【作者】 安文琪；

【导师】 张玉静；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 流感病毒属于正粘病毒科(Orthomyxoviridae),是一种含有8个基因片段的分节段RNA病毒。病毒分为甲(A)、乙(B)、丙(C)三型,其中A型对人类危害最严重,A型引起的并发症危害大,易造成神经-内分泌-免疫网络的紊乱。A型流感病毒的基因组是负链RNA,因而直接不具有感染性,各个RNA片段需要与聚合酶蛋白(PB2、PBI、PA,统称为P蛋白)及核蛋白(NP)结合在一起形成核糖核蛋白复合物即RNPs才有活性。流感病毒感染时,病毒首先与宿主细胞表面的特异性HA受体唾液酸(SA,N-乙酞神经氨酸)残基结合,通过融膜进入细胞后释放出RNPS,RNPS进入细胞核才开始病毒基因组的复制和转录,每个RNA片段单独组成一个转录单位,转录出mRNA和互补RNA(cRNA),mRNA翻译合成病毒蛋白,cRNA复制生成子代RNA病毒,然后在细胞浆中组装成完整的病毒粒子。流感病毒的反向遗传操作技术建立的难度较大,原因在于要同时在细胞内形成8个功能性核糖核蛋白复合物(RNPs)。流感病毒拯救技术的发展滞后于其它负义RNA病毒,流感病毒与大多数其它负义RNA病毒不同在于流感病毒基因组是在细胞核内复制。经过近10年的发展,在1999年Neumann等和Fodor等分别报道了改造后完全以质粒为基础的反向遗传技术,这是流感病毒拯救技术发展史上的转折点。其优点是不再像早期的方法那样感染细胞不再需要为RNA合成提供辅助病毒,从而避免了大量的筛选工作。本研究采用RG技术,通过8质粒流感病毒拯救系统,将冷适应、减毒病毒株A/AA/6/60(H2N2)的6个内部基因全片段克隆入pAD3000,其中的PB2、PB1、NP基因人工引入共5个氨基酸的突变。同时把2006～2007年流行株A/Wisconsin/67/2005(H3N2)的2个表面基因片段克隆入pAD3000,从而构建了8个转录/表达载体,用于拯救重配H3N2亚型人流感病毒减毒株,为流感减毒活疫苗的制备提供了保证。研究采用由美国St.Jude儿童研究医院的Robert Webster博士提供的上述PR8 8质粒系统,一一替换成构建好的用于重配病毒拯救的8个转录/表达质粒,共有8种7+1组合,验证其中每一个基因片段是否能够正常工作;同时用PR8的HA和NA表面基因以及H1N1的表面基因与验证好的冷适应株的6个内部骨架组合(6+2组合,重配病毒分别简写为rgAA-PR8和rgAA-H1N1),共转染细胞,验证冷适应株A/AA/6/60的6个内部基因是否能够协同发挥作用,同时对重配病毒的生物学特性进行了鉴定;在此基础上,接着把2006～2007年流行株A/Wisconsin/67/2005(H3N2)的表面基因与A/AA/6/60的6个骨架基因进行重排组合,对产生的重配病毒(6+2组合,简写为rgAA-H3N2)进行了初步鉴定,深入的研究还在进行之中。这些研究内容为我们弄清楚流感病毒基因组及其蛋白质在病毒复制及致病机制中的作用建立了平台,也为冷适应流感减毒活疫苗的研制以及粘膜免疫保护机制的研究提供了理论依据。本部分实验针对弱毒疫苗的特点,建立了rgAA-H3N2病毒株的三级毒种库并进行系统、全面鉴定,同时免疫BALB/c小鼠利用HI和ELISA等方法对其免疫原性进行了初步研究,为H3N2亚型流感减毒活疫苗的研发奠定了理论基础。这些研究为研制高效、安全的冷适应流感减毒活疫苗以及阐明流感病毒呼吸道粘膜免疫保护机制奠定了基础,为其它病毒减毒活疫苗研制奠定了基础。更多还原

【Abstract】 Influenza virus is the pathogen of influenza, which is a kind of highly contagious disease with the first incidence rate among all of the infectious diseases and hazardous infection which WHO declares to be strictly monitored. The three flu pandemics of the 20^th century were devastating. Especially in the 1918/1919 "Spanish flu", more than 20 million people died which exceeded the total number of people killed during the World War I and most of them were young people. Till now people could not control the disease due to the high variability of influenza virus in spite of the rapid progress of the modern scientific technology.Influenza virus, a member of the family Orthomyxoviridae, is a negative sense RNA virus with eight genomic segments. Influenza viruses are divided into types A, B and C. Influenza A viruses are the principal causes of influenza in humans which cause serious complications and disorder of immune nerve-endocrine-network. Moreover, it imposes a huge burden on the human health and the national economy.The antigens of the encoding influenza virus include HA, NA, NP and M polypeptides. HA and NA are two major antigens on the surface of influenza virus. HA is the major protective antigen of influenza virus to stimulate the production of neutralizing antibody. Comparison with HA, NA is conserved in a degree,which stimulates the production of antibody as same. As a result of seasonal antigenic variation of hemagglutinin（HA）and neuraminidase（NA）which decide the specificity of the pathogen, new virus strain appears annually, which pose a major obstacle to control influenza.To date, there is no effective therapeutic tool for influenza. Though influenza vaccines have been produced for over 50 years, vaccination is still the principal method of prophylaxis. Vaccines can be characterized as whole virus vaccines, split virus vaccines, surface antigen vaccines, DNA vaccines and live attenuated vaccines. Due to frequent antigenic shift, vaccine strain used in production needs to be changed annually. Commercially available trivalent inactivated vaccines suffer from limited efficacy in cross-protective immunity and routes of administration, given by intramuscular injection. Surface antigen vaccines can be used safely, but targeting mainly HA and NA antigen. They