2型登革病毒特异性单克隆抗体保护作用的实验研究
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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。
Dengue virus (DENV) is an enveloped virus belonging to mosquito-borne flaviviruses and possesses a positive stranded RNA genome. And DENV has 4 distinct serotypes (DENV1～4) derived from the different of envelop protein. DENV is transmitted by Aedes mosquitoes and Aedes aegypti, and it causes infections which leading to clinical symptoms ranging from self-limited, acute, febrile disease called dengue fever (DF) to life-threatening dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). Over 2.5 billion people, in more than 100 countries, are at risk of dengue infection, with millions of cases occurring around the world every year in the tropics and subtropics worldwide. Now WHO already put DHF/DSS, Hepatitis, Malaria and Tuberculosis into the list of the most four serious infectious diseases of the whole world. However, the pathogenesis of DENV infection is not clear, and there is not an efficient way to deal with the disease in the clinic therapy.
Epidemiological studies indicated that DHF/DSS was more commonly seen during secondary infection with different serotype of DENV from that caused the primary infection. Antibody dependent enhancement (ADE) in DENV infection was suggested as one of explanation for this phenomenon and became a barrier for development of DENV vaccine. And there are two more mechanisms about the pathogenesis of DHF/DSS: (1) the virulence or replication ability of the virus; (2) immuno-pathogenesis theory: the cross reactive T-cell results in the immuno-damage. Recently the high titer of virus, secondary infection with different virus serotype and DENV2 were thought to be the three main factors to cause the DHF/DSS.
In recent several years, various strategies for DENV vaccine development were being pursued, from attenuated viruses and inactivated viruses to DNA vaccine and recombinant preparations. Especially vaccine development focuses on tetravalencea vaccine to defend four serologic types. Although the live attenuated tetravalencea vaccine had been regarded as the most potential vaccine, unfortunately there is no an available and safe vaccine up to now.
Based on above-mentioned situation of DENV infection and their clinic therapy, researchers think about passive immunity or therapy-monoclonal antibodies once more. As early as 1907, sera from individuals recovering from rubeola (measles) were used to prevent infection from this highly contagious virus. During the 1940s, improvements in the quality of fractionated human immunoglobulin led to the widespread use of intramuscular injections of pooled human immunoglobulin to prevent and treat viral infections. Many of these products are still on the market today. However, the disadvantages associated with polyclonal antibody products are that the vast majority of their constituent virus-specific antibodies are non-neutralizing、batch-to-batch variation and risk of pathogen transmission.
With the development of monoclonal antibody (mAb) preparation since 1982, using specific mAb with neutralizing ability to protect and therapy virus diseases became new focus. These successes, in both industrial and academic laboratories, coupled with ongoing changes in the biomedical and regulatory environments, herald an era when the commercial development of human antiviral mAb therapies will likely surge. Thus it is a basic of the stratagem to obtain neutralizing antibodies, especially neutralizing mAbs.
In the present study, purified DENV serotype 2 (DENV2) obtained from density gradient centrifugation was used as antigen to immunize BALB/c mice. After cells fusion and identification by indirect enzyme-linked immunosorbent assay (ELISA), Western-blot and indirect immunofluorescence assay (IFA), ten mAbs (78#、1E7、2B10、6C1、6H3、7F7、8B6、8F12、8H1 and 8H4) were generated and they showed different neutralizing activity to DENV2 in vitro. 78# and 8F12 had neutralizing ability and could protect suckling mice from the virus challenge partly. Our results provided insight into potential application of these mAbs for prophylactic and therapeutic purposes. The main results were as follows:
RESULTS
1. Preparation and verification of purified virus
DENV2 (strain Tr1751) was propagated in C6/36 cells and the supernatant was obtained and stored at–80°C, and the titre of 3 litre liquid was about 1×107 PFU/ml. After PEG-8000 concentrated, the titre recovered around 2×107 PFU/ml. The suspension was loaded onto pre-formed gradients made up of 30 %, 40 %, 50 %, 55 % and 60 % (w/v) sucrose, and ultra-centrifuged at 110 000×g. At the end of sucrose density gradient centrifugation, the band between 55 %～60 % (w/v) was the main band which DENV sedimentation. The purified virus was collected for the studies behind after confirmed by SDS-PAGE and plaque assay (about 1×106 PFU/ml).
