抗HEV嵌合抗体的构建及在CHO细胞中的表达

通过RT-PCR方法从分泌戊型肝炎(戊肝)病毒中和性鼠源单克隆抗体(单抗)8C11的杂交瘤细胞中克隆出抗体基因的重链可变区(VH)、轻链可变区(VK)序列,并分别克隆到含有人gamma 1重链和kappa轻链恒定区序列的pcDNA3.1/Hygro和pcDNA3.1( )质粒中,共转染中华仓鼠卵巢癌细胞(CHO)细胞.RT-PCR结果表明,转染的CHO细胞转录了嵌合重链及轻链基因,间接ELISA及Western blot结果表明:翻译出的两种多肽在细胞内正确组装成嵌合抗体分子,并可分泌至细胞外,表达的嵌合抗体保留了原鼠单抗的抗原结合特异性及对8H3结合抗原的增强作用.8C11嵌合抗体的成功表达可降低鼠源性,为探讨戊肝抗体治疗的可能性奠定了基础.     

戊型肝炎病毒衣壳蛋白中和表位间的构象诱导

重组蛋白NE2包含了戊型肝炎病毒(HEV)衣壳蛋白(pORF2)的aa394～606片段.在NE2上已鉴定出了2个HEV中和表位,并获得了3个识别中和表位的单克隆抗体(MAb)8C11、13D8和8H3.这3个MAb间的交叉阻断ELISA实验发现,8C11和13D8可以彼此完全阻断,8H3对8C11和13D8均不能阻断,而8C11非但不能阻断8H3,反而显著增强了8H3与抗原的结合.用生物传感器进行的抗体与抗原结合的动力学分析也证实了这一现象.这些结果提示,在NE2上8H3表位区域受到抗原上某些结构的掩盖,而8C11与NE2的结合引起了抗原空间结构的改变,导致了掩盖8H3表位的结构的去除和8H3表位的充分暴露.免疫捕获RT-PCR发现,8C11同样可以显著增强8H3对天然HEV病毒的捕获能力,提示这种结合诱导的衣壳蛋白空间构象改变在天然HEV病毒颗粒上同样存在.     

颗粒化重组戊型肝炎病毒衣壳蛋白及其抗原性与免疫原性

过去的研究发现大肠杆菌表达的戊型肝炎病毒（HEV）衣壳蛋白ORF2的aa394-606片段NE2可以形成同源多聚体,并具有良好的免疫保护性,但纯化后的免疫原性较弱。这里表达了3个NE2蛋白的N端延伸突变体,发现对应于ORF2 aa368_606的重组蛋白HEV239在体外可以形成颗粒性抗原。HEV 239抗原颗粒与戊肝患者血清反应性良好,对中和性单克隆抗体8C11的反应性与NE2抗原相当,而对另一中和性单克隆抗体8H3的反应性较NE2抗原有显著提高,表明HEV 239抗原颗粒具有比NE2更好的抗原性。纯化后的HEV 239抗原颗粒直径约为15～30nm。铝佐剂吸附的HEV 239免疫Balb/c小鼠的半数有效剂量（ED50）在0.08～0.25μg之间,而同样以铝佐剂吸附的NE2抗原60μg剂量免疫的抗体阳转率仅25%,表明HEV 239抗原颗粒具有更好的免疫原性。     

戊型肝炎病毒细胞吸附模型的建立及病毒吸附区域初步研究

E.coli中表达的HEV衣壳蛋白片段P239(aa368~606)形成的类病毒颗粒与戊肝患者恢复期血清及中和单抗具有良好的反应性,较好地模拟了天然HEV病毒颗粒的表面空间结构。利用P239吸附HepG2细胞的模型来模拟HEV对宿主细胞的吸附,多株中和单抗对吸附的阻断验证了吸附的特异性。P239与多株传代细胞系的吸附结果则表明了这种特异性吸附的细胞选择性。对阻断P239吸附的线性单抗进行定位,初步确定了P239与细胞相互作用的区域:ORF2上的aa423~443很可能和病毒上的细胞膜受体结合部位非常靠近,或可能直接参与构成了病毒与细胞特异性识别的表位。此本研究为进一步研究HEV与宿主细胞的相互作用提供一定的线索。     

