染成为热点,部分制品至今还被用于临床治疗。而特异性不高、成分复杂和潜在的疾病传播途径等缺陷极大的限制了此类生物制剂的使用。1982年mAb技术的出现后,研发高中和特性的mAb用于预防和治疗病毒性疾病就受到研究者的青睐。特别是进入21世纪以来,生物技术和工业技术的进步,大量的治疗性mAb药物已进入临床实验或已应用于临床,成为生物制药中增长最快的部分。治疗性mAb成为防治病毒的新手段,新焦点。因此,无论从研究DENV病理机制角度,还是从研发治疗性mAb角度,获得多株特异性mAb具有重要意义。特别是具有高中和能力的mAb,是研究致病机制的免疫学工具和治疗性mAb的基本材料。本试验首先通过蔗糖密度梯度高速离心获得纯化的DENV2(Tr1751)株的具感染活性的病毒颗粒,并利用纯化的病毒颗粒免疫SPF级BALB/c小鼠。之后通过杂交瘤细胞技术获得分泌DENV特异性mAb的杂交瘤细胞株10株,78#、1E7、2B10、6C1、6H3、7F7、8B6、8F12、8H1、8H4。然后分别对10株mAb进行性质鉴定,其中具有中和能力的mAb有7株(2B10、78#、1E7、7F7、8B6、8F12、8H4)包括2株中和能力较高的mAbs(78#、8F12),并对其在体内外的保护活性进行验证。结果提示通过全病毒免疫来制备中和抗体具有一定的优势,获得的mAbs也为后续研究DENV的致病机理,以及治疗性mAb的研发提供了有利的免疫学工具。本研究主要结果与结论如下: 1.建立了纯化的DENV2的方法,获得具有感染性的纯化病毒本实验通过DENV2的天然中介宿主细胞白纹伊蚊C6/36细胞增殖出大量病毒,并测定病毒滴度。除去细胞碎片后,使用PEG-8000对病毒进行浓缩,测定浓度病毒滴度。最后通过蔗糖密度梯度高速离心来获得纯化的DENV2具感染性的病毒颗粒,并通过病毒滴度测定和SDS-PAGE来验证纯化病毒及其含量。通过不同蔗糖密度梯度比较,确定了DENV2主要沉降区带,建立了纯化DENV的方法,并制备了一定量的纯化病毒供下述实验使用。 2.通过杂交瘤技术获得10株鼠源性抗DENV2的mAbs 利用纯化的DENV2病毒颗粒免疫SPF级的BALB/c小鼠10只,细胞融合前取小鼠尾血进行细胞爬片间接免疫荧光染色,检测特异性抗体产生情况。无菌条件下取出免疫效果好的小鼠脾脏,机械分离为单个细胞后与小鼠骨髓瘤细胞株SP2/0在融合剂PEG-4000的作用下形成杂交瘤细胞。通过HAT培养筛选、间接ELISA阳性筛选和反复单克隆化等方法,获得了10株分泌DENV2特异性mAbs的稳定杂交瘤细胞株,命名为78#、1E7、2B10、6C1、6H3、7F7、8B6、8F12、8H1、8H4。以无血清1640培养液分别培养各杂交瘤细胞来获得浓度较低的mAb或通过小鼠腹腔注射培养来获得大量较高浓度的mAb腹水,供下述实验使用。 3. 10株mAbs性质的鉴定 (1)间接ELISA验证10株mAbs的特异性。以纯化的DENV2为抗原包被96孔板,同时用1 % BSA包被96孔板作为阴性对照。以mAb上清液或腹水为一抗,HRP-山羊抗小鼠IgG为二抗,OPD显色。终止显色后测定OD492nm。以吸光值大于对照值2.1倍为阳性。制备的10株mAbs上清或腹水反映强弱各不相同,但均显示为阳性。 (2)通过间接ELISA进行抗体饱和曲线的绘制。实验步骤同前,首先确定抗原最适包被浓度,然后将各株mAb腹水倍比稀释后作为一抗,显色后绘制OD492nm曲线,通过分析抗体饱和曲线得出各株抗体饱和稀释度。对于识别相同病毒蛋白,并且抗体亚型相同的mAbs,我们通过抗体叠加实验来验证其识别的抗原位点是否重叠。将这些mAbs稀释到各自的饱和浓度后,分别进行ELISA叠加实验,结果显示均出现较明显的叠加阳性,证实它们识别不同的病毒抗原位点。 (3)间接免疫荧光实验测定各mAb对天然病毒抗原的识别情况。将感染DENV2后发病的幼鼠脑制成组织切片进行间接免疫荧光测定,发现8株mAbs(78#、1E7、2B10、6C1、7F7、8B6、8F12、8H4)能出现特异性荧光,而2株mAbs(6H3、8H1)没有特异性反应出现。 (4)Western-blot确定各株mAb识别的病毒蛋白。纯化病毒经过SDS-PAGE后,半干转印到醋酸纤维膜上,按泳道剪条后,分别孵育各mAb,以HRP-山羊抗小鼠IgG为二抗,DAB显色。10株mAbs中有6株(78#、1E7、2B10、8B6、8F12、8H1)识别DENV2 E蛋白,1株(6C1)识别非结构蛋白NS3,2株(8H1、6H3)识别非结构蛋白NS5,1株(7F7)无特异性反应条带出现。 (5)其它指标:通过Sigma抗体分型试剂盒对各株mAb的抗体型和亚型进行鉴定。