2. Obtaining ten hybridoma cells which secrete the specific mAb against DENV2
BALB/c mice were immunized with purified DENV2 and an equal volume of complete Freund’s adjuvant. After booster immunizations and last immunization, the spleen cells from immunized mouse were fused with SP2/0 myeloma cells by PEG-4000. Then the hybridomas were selected by HAT culture medium. The supernatants of living cells were subjected to test antibody titer by indirect ELISA. Positive hybridomas were selected and cloned 5～8 times, then ten hybridomas named as 78#、1E7、2B10、6C1、6H3、7F7、8B6、8F12、8H1 and 8H4 were obtained. MAbs were harvested from hybridomas grown in 1640 medium without fetal bovine serum. The hybridomas were intraperitoneally injected to BALB/c mice for production ascites of mAbs respectively. The ascites were collected for the following examinations.
3. Identification of mAbs specificity
(1) Indirect ELISA
The 96-well plates were coated with purified DENV2 or bovine serum albumin. The supernatants of hybridomas were added as primary antibody respectively. Mouse anti-DENV2 sera and normal mouse sera (NMS) were used as positive and negative controls. HRP-conjugated goat anti-mouse IgG was used as secondary antibody, and coloration with o-phenylenediamine. The absorbencies were determined at 492nm. The absorbencies values of ten hybridomas were 2.1-fold higher than that in controls.
(2) Saturation curve of mAb
The protocol of this experiment is same as indirct ELISA. After optimization concentration of antigen was confirmed, mAb ascites were diluted into 1:10、1:20、1:40、1:80、1:160、1:320、1:640、1:1280、1:2560、1:5120、1:10240、1:20480 as primary antibodies respectively. Then the saturation curve of mAbs OD492nm were drew, and from the curve we got the mAbs’degrees of saturation.
We selected mAbs which recognized the same virus protein and belonged to the same subclass of Ig to undergo build-up assay. On the base of mAbs’degrees of saturation, 8F12 + 1E7 and 78# + 2B10 were chosen to build up. The results showed OD492nm step up after mAbs overlay and suggested that they recognized different epitopes.
(3) Indirect immunofluorescence assay (IFA)
Three-week-old female BALB/c mice were inoculated intracerebrally with DENV2. On the day of paralysis onset, the brains were taken out from the mice and then subjected to prepare freezen sections. The sections were incubated with mAb ascites as primary antibodies and then incubated with FITC-conjugated goat anti-mouse IgG as secondary antibody. The images were observed under fluorescence microscope. Anti-DENV2 sera and NMS were used as positive and negative controls respectively. Eight mAbs (78#、1E7、2B10、6C1、7F7、8B6、8F12、8H4) showed positive reaction with brain sections of DENV2-infected mice. Strong fluorescence intensity was observed in the cytoplasm of the cells in hippocampus, and the distribution of specific reaction was similar with that seen in section stained with mouse anti-DENV2 sera. However, the intensity of fluorescence of mAb was weak as compared with positive control. Specific reaction was rarely observed in section stained with two mAbs (6H3、8H1) and NMS.
(4) SDS-PAGE and Western-blot
The purified virus was denatured and applied onto the gels. After electrophoresis, the gels were stained with coomassie brilliant blue or transferred onto nitrocellulose membrane for western-blot analysis. In SDS-PAGE profile, main DENV2 proteins were detected with estimated molecular masses ranging from 40 to 100 Kd. Western-blot was carried out to identity the specificity of mAbs to DENV2 proteins. Six mAbs (78#, 1E7, 2B10, 8B6, 8F12 and 8H1) reacted specifically with DENV2 E protein which relative molecular mass was about 60 Kd. One mAb (6C1) reacted specifically with DENV2 NS3 protein which relative molecular mass was about 70 Kd. Two mAbs (8H1, 6H3) reacted specifically with DENV2 NS5 protein which relative molecular mass was about 100 Kd. MAb 7F7 had no reaction with DENV2 polypeptide. In order to confirm it, the same experiment was performed using lysates of Vero cells and DENV2 infected Vero cells. As expected, the similar results were obtained.