Clioquinol targets zinc to lysosomes in human cancer cells

A panel of 52 murine monoclonal antibodies was found to recognize antigenic determinants that had been conserved among all major genetic subgroups of the H5N1 avian influenza virus prevalent since 1997. We screened a phage display library for peptides recognized by one such antibody (8H5). We analysed the specificity of 8H5 for reactive peptides presented as fusion proteins of HBc (hepatitis B core protein) and HEV (hepatitis E virus) structural protein, p239. This was then related to the specificity of the native HA (haemagglutinin) molecule by virtue of the capacity of fusion proteins to compete for 8H5 binding with different strains of H5N1 virus and the reactivity of antisera generated against fusion proteins to bind native HA molecules, and to inhibit haemagglutination and arrest infection by the virus. Nine reactive peptides of different amino acid sequences were identified, six of which were also reactive with the antibody in association with HBc and four were in association with p239. Binding occurred with the dimeric form of the four p239-fusion proteins and one of the HBc-fusion proteins, but not with the monomeric form. The HBc-fusion proteins blocked 8H5 binding with four strains of H5N1 influenza virus. Mouse antisera generated against fusion proteins bound to HA molecules, but did not inhibit haemagglutination or arrest H5N1 infection. Our findings indicate that 8H5 recognizes discontinuous sites presented by secondary and possibly higher structural orders of the peptides in spatially favourable positions for binding with the antibody, and that the peptides partially mimic the native 8H5 epitopes on the H5N1 virus.     

抗CD3基因工程嵌合抗体IgG在哺乳动物细胞中的表达和活性测定

利用基因工程方法将鼠源性抗CD3抗体HIT3a的可变区和人源抗体(IgG)的完整的恒定区连接起来,构建全抗型抗CD3嵌合抗体,该型抗体具有较低的免疫源性可作为免疫抑制剂应用于器官移植,减少受体产生免疫排斥,提高移植器官的存活率。利用PCR方法从抗CD3 ScFv重组噬菌体表达载体pCANTAB 5E上扩增抗CD₃抗体的轻链和重链可变区,将轻链和重链可变区组装到含有人抗体（IgG）恒定区的表达载体中,构建抗CD3嵌合抗体IgG的轻链和重链表达载体PKN100和PG1D105,并用脂质体法共转染CHO细胞。结果证明,抗CD₃嵌合抗体的V_L和V_H与HIT3a抗体的V_L和V_H完全相符,ELISA和Western blot检测结果证实转染细胞的培养上清中含有抗CD₃嵌合抗体IgG的表达,表达产物能与Jurkat细胞结合,并能竞争性抑制HIT3a抗体和Jurkat细胞结合活性,³H-TdR掺入实验表明, 抗CD3嵌合抗体与亲代抗体HIT3a一样,具有促进外周血单核细胞增殖的作用。我室构建的全抗型抗CD3嵌合抗体分子表达载体可在CHO细胞中稳定表达,表达产物有较好生物活性,具有潜在的临床应用价值。     

戊肝病毒Th表位肽免疫可增强其载体蛋白的体液免疫应答

戊型肝炎病毒衣壳蛋白内包含一个强H-2d限制性Th表位P34。以该表位肽免疫BALB/c鼠,其脾细胞能够在体外识别重组戊型肝炎病毒衣壳蛋白,剔除实验表明应答细胞几乎完全是CD4 T细胞,证明P34表位肽能有效诱导产生特异性Th细胞。以P34肽初免小鼠,再以包含该表位的重组戊型肝炎病毒抗原(E2)免疫,结果表明,10μg、20μgE2免疫组在免疫后第1周即有部分小鼠产生抗体,到第3周所有小鼠均能够产生抗体;而对照肽P18初免的小鼠,以20μgE2加强免疫亦无法诱导小鼠产生抗体。这表明,Th表位肽P34初免诱导产生的Th细胞能够有效促进小鼠对携带该表位的载体蛋白的体液免疫应答。     

Hepatitis E virus DNA vaccine elicits immunologic memory in mice

Injection of an expression vector pJHEV containing hepatitis E virus (HEV) structural protein open reading frame 2 gene generates a strong antibody response in BALB/c mice that can bind to and agglutinate HEV. In this study, we tested for immunologic memory in immunized mice whose current levels of IgG to HEV were low or undetectable despite 3 doses of HEV DNA vaccine 18 months earlier. Mice previously vaccinated with vector alone were controls. All mice were administered a dose of HEV DNA vaccine to simulate an infectious challenge with HEV. The endpoint was IgG to HEV determined by ELISA. Ten days after the vaccine dose, 5 of 9 mice previously immunized with HEV DNA vaccine had a slight increase in IgG to HEV. By 40 days after the vaccine dose, the level of IgG to HEV had increased dramatically in all 9 mice (108-fold increase in geometric mean titer). In contrast, no control mice became seropositive. These results indicate that mice vaccinated with 3 doses of HEV DNA vaccine retain immunologic memory. In response to a small antigenic challenge delivered as DNA, possibly less than delivered by a human infective dose of virus, mice with memory were able to generate high levels of antibody in less time than the usual incubation period of hepatitis E. We speculate that this type of response could protect a human from overt disease.     