are not sufficiently cross-reactive to protect against antigenic variants, vaccine production is time-consuming and they are not fit for seasonal influenza. DNA vaccine developed late and aimed at protective antigen formulation. There are still some problems such as large antigen dose and gene integration with chromosomal which are difficult to solve. However, live attenuated vaccine is able to induce strong systemic immunity and humoral responses and local mucosal immune responses, but remains invariably virulence and potential rebound of toxicity. In view of the status quo of influenza vaccines, much attention has been given to the development of effective and neotype influenza vaccines. Recently, the reverse genetics provided a favourable opportunity for live attenuated vaccines. Efforts are currently under way to develop a live attenuated influenza vaccine which based on reverse genetics. Since 1999, Neumann and Hoffmann et al have developed an eight-plasmid DNA transfection system for the rescue of infectious influenza A virus entirely from cloned cDNA. The development opens the way for the study of live attenuated influenza vaccine. Rescuing recombinant influenza virus vaccine on the MDV-A background is a focus of influenza vaccine. In this dissertation, we used the eight-plasmid rescue system and selected 6 internal gene of influenza strain A/Ann Arbor/6/60（H2N2） as the genetic backbone for generation of recombinant H1N1 subtype influenza virus and 6+2 reassortants expressing the HA and NA from the 2006～2007 circulating strain A/New Caledonia/20/99（H1N1）. Finally, we successfully established a cold-adapted H1N1 subtype influenza virus rescue system, this reverse genetics platform is strategically important for vaccine development and influenza prevention.The whole dissertation consists of three parts:1. Construction of eight-plasmid system for influenza virusBased on functional bi-directional transcription/expression vector pAD3000, influenza virus strain A/AnnArbor/6/60 （H2N2）, a cold-adapted （ca）, temperature sensitive （ts）, live attenuated was used as the master donor virus （MDV） for virus rescue, in which six internal gene fragments were fully synthesized. Meanwhile, five amino acid substitutions have been artificially altered by human intervention and six pairs specific primers were designed according to references. The 2006～2007 circulating influenza virus strain A/Wisconsin/67/2005（H3N2） was propagated in 10-day-old embryonated chicken eggs and concentrated by density gradient centrifugation on sucrose. Total RNA was extracted from infected allantoic fluid. The HA and NA gene were amplified using the universal primer pairs according to Hoffmann. Expand High Fidelity PCR System was used and 8～10 clones were sequenced in order to confirm the authenticity of 5’ and 3’ ends. Eight transcription /expression plasmids were obtained and named as pMDV-A-PB2, pMDV-A-PB1, pMDV-A-PA, pMDV-A-NP, pMDV-A-M, pMDV-A-NS, pHW214-H and pHW214-NA, respectively. Finally, transcription and expression of vRNA and mRNA were realized by using one vector and pol I /pol II RNA polymerase of cells after transfection, which contribute to the construction of cold-adapted influenza virus rescue system.2. Preparation of reassortant H1N1 subtype influenza virus2.1 Validation of cold-adapted influenza virus rescue systemSome desired infectious H1N1 subtype recombinant viruses were produced by co-transfection a single gene segment from MDV-A together with the complementary seven segments from control A/PR/8/34 strain. In addition, HA and NA derived from strain A/New Caledonia/20/99 or A/PR/8/34 and other internal gene segments from A/Ann Arbor/6/60, in other words, there are eight kinds of 7+1 and a kind of 6+2 combinations. The results revealed that each of the 8 genomic segments cloned in pAD3000 