(5) Ten hybridomas were isotyped by Mouse mAb Isotyping Kit (Sigma, USA). 8F12, 1E7 and 6H3 were of subclass IgG2a; 78#, 6C1 and 2B10 were of subclass IgG2b; 8H1 and 8B6 were of subclass IgG3; 7F7 and 8H4 were of subclass IgG1. Giemsa`s staining confirmed the karyotypes of hybridomas (about 100 chromosomes).
4. Plaque reduction neutralization test (PRNT)
2 mg of mAb ascites (or NMS as control) were serially diluted into 1:10, 1:20, 1:40, 1:80, 1:160 and 1:320 in Minimum Essential Medium (MEM). After heat inactivation at 56°C for 30 min, all dilutions were incubated with an equal volume of DENV2 solution containing about 100 PFU/200μl at 37°C for 1 h. Then samples (DENV2-mAb or DENV2-NMS, 200μl/well) were assayed on Vero cell monolayer under overlay medium containing 5 % FBS and 1.2 % methylcellulose. After 7 days of incubation at 37°C with 5 % CO2, cells were stained with 0.5 % crystal violet and the numbers of plaque were counted. Neutralizing titers of mAbs were expressed as the reciprocal of the maximum dilution of mAb that yielded >50 % plaque reduction (PRNT50) in the virus inoculum.
According to results of PRNT, 8F12 and 78# showed neutralizing ability with PRNT50 titer 40 and 60 respectively. 8H4, 7F7, 1E7, 8B6 and 2B10 had neutralizing ability with PRNT50 titer 15, 20, 30, 30 and 35 respectively. However, 8H1, 6C1 and 6H3 had rarely neutralizing effect. When 8F12 and 78# were mixed with equal volume, the combination had strong neutralizing effect about PRNT50 titer 80 at the same experimental condition. In contrast, DENV2-NMS groups had only a little non-specific interference to DENV2 infection.
5. Protection assay in suckling mice
78# and 8F12 mAbs were chosen for the protection assay. Firstly, DENV2 was incubated with each mAb (or NMS, or combination of two mAbs) for 1 h at 37°C water-bath. NMS was used as negative control. Then each newborn BALB/c mouse (on the second day after delivery) was intracerebrally inoculated with 20μl mixture of DENV2-mAb or DENV2-NMS containing 500 or 104 PFU of DENV2 and 80μg mAb or NMS. And the morbidity and mortality were recorded daily. 169 suckling mice were divided into five main groups. Group A and C, and group B and D were used for the protection assay of 8F12 or 78# in either 500 or 104 PFU of DENV2 respectively. Group E test the protection of combination of 8F12 and 78# in 500 PFU of DENV2. Each main group was further divided into two subgroups for mAb and control (NMS) respectively. All infected mice of five groups were observed for two weeks.
All mice in NMS groups showed disease at day 6 and dead at day 8. When each mouse was inoculated intracerebrally with DENV2-mAb mixture containing 104 PFU, they became paralysis, ruffled, slowing of activity and kyphoscoliosis during 6～8 days. And with progression of DENV2 infection, most of them showed severe sickness with anorexia, asthenia and dead at day 9. When each mouse was inoculated with DENV2-mAb mixture containing 500 PFU, they showed sickness gradually and dead at day 11. Although no mouse survived, the time to fall ill and dead in mAbs group mice were obviously delaied to control mice. Moreover, when mixture of 8F12 and 78# was incubated with 500 PFU of DENV2, the mice were protected markedly from the virus challenge. Not only survival time was longer than all the groups above, but also five mice can totally be protected in observed period (survival for 14 days).
In conclusion, ten mAbs were obtained from this study and they recognize natural and denatural DENV proteins. The mAbs showed different neutralizing ability and could protect newborn mice from DENV2 challenge partly. The results demonstrated that our mAbs might be useful tools for studying the mechanism of DENV infection and basic materials for investigating therapy mAb to diseases caused by DENV.
