WO2018157454A1 - 一种流感病毒弱毒活疫苗毒株的筛选和鉴定方法 - Google Patents

一种流感病毒弱毒活疫苗毒株的筛选和鉴定方法 Download PDF

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WO2018157454A1
WO2018157454A1 PCT/CN2017/080560 CN2017080560W WO2018157454A1 WO 2018157454 A1 WO2018157454 A1 WO 2018157454A1 CN 2017080560 W CN2017080560 W CN 2017080560W WO 2018157454 A1 WO2018157454 A1 WO 2018157454A1
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virus
gene
mice
attenuated
influenza virus
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程根宏
秦晓峰
王路岚
徐娟
刘舒扬
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苏州系统医学研究所
美国加利福尼亚大学
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  • the invention relates to a method for screening and identifying a live virus vaccine strain of influenza virus, belonging to the technical field of biopharmaceuticals.
  • influenza is a respiratory infection that poses a serious threat to human health and public health.
  • the current main method of preventing and treating influenza is to treat patients with antiviral drugs and to immunize the normal population.
  • influenza viruses due to the widespread drug resistance of influenza viruses that have been popular in recent years, the efficacy of currently used anti-influenza drugs is very limited, and the treatment of influenza faces a situation where no medicine is available. Therefore, vaccination has become the most basic and effective means of the current influenza prevention system.
  • influenza vaccines There are two types of influenza vaccines currently in use, one is an influenza virus inactivated vaccine and the other is a live attenuated influenza virus vaccine. China uses an influenza inactivated vaccine.
  • the chicken embryo production system using the current vaccine generally takes nearly six months to produce a new vaccine, which often prevents us from producing in time when it is facing an influenza epidemic or pandemic. And reserve a sufficient amount of vaccine.
  • some bird flu viruses may not be able to produce vaccines for such viruses at all through chicken embryos due to their high lethality to chicken embryos.
  • the live attenuated influenza vaccine can provide a very good immune protection effect to the human body because of its superiority in mediating cellular immunity and mucosal immunity.
  • cold-adapted attenuated vaccines are used to continuously pass the wild-type influenza virus under non-viral physiological conditions, so that the virus obtains a certain mutation in the process of cold adaptation, and then screens out the mutant virus as a live attenuated vaccine.
  • Strain This method is very time consuming, and the amount of mutant virus that can be obtained by cold adaptation is very limited, resulting in little selectivity in the candidate screening process.
  • the mutant virus obtained by cold adaptation often has one or several point mutations in the genome of the virus. This mutation is very prone to reversion mutation in the subsequent vaccine production and immunization process, which may cause the vaccine to return to the ancestor. Get sick.
  • the existing cold-adapted attenuated vaccines have been developed by relevant foreign units.
  • the present invention is a method for obtaining a vaccine strain of attenuated live vaccine by constructing and screening a high-density random insertion mutant library mediated by a Mu phage transposon of a viral gene, and developing a set of obtained A systematic system for systematic evaluation of attenuated live vaccine strains. These methods can be directly applied to the screening and evaluation of influenza virus attenuated live vaccine strains, and have extensive reference significance for the development and development of other virus attenuated live vaccines.
  • the present invention adopts the following technical solutions:
  • the invention relates to a method for screening and identifying a live attenuated influenza virus vaccine strain, which comprises the following steps:
  • the 15 nt long oligonucleotide sequence of 5'-NNNNNTGCGGCCGCA-3' was inserted into each base of the influenza A/WSN/1933M gene using the Finnzymes Mu phage transposon-mediated random insertion mutation kit. a high-density mutant library of influenza virus M genes;
  • the plasmid carrying the M gene mutant was transformed into E. coli DH10B receptor cells by electroporation, and the M gene mutant library plasmid was extracted from the recombinant strain, and then reversed by A/WSN/33H1N1 influenza virus 8 plasmid virus. Learning the operating system to obtain a library of virus mutants;
  • the virus mutant library virus obtained in the step (2) is passaged on the MDCK cells, and then the viral RNA is extracted by the TRIzol reagent, and each RNA is reverse-transcribed according to the reverse transcription kit iScriptTM cDNA Synthesis kit to generate the corresponding cDNA.
