【作者】 王浩；

【导师】 巴德年； 何维； 崔莲仙；

【作者基本信息】 北京协和医学院 ， 免疫学， 2011， 博士

【摘要】 流感病毒作为最主要人类感染病毒之一,对人类的危害巨大。曾在上个世纪引起了三次全球性的流感大流行,造成数千万人的死亡。进入新世纪以来,在2009年爆发了全球性的流感大流行,给世界的社会与经济发展造成了一定程度的负面影响。尽管已经研发出灭活流感疫苗,较好的控制了该病毒株的持续流行和毒性加剧。但是,目前针对新型甲型H1N1流感病毒的免疫学及其致病机理仍了解不多。血凝素(haemegglutinin, HA),作为最主要的参与病毒感染的表面抗原之一,对于H1N1病毒的研究具有重要意义。本研究利用杆状病毒昆虫表达系统表达了具有生物学活性的新型甲型H1N1 HA蛋白和NA蛋白,以此为基础,开展了如下两方面研究：首先,开展了新型甲型H1N1 HA蛋白人中和单克隆抗体的制备。利用EBV转化记忆性B细胞,在CpG和饲养细胞的存在下,培养转化B细胞；经有限稀释后筛选能分泌HA蛋白特异性抗体的细胞克隆,共筛选96U孔板135块包含12960株B细胞样品；最终,经多次筛选获得45株阳性克隆细胞。这些阳性细胞克隆中有36株阳性细胞克隆在随后1-2月中死亡,提示EBV转化的B细胞中绝大部分是短寿命的,而仅有少数才能长期存活(>2月)。为避免阳性克隆细胞进一步丢失,我们将阳性细胞与K6H6/B5细胞系进行PEG融合以获得永生的阳性细胞。然而经多次实验,但却无法筛选到杂交瘤细胞。在不能获得永生阳性细胞的情况下,本研究改变研究策略,通过单细胞克隆团5’-RACE方法获得4对阳性细胞轻、重链可变区cDNA基因,进而利用杆状病毒昆虫细胞表达该基因工程抗体。结果表明,昆虫细胞不是基因工程抗体表达的合适宿主细胞,尽管本研究在采用昆虫表达时也有文献支持。下一步研究中需要使用更合适的CHO细胞表达上述HA特异性人基因工程抗体。其次,本课题进行了新型甲型流感病毒HA表位肽疫苗研究。流感病毒利用连续点突变和基因重组导致不断变异,进而逃避机体的记忆免疫应答。目前的疫苗往往只是针对某一特定的病毒株或亚型,不能保护机体不被其他病毒株感染。而针对表位疫苗的设计,通常利用的是线性表位,无论是B细胞表位,还是T细胞表位。本部分课题通过设计能模拟构象表位的肽疫苗,进而免疫小鼠,利用ELISA、血凝抑制实验、中和实验以及肽阻断ELISA实验,评价其是否能够引起抗体免疫应答。结果显示,该HA肽疫苗免疫接种的小鼠血清能够结合肽、重组H1N1 HA蛋白、重组H5N1 HA蛋白以及H1N1灭活疫苗；能够抑制血凝实验,同时还能够中和非同种流感病毒亚型的H1N1和H5N1病毒。上述结果表明,新型HA肽疫苗能够诱导机体产生相应的体液免疫应答,并且具有交叉保护作用。综上所述,本研究取得了以下主要成果：1、利用杆状病毒表达系统成功表达出具有生物学活性的HA蛋白和NA蛋白,对BaculoGold system和Bac-to-Bac system这两种杆状病毒昆虫表达系统进行比较,结果表明后者更适合表达HA蛋白。2、EBV转化B细胞,筛选到9株能够分泌HA特异性抗体的B细胞,进一步获得4对阳性细胞轻、重链可变区cDNA基因,为进一步制备基因工程抗体打下了基础。3、首次改进了国外文献中报道的单细胞5’-RACE获取轻、重链可变区cDNA的方法,使用挑取单细胞克隆团进行5’-RACE,提高了效率的同时降低了操作的难度。4、新型流感HA肽疫苗能够诱导机体产生保护性抗体免疫应答,对不同流感病毒亚型(H1N1和H5N1)具有交叉保护性。更多还原