通过Giemsa染色确定杂交瘤细胞的染色体数量,检测杂交瘤细胞传代或复苏后的细胞稳定状态。 4. PRNT测定10株mAbs的中和活性以非洲绿猴Vero细胞为测试细胞,mAb-DENV2混合物为感染源,正常鼠血清(normal mouse sera,NMS)-DENV2混合物为阴性对照,测定50 %病毒中和浓度——PRNT50。待Vero细胞在24孔板中单层覆盖生长后,将mAb(或NMS)倍比稀释1:10、1:20、1:40、1:80、1:160、1:320。加入等体积的病毒稀释液后,mAb(或NMS)的实际稀释度为1:20、1:40、1:80、1:160、1:320、1:640。控制加入的病毒量,1 ml混合物中含有约1000 PFU的DENV2。mAb-DENV2(或NMS- DENV2)混合物37°C水浴1 h后,每孔200μl加入各孔中37°C孵育1.5 h。每孔约含有200 PFU的病毒和不同稀释度的抗体(或NMS)。之后以含1.2 %的甲基纤维素的MEM培养基培养7天,再通过结晶紫染色对病毒形成的噬斑(Plaque)进行计数。通过比对相同稀释度NMS的阴性孔,观察各株mAb有无中和作用,并计算中和抗体的PRNT50值。具有中和能力的mAb有7株:78#(1:60)、1E7(1:30)、2B10(1:35)、7F7(1:20)、8B6(1:30)、8F12(1:40)、8H4(1:15)。其中具有较高中和能力的2株mAbs:78#和8F12,两者混合后的PRNT50达到1:80。 5. BALB/c乳鼠保护实验以BALB/c新生乳鼠(生后第2天)为DENV2挑战及抗体体内保护的研究对象,以体外实验中具有较高中和能力的78#和8F12 mAbs为实验对象,NMS为阴性对照。将mAb-DENV2或NMS-DENV2混合物37°C水浴1 h后,通过乳鼠脑内注射,进行病毒挑战实验。在对高低两个病毒进入量104 PFU和500 PFU进行比较的同时,分别对两株mAbs的体内保护性进行研究。发现它们均在一定程度上起到保护作用,乳鼠发病和死亡时间均比对照组延后。特别是在病毒进入量较低时,保护作用更为明显。在上述实验的基础上,将两株mAbs进行“cocktail”组合实验。取500 PFU的病毒进入量,mAbs-DENV2于37°C水浴1 h后注射。结果显示抗体的保护作用大幅度提高。乳鼠发病和死亡时间比对照组延后更显著,并且有16.7 %的乳鼠被彻底保护(存活超过2周的观察期)。总之,本实验建立了通过蔗糖密度梯度高速离心来纯化DENV2的方法,并以纯化病毒为抗原免疫BALB/c小鼠。通过免疫小鼠脾细胞与SP2/0细胞融合、筛选,获得了10株DENV2特异性的mAbs。经过性质鉴定,我们对10株mAbs所识别的病毒蛋白、抗体饱和度、免疫学活性等多方面进行性质鉴定。同时,通过PRNT对它们的中和特性进行检测。在7株具有中和能力的mAbs中,78#和8F12具有较高的中和能力。进而又对这2株mAbs进行了BALB/c乳鼠保护实验,结果提示它们具有一定的保护作用,而两者的混合物保护作用更明显。因此,10株mAbs是进一步研究DENV2感染机制的有效免疫学工具。同时,具有较好中和能力的mAbs也为后续研究治疗性mAb的提供了初步的材料。
[Abstract]:Dengue Virus (DENV) is the genus of the yellow virus and the membrane of the single strand RNA virus. Because of the antigenicity of the envelope protein, it is divided into four serotypes. That is, DENV1 to 4.DENV is widely prevalent in the tropics and subtropics in the medium of Aedes aegypti and Aedes albopictus. Every year, DENV causes millions of people to infect and cause dengue. Dengue classical fever (DF) and dengue hemorrhagic fever / shock syndrome (dengue hemorrhagic fever/dengue shock syndrome, DHF/DSS) are self limiting febrile diseases; DHF/DSS is a serious threat to patients' life, the main