  • the specific forward primers of 3 M genes were used, respectively, 5'-AGCAAAAGCAGGTAGATATT-3', 5'-GGGGCCAAAGAAATAGCACT-3', 5'-TCCTAGCTCCAGTGCTGGTC-3' and the insertion sequence specificity of the Vic marker.
  • the fluorescently-labeled PCR product obtained by PCR was set up for one-time repetition and the Liz-500 molecular weight standard was sequenced using a 96-capillary 3730xl DNA analyzer.
  • the generated data was analyzed by the ABI software according to the following criteria to remove the PCR process, primers and sequencing instruments. Non-specific data;
  • the mutant library virus was concentrated by ultracentrifugation, and the virus titer was measured for subsequent mouse infection experiments.
  • the virus was infected with C57/B6 mice of 6-8 weeks old by intranasal drip, respectively.
  • the lung tissues of the mice were collected and homogenized on the second, fourth, sixth and eighth days.
  • Total RNA was extracted from the lung homogenate with TRIzol reagent, and the samples were sampled according to the above method (3).
  • the viral M gene was sequenced and qualitatively and quantitatively analyzed. According to the presence of different M gene mutant viruses in each sample, attenuated live vaccine candidate strains were determined;
  • Attenuated live vaccine genetic stability test The W7-791 virus was passaged in MDCK cells and mice, and the sequence of the M gene in the gene sequence obtained from the cell or mouse lung homogenate was determined. Determining that the mutation of the W7-791 virus M gene can be stably inherited;
  • the conditions of the electrotransformation in the step (2) are 2.0 kV, 200 ⁇ , and 25 ⁇ F.
  • the specific method for obtaining the virus mutant library in the step (2) is: the cultured HEK293T cells are transferred to the 6-well culture plate, and when the cell confluence reaches 80-90%, according to the transfection reagent operation instructions, The plasmid inserting the mutant M gene and the plasmid containing the other 7 gene fragments of influenza virus were mixed in equal amounts, mixed with the transfection reagent in proportion, incubated at room temperature for 15 min, added dropwise to HEK293T cell culture medium, and cultured at 37 ° C, 5% CO 2 . The cells were cultured for 48 h, and the supernatant of the transfected cells was collected and the virus was inoculated. The MDCK cells were expanded, and the virus was collected 48 hours after infection, and some of the virus was frozen for later use.
  • the PCR in step (3) uses Novagen's PCR enzyme KOD Hot-Start polymerase, the reaction conditions of the PCR are pre-denaturation 95 ° C, 10 min; denaturation 95 ° C, 45 s; annealing 52 ° C, 30 s; extension 72 ° C, 90 s; Run 30 cycles; finally extend at 72 ° C for 10 min.
  • the method for removing non-specific data in step (3) is: (a) all data meet the standard default detection level; (b) the initial 70 bp of the sequence has a strong non-specific background, and is removed; c) all sequences are aligned with the DNA sequence corresponding to the influenza virus M gene; (d) the sequencing data are compared with wild-type A/WSN/1933 virus-infected cells, uninfected virus cells, and different gene libraries. Normalized processing.
  • the criterion for determining the attenuated vaccine candidate strain in the step (4) is that the virus can be efficiently replicated after infection, and is cleared by the body at 6-8 days.
  • the titer in the step (5) is 10 6 - 10 8 TCID50.
  • influenza virus attenuated live vaccine strain screened by the method of the present invention was deposited at the General Microbial Culture Collection of the China Microbial Culture Collection Management Committee, and the deposit number was CGMCC No. 13784. Its M gene has the sequence shown as SEQ ID No. 1.
  • Mu phage transposon-mediated random insertion technology enables rapid and high-throughput access to a library of high-density mutants of any gene, combined with the influenza virus reverse genetics operating system, to obtain a large library of influenza virus mutants.
  • the virus mutant library provides a material basis for screening influenza virus attenuated vaccine strains. This is superior to traditional methods for constructing attenuated vaccine strains by cold-adapted mutations.
  • the invention utilizes the emerging second-generation high-throughput sequencing technology and the screening of viral mutant libraries in mice to obtain a vaccine screening technique for attenuated live vaccine candidate strains, which also has high throughput and high speed. And the reliability of the results, can greatly shorten the development cycle of attenuated vaccines.