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。
Dengue virus (DENV) is an enveloped virus belonging to mosquito-borne flaviviruses and possesses a positive stranded RNA genome. And DENV has 4 distinct serotypes (DENV1～4) derived from the different of envelop protein. DENV is transmitted by Aedes mosquitoes and Aedes aegypti, and it causes infections which leading to clinical symptoms ranging from self-limited, acute, febrile disease called dengue fever (DF) to life-threatening dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). Over 2.5 billion people, in more than 100 countries, are at risk of dengue infection, with millions of cases occurring around the world every year in the tropics and subtropics worldwide. Now WHO already put DHF/DSS, Hepatitis, Malaria and Tuberculosis into the list of the most four serious infectious diseases of the whole world. However, the pathogenesis of DENV infection is not clear, and there is not an efficient way to deal with the disease in the clinic therapy.
Epidemiological studies indicated that DHF/DSS was more commonly seen during secondary infection with different serotype of DENV from that caused the primary infection. Antibody dependent enhancement (ADE) in DENV infection was suggested as one of explanation for this phenomenon and became a barrier for development of DENV vaccine. And there are two more mechanisms about the pathogenesis of DHF/DSS: (1) the virulence or replication ability of the virus; (2) immuno-pathogenesis theory: the cross reactive T-cell results in the immuno-damage. Recently the high titer of virus, secondary infection with different virus serotype and DENV2 were thought to be the three main factors to cause the DHF/DSS.
In recent several years, various strategies for DENV vaccine development were being pursued, from attenuated viruses and inactivated viruses to DNA vaccine and recombinant preparations. Especially vaccine development focuses on tetravalencea vaccine to defend four serologic types. Although the live attenuated tetravalencea vaccine had been regarded as the most potential vaccine, unfortunately there is no an available and safe vaccine up to now.
Based on above-mentioned situation of DENV infection and their clinic therapy, researchers think about passive immunity or therapy-monoclonal antibodies once more. As early as 1907, sera from individuals recovering from rubeola (measles) were used to prevent infection from this highly contagious virus. During the 1940s, improvements in the quality of fractionated human immunoglobulin led to the widespread use of intramuscular injections of pooled human immunoglobulin to prevent and treat viral infections. Many of these products are still on the market today. However, the disadvantages associated with polyclonal antibody products are that the vast majority of their constituent virus-specific antibodies are non-neutralizing、batch-to-batch variation and risk of pathogen transmission.
With the development of monoclonal antibody (mAb) preparation since 1982, using specific mAb with neutralizing ability to protect and therapy virus diseases became new focus. These successes, in both industrial and academic laboratories, coupled with ongoing changes in the biomedical and regulatory environments, herald an era when the commercial development of human antiviral mAb therapies will likely surge. Thus it is a basic of the stratagem to obtain neutralizing antibodies, especially neutralizing mAbs.
In the present study, purified DENV serotype 2 (DENV2) obtained from density gradient centrifugation was used as antigen to immunize BALB/c mice. After cells fusion and identification by indirect enzyme-linked immunosorbent assay (ELISA), Western-blot and indirect immunofluorescence assay (IFA), ten mAbs (78#、1E7、2B10、6C1、6H3、7F7、8B6、8F12、8H1 and 8H4) were generated and they showed different neutralizing activity to DENV2 in vitro. 78# and 8F12 had neutralizing ability and could protect suckling mice from the virus challenge partly. Our results provided insight into potential application of these mAbs for prophylactic and therapeutic purposes. The main results were as follows:
RESULTS
1. Preparation and verification of purified virus
DENV2 (strain Tr1751) was propagated in C6/36 cells and the supernatant was obtained and stored at–80°C, and the titre of 3 litre liquid was about 1×107 PFU/ml. After PEG-8000 concentrated, the titre recovered around 2×107 PFU/ml. The suspension was loaded onto pre-formed gradients made up of 30 %, 40 %, 50 %, 55 % and 60 % (w/v) sucrose, and ultra-centrifuged at 110 000×g. At the end of sucrose density gradient centrifugation, the band between 55 %～60 % (w/v) was the main band which DENV sedimentation. The purified virus was collected for the studies behind after confirmed by SDS-PAGE and plaque assay (about 1×106 PFU/ml).