feature of which is the significant increase in vascular permeability, resulting in plasma leakage. About 500 thousand of the patients are required each year. Hospitalization should be timely, or the mortality rate is as high as 50%. However, the pathogenesis of DHF/DSS and the mechanism of interaction between DENV and host cells are not yet clear.
Although in the past few decades, researchers have developed vaccines for DENV from multiple ways and multiple angles, but there are still no vaccines and drugs to prevent and treat DENV infection. Both live attenuated vaccines, polyvalent and nucleic acid vaccines, protein components vaccines meet the DENV epidemic strain variation, the antibody enhancement effect (antibody dependent enha) Ncement, ADE) and other obstacles have progressed slowly.
Based on the recent increase in the number of patients with DHF/DSS and the present situation, passive immunity or therapeutic monoclonal antibodies (Monoclonal antibodies, mAb) have attracted the attention of researchers again. People who were used to treat measles virus infection early in 1907 were raised from the high immune sera in the 40s of the.20 century. The immunoglobulin to prevent and treat viral infection has become a hot spot, and some of the products are still used for clinical treatment. But the specificity is not high, the complexity of the ingredients and the potential for the spread of the disease are greatly limited in the use of such biological agents in the use of.1982 mAb Technology, and the development of high school and characteristic mAb for prevention and treatment. Viral diseases are favored by researchers, especially since the twenty-first Century, the progress in biotechnology and industrial technology, a large number of therapeutic mAb drugs have entered clinical trials or have been applied to the clinic, becoming the fastest growing part of biopharmaceuticals. Therapeutic mAb has become a new and new focus for the prevention and treatment of viruses.