was showed to be functionally expressed in a reassortant experiment and 6 internal gene backbone work synergialy. Meanwhile, it prepared for further screening and construction cold-adapted reassortant virus. The transfection reaction using the 8 A/PR/8/34 plasmids was used as a positive control. During these courses, progeny virus for eggs propagation is produced in 35mm dishes by optimizing rescue systems.Then, we identified and analyzed the partial biological properties of the recoveried viruses PB2/PR8, PR8 and PR8/rMDV-A. The rescued viruses had similar morphology to wild-type virus strain by electronic microscope. Amino acid substitutions were present in progeny virus by RT-PCR. The reassortant virus was identified by HA, HI and IFA experiment. It showed that reassortant virus propagation prefer to lower temperature such as 33 degree by PFU of PR8/rMDV-A. PR8/rMDV-A grew to high titer and weak virulence in embryonic eggs during passage of the virus and the cytoplasmic effect on infected MDCK cells were correlated with the strain A/New Caledonia/20/99, virulence and propagation in lower temperature were correlated with the master donor virus strain A/Ann Arbor/6/60. These results showed that the cold-adapted rescue system worked well.2.2 Generation and identification of H3N2 subtype attenuated influenza virus vaccine Based on the functional cold-adapted virus rescue system, we reassorted HA and NA gene of the 2006～2007 circulating influenza virus strain A/Wisconsin/67/2005（H3N2）. A positive H3N2 subtype recombinant virus was obtained successfully by co-transfecting COS-1 cells. A HA titer of approximately 1:512～1:1024 were detected in the first passage of the rescued viruses. Virus titer was stable and the homology of HA and NA gene were above 99.9% following four passages. The virion morphology of the rescued viruses was verified to be identical to that of the wild-type PR8-strain. Preliminary identification was carried out and further study is in progress. The establishment of cold-adapted H1N1 subtype rescue system by RG will contribute to development of cold-adapted human influenza virus vaccine candidates and mucosal immune mechanisms against influenza.3. Construction of Three-tier Virus Seed Library and immunogenicity test of rgAA-H3N23.1 Construction of Three-tier Virus Seed Library of rgAA-H3N2The three-tier virus seed library of rgAA-H3N2 was constructed which contained the primary seed lots, the master seed lots and working seed lots. The titers of seeds from working seed lots were above 7.2 LogTCID₅₀/ml. The seeds were qualified in sterility and mycoplasma tests, and negative in haemadsorption and non-haemadsorption tests. In test for adventitious agents, both the survival rates of mice and suckling mice were more than 86 %. All the quality indexes of rgAA-H3N2 strain complied with the Chinese Requirements for Biologics 2005. It may be used as the seeds for production of cold-adapted attenuated influenza vaccine.3.2 Immunogenicity of reassortment virus rgAA-H3N2The working seed lots of rgAA-H3N2 were injected into allantoic cavity of 10 days embryonated chick eggs. The allantoic fluid was collected which HA titre≥2⁸ after incubation. The purified antigen was got by ultracentrifugation. Six-eight week old BALB/c mice were immunized with rgAA-H3N2 intranasally and subcutaneous injection. The immune dose was 10⁵TCID50 and the internal time was 28d between two immune. The serum were collected after 28d of primary immune, and 14d of second immune, neutral antibodies were detected by HI test. Specificity sera IgG were detected by ELISA. At the same time ,the local sIgA in lung wash and nasal wash were detected by ELISA. These results opens the way for farther research on gene function and novel vaccine candidate of cold-adapted, live attenuated human influenza virus.更多还原