应用噬菌体随机肽库技术筛选戊型肝炎病毒中和表位模拟肽

以识别戊型肝炎病毒（HEV）构象依赖性中和表位的单克隆抗体8C11、8H3作为固相筛选分子,对噬菌体随机7肽库进行4轮筛选后,随机挑取单克隆噬菌体进行测序。合成优势7肽序列基因,将其插入HBcAg-AA78-83位置之中,进行原核表达,所获重组蛋白经蛋白印迹实验证实可与相应单抗结合,电镜下可见重组蛋白能形成与HBcAg相似的类病毒颗粒。化学合成单抗8H3筛选出的优势7肽,所获7肽经生物传感器结合实验证实与单抗8H3结合。这些结果提示用噬菌体7肽库可以筛选出部分模拟构象性表位的短肽,为亚单位疫苗的研制提供了新的思路。     

A murine–human chimeric IgG antibody against vascular endothelial growth factor receptor 2 inhibits angiogenesis in vitro

Vascular endothelial growth factor receptor 2 (VEGFR2) has been reported to play an important role in angiogenesis and tumorigenesis. A murine anti-VEGFR2 mAb (A8H1) has been established in a previous study. To reduce the incompatibility of the murine mAb for human use, the chimeric anti-VEGFR2-IgG was developed by genetic recombination of the variable regions of the A8H1 antibody and the constant regions of human IgG, and was expressed in Sp2/0 cells transfected with the two recombinant vectors containing the heavy chain and the light chain regions. After screening, clone 2F12 was selected and was found to stably secrete the murine–human chimeric anti-VEGFR2-IgG (coded 2F12). This chimeric IgG maintained the specificity and the affinity of the parental murine antibody against VEGFR2, and effectively identified VEGFR2 expressed on the surface of HUVECs and BEL-7402 cells. Furthermore, the 2F12 antibody demonstrated inhibition of angiogenesis in vitro, such as proliferation, migration, invasion and tube formation of HUVECs. This murine–human chimeric IgG may be considered for further development as an anti-angiogenesis and anti-tumor agent.     

合成肽抗原在戊型肝炎病毒感染诊断中的应用

An ELISA for the detection of anti HEV using synthetic peptide antigens was developed. The synthetic antigens were encoded by OFR2 and OFR3 genes of HEV. The purpose of this study was to determine the applicability of the synthetic antigens in the serodiagnosis of hepatitis E. The anti HEV detection using synthetic antigens was carried out in 47 healthy subjects and 89 patients with acute or chronic viral hepatitis. The results showed that the positive rate of anti HEV IgG in healthy subjects was 4.2%(2/47), and no IgM antibody to HEV was found. The positive rates of IgG and IgM antibodies to HEV in the hepatitis patients were 8.9% and 10% respectively. In addition, we compared the detecting efficacy of the synthetic antigens with that of the market reagent in 57 serum samples, the total coincident rate was 87.7% (50/57). All of the results accorded with the literatures reported. This study suggests that the ELISA based on the synthetic peptide antigens was specific, sensitive and convenient in diagnosis of HEV infection, it can be widely used in both clinical and epidemiological reseaches.     