Therefore, it is of great significance to obtain multiple specific mAb from the study of DENV's pathological mechanism or from the perspective of developing therapeutic mAb. Especially, the mAb with high school and ability is an immunological tool to study the pathogenesis and the basic material for the therapeutic mAb. This experiment first passed the sucrose density gradient centrifugation to obtain the purified DENV. 2 (Tr1751) strain of the infected virus particles, and immunized SPF BALB/c mice with purified virus particles. After the hybridoma cell technique, 10 hybridoma cells secreting DENV specific mAb, 78#, 1E7,2B10,6C1,6H3,7F7,8B6,8F12,8H1,8H4., were then identified to 10 strains of mAb, which had neutralization ability. 7 strains of mAb (2B10,78#, 1E7,7F7,8B6,8F12,8H4) including 2 neutralized mAbs (78#, 8F12) and their protective activity in vivo and in vivo are verified. The results suggest that the neutralization antibody is prepared through the whole virus immunity. The obtained mAbs is also a continuation of the pathogenesis of DENV, as well as the development of therapeutic mAb. A beneficial immunological tool.
The main results and conclusions of this study are as follows:
1. a method of purifying DENV2 was established to obtain infectious purified virus.
In this experiment, a large number of viruses were proliferated by DENV2's natural medium host cells of Aedes albopictus C6/36 cells and the virus titer was measured. After removing the cell fragments, the virus was concentrated by PEG-8000 to determine the titer's titer. Finally, the purified DENV2 infected virus particles were obtained by sucrose density gradient high speed centrifugation. The virus titer and SDS-PAGE were used to verify the purified virus and its content. Through the comparison of different sucrose density gradient, the main subsidence zone of DENV2 was determined, the method of purifying DENV was established, and a certain amount of purified virus was prepared for the following experiment.
2. 10 strains of murine anti DENV2 mAbs were obtained by hybridoma technique.
The purified DENV2 virus particles were used to immunization 10 mice of SPF grade BALB/c mice. The mouse tail blood was taken to detect the specific antibody production by indirect immunofluorescence staining of the mouse tail blood before fusion. The spleen of mice with good immune effect was removed under aseptic conditions, and the machine was separated into a single cell and mouse myeloma cell line SP2/0 in the fusion agent P. Hybridoma cells were formed under the action of EG-4000. Through HAT culture screening, indirect ELISA positive screening and repeated monoclonal, 10 stable hybridoma cell lines secreting DENV2 specific mAbs were obtained, named 78#, and 1E7,2B10,6C1,6H3,7F7,8B6,8F12,8H1,8H4. was cultured in serum-free 1640 culture solution to obtain the hybridoma cells respectively. A high concentration of mAb ascites was obtained by low concentration of mAb or by intraperitoneal injection in mice for use in the following experiments.
Identification of the properties of 3.10 strains of mAbs
(1) the specificity of 10 strains of mAbs was verified by indirect ELISA. The purified DENV2 was coated with 96 orifice plates, and 1% BSA was used as a negative control with 96 orifice plates. MAb supernatant or ascites was a resistance, HRP- Goat anti mouse IgG was two, OPD coloured. The determination of OD492nm. was positive with 2.1 times more than 2.1 times the control value. The 10 mAbs prepared. Supernatants or ascites reflect strong or weak, but all show positive.
(2) draw the antibody saturation curve through indirect ELISA. The experiment step is first to determine the optimum concentration of the antigen, then the mAb ascites of each strain is diluted as one anti, and then the OD492nm curve is drawn after coloring, and the antibody saturation curve is analyzed. With the same subtype of mAbs, we tested the overlap of the antigens identified by the antibody superposition experiment. After diluting these mAbs to their respective saturated concentrations, the ELISA superposition experiment was carried out respectively. The results showed that there were obvious superposition positive, which confirmed the identification of different viral antigen loci.
(3) indirect immunofluorescence test was used to determine the identification of mAb for natural virus antigen. Indirect immunofluorescence was used to make tissue slices of young mice infected with DENV2 after infection. It was found that 8 strains of mAbs (78#, 1E7,2B10,6C1,7F7,8B6,8F12,8H4) could have specific fluorescence, and 2 strains of mAbs (6H3,8H1) had no specific reaction.