【Abstract】 Influenza virus, as an ancient virus, presents a significant and persistent threat to public health and killed millions of people worldwide, especially three global influenza pandemic in the last century. In this century, the outbreak of novel swine-origin H1N1 influenza A virus pandemic worldwide, resulted in global panic.Despite the inactivated influenza vaccine developed for control and prevention of this novel virus strain. However, at present the learning for the novel influenza H1N1 virus and its pathogenic mechanism of the immunological research is still poorly understood. Haemegglutinin (HA), as one of the most major surface antigen for viral infection, has a great significance for H1N1 virus research.In this study, recombinant H1N1 HA and NA proteins were expressed in two baculovirus insect expression system. As the basis of the following research:First of all, make human monoclonal antibodies from memory B cells:potent neutralization of 2009 novel H1N1 influenza virus. Using EBV transformed memory B cells, under feeder cells in the presence of CpG, culture the transformed B cells; after limited dilution, screened HA-protein specific antibody-secreting cell clones; 96U-platex 135 were screened totally, that were 12960 B cell samples; the results showed that 45 positive clones were obtained, but 36 of which were died during the culture in vitro. It indicated that EBV-transformed B cells in a large part were short-lived, and only a small part lived for a long survival (>two months). In order to avoid further loss of the positive clone cells, we try to fuse the positive cells and K6H6/B5 using PEG to obtain immortal positive cells. The hybridomas couldn’t be obtained in repeated experiments. In the case of failure of fusion experiments,4 pairs of variable region cDNA of light and heavy chain were obtained from positive cell clones by single-cell clone 5’-RACE method, and then genetic engineering antibody was expressed in insect cells by the baculovirus expression system. The result showed that there’s no antibody production and insect cells are not suit as host cell to produce antibody. And the better is CHO cells, which become ideal host cells for antibody expression in next step.Second, we research on novel influenza A virus hemmagglutinin-peptide based vaccine. The continuous antigenic drifts and shifts of Influenza virus HA leads to the continuous variation of the virus, and can evade the immune response. Current vaccines only provide immunity to viral isolates similar to the vaccine strain and always based the whole HA proteins, which could not protect against the infection of devise influenza virus strains. At present, the design of epitope-based vaccine is utilizing the linear epitope, including both the B-cell epitope and T-cell epitope. In this part of the study, we provided a novel strategy to mimic conformational epitope as a linear peptide-based vaccine to induce antibody response of mice. The immunogenicity and protective efficiency of the peptides were examined after vaccination using EIISA, haemegglutination inhibition (HI) and neutralization assays. We found that this novel HA peptide-based vaccine could induce strong humoral responses against H1N1 and H5N1 in mice after immunization. This novel peptide is a promising strategy for vaccine design and a candidate for eliciting antibody response against diverse subtypes of influenza virus.In summary, our findings:1. Functional HA and NA proteins were successfully expressed in two distinct bacularvirus expression systems (BaculoGold systema and Bac-to-Bac system), of which the Bac-to-Bac bacularvirus expression system is more suitable for expression of A/Sichuan/1/2OO9(H1N1) HA protein.2.9 HA-specific antibody-secreting cell clones were obtained and 4 pairs of variable region cDNA of light and heavy chains were obtained by 5’-RACE; This is the base of the next genetic engineering antibody production.3. Firstly improved single-cell clone 5’-RACE method has been used to obtain the cDNA of variable region of antibody and more efficient, compare with the single-cell 5’-RACE method.4. Novel HA peptide-based vaccine could induce strong humoral responses against H1NI and H5N1 in mice after immunization. This novel peptide is a promising strategy for vaccine design and a candidate for eliciting antibody response against diverse subtypes of influenza virus.更多还原