2. Obtaining ten hybridoma cells which secrete the specific mAb against DENV2
BALB/c mice were immunized with purified DENV2 and an equal volume of complete Freund’s adjuvant. After booster immunizations and last immunization, the spleen cells from immunized mouse were fused with SP2/0 myeloma cells by PEG-4000. Then the hybridomas were selected by HAT culture medium. The supernatants of living cells were subjected to test antibody titer by indirect ELISA. Positive hybridomas were selected and cloned 5～8 times, then ten hybridomas named as 78#、1E7、2B10、6C1、6H3、7F7、8B6、8F12、8H1 and 8H4 were obtained. MAbs were harvested from hybridomas grown in 1640 medium without fetal bovine serum. The hybridomas were intraperitoneally injected to BALB/c mice for production ascites of mAbs respectively. The ascites were collected for the following examinations.
3. Identification of mAbs specificity
(1) Indirect ELISA
The 96-well plates were coated with purified DENV2 or bovine serum albumin. The supernatants of hybridomas were added as primary antibody respectively. Mouse anti-DENV2 sera and normal mouse sera (NMS) were used as positive and negative controls. HRP-conjugated goat anti-mouse IgG was used as secondary antibody, and coloration with o-phenylenediamine. The absorbencies were determined at 492nm. The absorbencies values of ten hybridomas were 2.1-fold higher than that in controls.
(2) Saturation curve of mAb
The protocol of this experiment is same as indirct ELISA. After optimization concentration of antigen was confirmed, mAb ascites were diluted into 1:10、1:20、1:40、1:80、1:160、1:320、1:640、1:1280、1:2560、1:5120、1:10240、1:20480 as primary antibodies respectively. Then the saturation curve of mAbs OD492nm were drew, and from the curve we got the mAbs’degrees of saturation.
We selected mAbs which recognized the same virus protein and belonged to the same subclass of Ig to undergo build-up assay. On the base of mAbs’degrees of saturation, 8F12 + 1E7 and 78# + 2B10 were chosen to build up. The results showed OD492nm step up after mAbs overlay and suggested that they recognized different epitopes.
(3) Indirect immunofluorescence assay (IFA)
Three-week-old female BALB/c mice were inoculated intracerebrally with DENV2. On the day of paralysis onset, the brains were taken out from the mice and then subjected to prepare freezen sections. The sections were incubated with mAb ascites as primary antibodies and then incubated with FITC-conjugated goat anti-mouse IgG as secondary antibody. The images were observed under fluorescence microscope. Anti-DENV2 sera and NMS were used as positive and negative controls respectively. Eight mAbs (78#、1E7、2B10、6C1、7F7、8B6、8F12、8H4) showed positive reaction with brain sections of DENV2-infected mice. Strong fluorescence intensity was observed in the cytoplasm of the cells in hippocampus, and the distribution of specific reaction was similar with that seen in section stained with mouse anti-DENV2 sera. However, the intensity of fluorescence of mAb was weak as compared with positive control. Specific reaction was rarely observed in section stained with two mAbs (6H3、8H1) and NMS.
(4) SDS-PAGE and Western-blot
The purified virus was denatured and applied onto the gels. After electrophoresis, the gels were stained with coomassie brilliant blue or transferred onto nitrocellulose membrane for western-blot analysis. In SDS-PAGE profile, main DENV2 proteins were detected with estimated molecular masses ranging from 40 to 100 Kd. Western-blot was carried out to identity the specificity of mAbs to DENV2 proteins. Six mAbs (78#, 1E7, 2B10, 8B6, 8F12 and 8H1) reacted specifically with DENV2 E protein which relative molecular mass was about 60 Kd. One mAb (6C1) reacted specifically with DENV2 NS3 protein which relative molecular mass was about 70 Kd. Two mAbs (8H1, 6H3) reacted specifically with DENV2 NS5 protein which relative molecular mass was about 100 Kd. MAb 7F7 had no reaction with DENV2 polypeptide. In order to confirm it, the same experiment was performed using lysates of Vero cells and DENV2 infected Vero cells. As expected, the similar results were obtained.