(4) Western-blot determined the virus protein identified by mAb. After the purification of the virus, the virus was transferred to the cellulose acetate membrane by SDS-PAGE, and after the strip was cut, the mAb was incubated respectively. The HRP- Goat anti mouse IgG was two, and 6 strains of DAB color.10 strain (78#, 1E7,2B10,8B6,8F12,8H1) identified the DENV2 protein, and 1 strains identified the non structural protein. 2 strains (8H1,6H3) identified non structural protein NS5,1 strain (7F7) with no specific reaction bands.
(5) other indicators: the Sigma antibody typing kit was used to identify the type and subtype of each strain of mAb. The number of chromosomes of hybridoma cells was determined by Giemsa staining, and the cell stability of hybridoma cells was detected and after resuscitation.
Determination of neutralization activity of 10 strains of mAbs by 4. PRNT
The mAb-DENV2 mixture was the source of infection, and the normal rat serum (normal mouse sera, NMS) -DENV2 mixture was negative control, and the neutralization concentration of 50% virus - PRNT50. was determined - PRNT50., taking the Vero cell of African green monkey as the test cell.
After the growth of a single layer of Vero cells in a 24 orifice plate, after adding mAb (or NMS) times the dilution 1:10,1:20,1:40,1:80,1:160,1:320. to the equal volume of viral diluents, the actual dilution of mAb (or NMS) is the amount of virus added by 1:20,1:40,1:80,1:160,1:320,1:640. control, and the 1 ml mixture contains about 1000 PFU DENV2.mAb-DENV2 (or NMS- DENV2). After the mixture of 37 C water bath 1 h, 200 mu l per pore was incubated at 37 degrees in each hole and 1.5 h. containing 200 PFU virus and different dilution antibodies (or NMS). Then, the MEM culture medium containing 1.2% methyl cellulose was cultured for 7 days, and then the virus plaque (Plaque) was counted by crystal violet staining. By comparison of the same dilution NMS. The negative pores were used to observe whether mAb had neutralization and calculated the PRNT50 value of neutralization antibody. The neutralizing mAb had 7 strains: 78# (1:60), 1E7 (1:30), 2B10 (1:35), 7F7, 8F12, 8F12, 8H4, of which 2 plants with higher neutralization capacity were mixed.
5. BALB/c milk rat protection experiment
The BALB/c neonatal rats (second days after birth) were the subjects of DENV2 challenge and antibody in vivo protection. The 78# and 8F12 mAbs with high neutralization ability in the experiment were tested and NMS was negative control. After the mixture of mAb-DENV2 or NMS-DENV2 mixture 37 degree C water bath 1 h, the test of virus challenge was carried out by intramuscular injection of the rat brain. The high and low two were high and low. In comparison with 104 PFU and 500 PFU, two strains of mAbs were studied in vivo. They were found to play a protective role to some extent, and the onset and death time of the mice were all longer than those of the control group. Two strains of mAbs were carried out in the "cocktail" combination test. The virus entry of 500 PFU was taken and mAbs-DENV2 was injected after 1 h in 37 C water bath. The results showed that the protective effect of the antibody was greatly improved. The onset and death time of the mice were more significant than that of the control group, and 16.7% of the mice were thoroughly protected (surviving for more than 2 weeks of observation).
In this experiment, the method of purifying DENV2 by sucrose density gradient high speed centrifugation was established and BALB/c mice were immunized with the purified virus as antigen. Through the fusion of mouse spleen cells and SP2/0 cells, 10 DENV2 specific mAbs. were identified. The virus proteins identified by 10 strains of mAbs and the saturation of antibody were obtained. They were identified in many aspects of immunological activity. Meanwhile, their neutralization properties were detected by PRNT. In 7 mAbs with neutralization capacity, 78# and 8F12 had higher neutralization ability. Then, the 2 mAbs was protected by BALB/c milk mice. The results suggest that they have a certain protective effect and a mixture of both. The protective effect is more obvious. Therefore, 10 mAbs strains are an effective immunological tool to further study the mechanism of DENV2 infection. At the same time, the better neutralizing mAbs also provides preliminary materials for the follow-up study of therapeutic mAb.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

【共引文献】