The design, construction and function of a new chimeric anti-CD20 antibody

A novel murine IgM-type anti-human CD20 monoclonal antibody (mAb) 1-28 was prepared in our Lab, which can induce apoptosis and inhibit proliferation of Daudi and Raji cells. However, the efficacy of 1-28mAb in human cancer therapy is likely to be limited by human anti-mouse antibody responses. A chimeric antibody, C1-28, containing 1-28mAb variable region genes fused to human constant region genes (gamma 1, kappa) was constructed. However, C1-28 lost the antigen-binding activity. Here, using sequence similarity and known 3D structure of antibody variable regions as template, the spatial conformations of 1-28 variable regions (i.e. V(H) and V(L)) were analyzed with computer-guided homology modeling methods. According to the surface electrostatic distribution and interaction free energy analysis, the relationship between structure and stability of 1-28 variable regions was studied theoretically and a new chimeric anti-CD20 antibody scFv-Ig named 5S was designed. Expression level of 5S in the culture supernatant was determined to be around 50mug/mL using sandwich ELISA method with chimeric antibody Rituxan as reference. 5S retained its murine counterpart's binding activity by fluorescence-activated cell-sorting analysis. Furthermore, it could kill CD20 positive Daudi and Raji cells by complement-dependent cytotoxicity. For binding affinity often decreased even lost when IgM antibody was constructed into chimeric IgG1 form, our success give a hint about how to construct a IgG1-type chimeric antibody from IgM-type murine antibody to preserve its binding activity.     

HepG2细胞中与戊型肝炎病毒衣壳蛋白相互作用蛋白的初步研究

利用重组戊型肝炎病毒(HEV)衣壳蛋白片段p239(368~606 aa)形成的类病毒颗粒作为亲和层析诱饵蛋白,HepG2为细胞模型,筛选与p239类病毒颗粒特异性相互作用蛋白。经过二维电泳分离,MALDI-TOF-MS分析鉴定得到GRP78/Bip,HSP90,alpha-tubulin及P43四个与p239有相互作用的候选蛋白。GRP78/Bip为热休克蛋白家族成员,体外免疫共沉淀实验结果证实,其与p239有特异性的结合。此研究结果为深入研究HEV的感染过程如吸附、入胞,以及HEV的致病机理提供了有益线索。     

Seroprevalence and Incidence of hepatitis E in Blood Donors in Upper Austria

Background

In recent years various studies showed, that hepatitis E virus (HEV) is a growing public health problem in many developed countries. Therefore, HEV infections might bear a transmission risk by blood transfusions. The clinical relevance still requires further investigations. The aim of this study was to provide an overview of acute HEV infections in Upper Austrian blood donors as well as a risk estimation of this transfusion-related infection.

Methods and Findings

A total of 58,915 blood donors were tested for HEV RNA using a commercial HEV RT-PCR Kit. 7 of these donors (0.01%) were PCR-positive with normal laboratory parameters in absence of clinical signs of hepatitis. Viral load determined by quantitative real-time PCR showed a HEV nucleic acid concentration of 2,217 293,635 IU/ml. At follow-up testing (2–11 weeks after donation) all blood donors had negative HEV RNA results. Additionally, genotyping was performed by amplification and sequencing of the ORF1 or ORF2 region of the HEV genome. All HEV RNA positive donor samples revealed a genotype 3 isolate. For the antibody screening, anti-HEV IgM and IgG were detected by ELISA. Follow up serological testing revealed that no donor was seropositive for HEV IgM or IgG antibodies at time of donation. Moreover, we verified the prevalence of anti-HEV IgG in 1,203 of the HEV RNA negative tested blood donors. Overall 13.55% showed positive results for anti-HEV IgG.

Conclusions

In the presented study, we investigated HEV infections in blood donations of Upper Austria over 1 year. We concluded that 1 out of 8,416 blood donations is HEV RNA positive. Seroprevalence of anti HEV IgG results in an age-related increase of 13.55%. Therefore, based on this data, we recommend HEV-PCR screening to prevent transmission of hepatitis E virus by transfusion.     

抗FGF2人鼠嵌合抗体在HEK293T细胞中的表达优化

目的：FGF2是肿瘤血管新生过程中最重要的因子之一,因此通过制备抗FGF2人鼠嵌合抗体中和其发挥作用,以达到抑制肿瘤生长的目的。方法：利用分泌抗FGF2抗体的杂交瘤细胞株IgG9B9和人B淋巴细胞,分别克隆抗体轻链可变区V_L、重链可变区V_H和人重链恒定区C_H基因,从pComb3λ载体中扩增出人λ 链恒定区C_L基因,通过重叠PCR,将V_L,V_H和C_L,C_H片段分别连接形成嵌合抗体的轻链L和重链H,将L/H链以单独构建或串联于同一载体的方式,构建抗FGF2嵌合抗体表达载体,并通过调控元件WPRE优化载体、共转染促生长因子aFGF以及调整表达温度等方式提高嵌合抗体在真核细胞中的表达。结果：成功构建了优化表达载体PLexm-WPRE、PLexm-aFGF;L、H链基因也成功构建,并以L、H或L-F2A-H（2A连接肽将L和H连接起来）的方式分别成功连接到PLexm,PLexm-WPRE载体中。转染细胞上清的ELISA鉴定结果表明,L/H链单独构建要比串联构建的方式具有更高的表达水平,WPRE能有效促进抗体的表达而aFGF并不能促进其表达,与31、37℃相比,33℃时抗体的表达量最高,同时嵌合抗体表现出了很好的结合活性及中和活性,竞争IC50=6.25μg/ml。通过亲和层析获得了高纯度的抗FGF2嵌合抗体。结论：在33℃下,人鼠嵌合抗体基因在WPRE存在下表达量最高,且与抗原FGF2有很好的结合活性及中和活性,为临床应用奠定了基础。     