  • the present invention develops a systematic and comprehensive vaccine immune effect evaluation system, which not only can scientifically evaluate the immune protection provided by the vaccine, but also can clarify the specific mechanism of the vaccine providing immune protection to a certain extent. Further improvements and optimizations are available to help.
  • Figure 1 is a schematic diagram showing the process of establishing a transposon-mediated M gene fragment mutation influenza virus library
  • Figure 2 is a genetic analysis map of the MM mutant library
  • FIG. 1 Distribution profile of different mutant clones isolated by primary mutation on M gene (A); schematic diagram of virus titer change in transfected M gene mutant plasmid into 293T cells (B);
  • FIG. 1 Schematic diagram of the effect of influenza virus on the viability of MDCK cells
  • Figure 6 Schematic diagram of virus titer changes in infected MDCK cells
  • FIG. 7 Schematic diagram of the effects of influenza virus strain (W7-791) and wild influenza virus strain (WT-WSN) of the present invention on mice;
  • FIG. 1 Schematic diagram of the resistance of mice to the same subtype influenza virus after inoculation of the influenza virus strain (W7-791) of the present invention.
  • FIG. 9 Schematic diagram of the immune response of mice to the influenza virus strain (W7-791) of the present invention.
  • FIG. 1 (A) a transposon-mediated mutation was randomly inserted into 15 bases on the influenza virus (A/WSN/1933) M gene fragment to obtain a high-density mutant M gene plasmid library.
  • the obtained M gene mutant plasmid library and the other seven wild type influenza virus gene reverse genetics manipulation plasmids were co-transfected to establish a mutant virus library.
  • Cell supernatants containing a mutated virus pool are continued to infect MDCK cells or directly infect mice. The lungs and lymphoid organs of infected mice were collected at different time points to isolate the virus.
  • Mutations that cause slower growth of the virus are identified by genotyping and further evaluated for in vivo and in vitro infection;
  • B PCR amplification is performed using gene-specific primers and primers that recognize the 15 nt insert, and the obtained PCR product is sequenced. The location of all the mutation points in the library can be obtained.
  • C Influenza mutation libraries can be screened in vivo or in vitro. The necessary and non-essential regions of the genome can be determined by comparing the results of the genotyping of the unscreened and screened libraries.
  • Figure 2 is a summary of the results of genetic analysis of the influenza virus A WSN (H1N1) M1 and M2 protein populations. Columns indicate insertion sites in the gene. All mutant libraries were transfected into 293T cells and expanded in MDCK cells for several rounds, labeled 1-4 generations. The fluorescence intensity represented by the peak reflects the amount of viral RNA (lower part).
  • Fig. 3 8 6-8 week old C57/B6 mice were intranasally infected with the M gene fragment mutant virus library. The lungs were collected at different time points and genotyped. Each peak represents the amount of viral RNA at the insertion site. PBS-treated and wild-type WSN-infected lung homogenates served as positive controls.
  • a single viral clone (A) screened from the M gene mutation library has an overview of the distribution of 67 different mutant clones separated by the primary mutation on the M gene.
  • MDCK cells were infected with 0.25 MOI of wild-type WSN virus and W7-791 virus to detect viral titers at different time points.
  • FIG 8 (A) Schematic diagram of mouse immunization and viral infection process;
  • BC 5 mice per group were intranasally immunized with 10 5 PFU of W7-791 or the same volume of PBS, and immunized with 4 times MLD50 WSN after one month of immunization.
  • Virus, the body weight and survival status of the mice were detected regularly after virus infection;
  • DE 5 mice per group were intranasally immunized with 10 5 PFU of W7-791 or PBS, and immunized with 4 times MLD50 of PR8 virus one month after immunization, virus The body weight and survival status of the mice were measured periodically after infection.
  • *** represents a P-value ⁇ 0.001.