(5) Ten hybridomas were isotyped by Mouse mAb Isotyping Kit (Sigma, USA). 8F12, 1E7 and 6H3 were of subclass IgG2a; 78#, 6C1 and 2B10 were of subclass IgG2b; 8H1 and 8B6 were of subclass IgG3; 7F7 and 8H4 were of subclass IgG1. Giemsa`s staining confirmed the karyotypes of hybridomas (about 100 chromosomes).
4. Plaque reduction neutralization test (PRNT)
2 mg of mAb ascites (or NMS as control) were serially diluted into 1:10, 1:20, 1:40, 1:80, 1:160 and 1:320 in Minimum Essential Medium (MEM). After heat inactivation at 56°C for 30 min, all dilutions were incubated with an equal volume of DENV2 solution containing about 100 PFU/200μl at 37°C for 1 h. Then samples (DENV2-mAb or DENV2-NMS, 200μl/well) were assayed on Vero cell monolayer under overlay medium containing 5 % FBS and 1.2 % methylcellulose. After 7 days of incubation at 37°C with 5 % CO2, cells were stained with 0.5 % crystal violet and the numbers of plaque were counted. Neutralizing titers of mAbs were expressed as the reciprocal of the maximum dilution of mAb that yielded >50 % plaque reduction (PRNT50) in the virus inoculum.
According to results of PRNT, 8F12 and 78# showed neutralizing ability with PRNT50 titer 40 and 60 respectively. 8H4, 7F7, 1E7, 8B6 and 2B10 had neutralizing ability with PRNT50 titer 15, 20, 30, 30 and 35 respectively. However, 8H1, 6C1 and 6H3 had rarely neutralizing effect. When 8F12 and 78# were mixed with equal volume, the combination had strong neutralizing effect about PRNT50 titer 80 at the same experimental condition. In contrast, DENV2-NMS groups had only a little non-specific interference to DENV2 infection.
5. Protection assay in suckling mice
78# and 8F12 mAbs were chosen for the protection assay. Firstly, DENV2 was incubated with each mAb (or NMS, or combination of two mAbs) for 1 h at 37°C water-bath. NMS was used as negative control. Then each newborn BALB/c mouse (on the second day after delivery) was intracerebrally inoculated with 20μl mixture of DENV2-mAb or DENV2-NMS containing 500 or 104 PFU of DENV2 and 80μg mAb or NMS. And the morbidity and mortality were recorded daily. 169 suckling mice were divided into five main groups. Group A and C, and group B and D were used for the protection assay of 8F12 or 78# in either 500 or 104 PFU of DENV2 respectively. Group E test the protection of combination of 8F12 and 78# in 500 PFU of DENV2. Each main group was further divided into two subgroups for mAb and control (NMS) respectively. All infected mice of five groups were observed for two weeks.
All mice in NMS groups showed disease at day 6 and dead at day 8. When each mouse was inoculated intracerebrally with DENV2-mAb mixture containing 104 PFU, they became paralysis, ruffled, slowing of activity and kyphoscoliosis during 6～8 days. And with progression of DENV2 infection, most of them showed severe sickness with anorexia, asthenia and dead at day 9. When each mouse was inoculated with DENV2-mAb mixture containing 500 PFU, they showed sickness gradually and dead at day 11. Although no mouse survived, the time to fall ill and dead in mAbs group mice were obviously delaied to control mice. Moreover, when mixture of 8F12 and 78# was incubated with 500 PFU of DENV2, the mice were protected markedly from the virus challenge. Not only survival time was longer than all the groups above, but also five mice can totally be protected in observed period (survival for 14 days).
In conclusion, ten mAbs were obtained from this study and they recognize natural and denatural DENV proteins. The mAbs showed different neutralizing ability and could protect newborn mice from DENV2 challenge partly. The results demonstrated that our mAbs might be useful tools for studying the mechanism of DENV infection and basic materials for investigating therapy mAb to diseases caused by DENV.
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