Management of Hepatitis E Virus (HEV) Zoonotic Transmission: Protection of Rabbits against HEV Challenge following Immunization with HEV 239 Vaccine

Hepatitis E virus (HEV) constitutes a significant health burden worldwide, with an estimated approximately 33% of the world’s population exposed to the pathogen. The recent licensed HEV 239 vaccine in China showed excellent protective efficacy against HEV of genotypes 1 and 4 in the general population and pregnant women. Because hepatitis E is a zoonosis, it is also necessary to ascertain whether this vaccine can serve to manage animal sources of human HEV infection. To test the efficacy of the HEV 239 vaccine in protecting animal reservoirs of HEV against HEV infection, twelve specific-pathogen-free (SPF) rabbits were divided randomly into two groups of 6 animals and inoculated intramuscularly with HEV 239 and placebo (PBS). All animals were challenged intravenously with swine HEV of genotype 4 or rabbit HEV seven weeks after the initial immunization. The course of infection was monitored for 10 weeks by serum ALT levels, duration of viremia and fecal virus excretion and HEV antibody responses. All rabbits immunized with HEV 239 developed high titers of anti-HEV and no signs of HEV infection were observed throughout the experiment, while rabbits inoculated with PBS developed viral hepatitis following challenge, with liver enzyme elevations, viremia, and fecal virus shedding. Our data indicated that the HEV 239 vaccine is highly immunogenic for rabbits and that it can completely protect rabbits against homologous and heterologous HEV infections. These findings could facilitate the prevention of food-borne sporadic HEV infection in both developing and industrialized countries.     

戊型肝炎病毒DNA免疫的研究

利用PCR方法获得1 163 bp的戊型肝炎（Hepatitis E Virus,HEV）开放读码框架(Open Reading Frame,ORF)ORF2之3’大片段和369bp ORF3的完整片段,分别克隆到真核表达载体pcDNA3中,构建两种含有HEV主要抗原表位的质粒DNApcE2和pcE3,分别或混合免疫Swiss小鼠三次（0时,第2周,第4周）,观察其在小鼠体内诱发的体液免疫应答。ELISA检测结果表明,pcE2和pcE3在小鼠体内均可诱导出一定水平的HEVIgG抗体,且在第三次免疫接种两周后,100%的小鼠抗体阳转。与两种质粒单独免疫相比,两者同时注射的抗体水平较高。本研究为HEVDNA疫苗的研究打下一定基础。     

Seroprevalence of antibodies to hepatitis A and E viruses in pediatric age groups in Turkey

Hepatitis A and hepatitis E are enteric transmitted viral diseases occurring in epidemic and sporadic forms especially in developing countries. Previous studies in Turkey showed that most residents are infected with HAV by the second decade of life. Since HEV is generally transmitted by the same route as HAV we conducted a community-based seroprevalence study for HAV and HEV infection in Ahatli area in Antalya, Turkey where socioeconomic conditions are low. Anti-HAV total immunoglobulin was tested by using a microparticle EIA (Axsym-Abbott Lab). Anti-HEV IgG was assayed by a micro ELISA method (Genelabs-Singapore). Of the 338 sera tested, 112 (33.1%) were positive for anti-HAV total antibody. Anti-HEV IgG was detected in three (0.89%) of the serum samples. Seropositivity rates of HAV in preschool and school children were 19.9% and 43.9% respectively (p < 0.001). No antibody to HEV was detected in preschool children, while the prevalence of anti-HEV IgG was 1.6% in children attending school. Our data showed that seroprevalence of anti-HAV is high among children samples but HEV infection appears to be relatively rare in pediatric age groups.