  • FIG. 9 (A) detection of viral titer in mouse lung homogenate; (B) detection of serum HAI activity in immunized mice; (C) detection of serum anti-influenza antibodies in immunized mice; (D) micro-neutralization assay Neutralizing antibody titer in serum of W7-791 immunized mice; (EF) passed the serum of W7-791 immunized mice to unimmunized mice, and after 24 hours, inoculated lethal doses of WSN and HK68/H3 virus, observed and The survival rate of mice at each time point was recorded; (GH) T cells from W7-791-immunized mice were passed to unimmunized mice, and lethal doses of WSN and HK68/H3 virus were inoculated 24 hours later, and the time points were observed and recorded. Rat survival rate.
  • FIG. 1 The specific technical scheme for the screening and evaluation method of the novel influenza virus attenuated live vaccine established by the invention is shown in FIG. 1 , and the specific description is as follows:
  • the plasmid carrying the M gene mutant was transformed into E. coli DH10B receptor cells by electroporation, and the conditions for electroporation were 2.0 kV, 200 ⁇ , 25 ⁇ F (ElectroMaxTM DH10B, Invitrogen).
  • the M gene mutant library plasmid was extracted from the recombinant strain, and then the virus mutant library was obtained using the A/WSN/33 (H1N1) influenza virus 8 plasmid virus reverse genetics operating system established by Hoffmann et al. (PNAS, 2000).
  • the specific method is: the cultured HEK293T cells are transferred to a 6-well culture plate, and when the cell confluence reaches 80-90%, according to the transfection reagent operation instructions, the plasmid containing the inserted mutation M gene and the other 7 genes containing the influenza virus are included.
  • the plasmids of the fragments were mixed in equal amounts, mixed with the transfection reagents in proportion, incubated at room temperature for 15 min, added dropwise to HEK293T cell culture medium, cultured at 37 ° C, 5% CO 2 incubator for 48 h, and the transfected cell supernatants were collected and The virus was inoculated on MDCK cells for amplification. The virus was collected 48 hours after infection, and some of the virus was frozen for later use (Fig. 1A).
  • the virus mutant library virus obtained above was passaged on MDCK cells, and then the viral RNA was extracted using TRIzol reagent (Invitrogen), and each RNA was subjected to the operation of the reverse transcription kit iScriptTM cDNA Synthesis kit (Bio-Rad). Reverse transcription is required to generate the corresponding cDNA.
  • the specific forward primers of each of the three M genes (5'-AGCAAAAGCAGGTAGATATT-3', 5'-GGGGCCAAAGAAATAGCACT-3', 5'-TCCTAGCTCCAGTGCTGGTC-3') were specifically specific to the inserted sequence of the Vic marker.
  • the reverse primer was used for PCR, and the PCR enzyme KOD Hot-Start polymerase of Novagen was used for PCR.
  • the reaction conditions of the PCR were pre-denaturation at 95 ° C for 10 min (1 cycle); denaturation at 95 ° C for 45 s; annealing at 52 ° C for 30 s; extension at 72 ° C for 90 s; run for 30 cycles; and finally 72 ° C for 10 min (1 cycle).
  • the fluorescently-labeled PCR product obtained by PCR (provided with one-time repetition) and Liz-500 molecular weight standard (Applied Biosystem) were sequenced using a 96-capillary 3730xl DNA analyzer (3730xl DNA Analyzer, Applied Biosystems) (Fig. 1B).
  • the generated data was analyzed using the ABI software according to the following criteria: (1) all data met the standard default detection level; (2) the original 70 bp of the sequence was removed because of its strong non-specific background; (3) all The sequences are all aligned with the DNA sequence corresponding to the influenza virus M gene; (4) the sequencing data are normalized to the wild-type A/WSN/1933 virus-infected cells, uninfected virus cells, and different gene library controls.
  • the treatment eliminates the non-specific data generated by the PCR process, primers, and sequencing equipment.
  • a mouse model and the above-described second generation sequencing technology to identify a viral mutant library group.
  • the method of sub-screening screened the vaccine strain of influenza virus attenuated live vaccine from the M gene mutant virus library (Fig. 1A).
  • the mutant library virus was first concentrated by ultracentrifugation, and the virus titer was measured for subsequent mouse infection experiments.
  • the virus infects 6-8 weeks old C57/B6 mice by intranasal administration, 8 rats in each group, and the lung tissues of the mice are collected on the second, fourth, sixth and eighth days after infection, respectively.
  • total RNA extracted from lung tissue of mice infected with PBS and wild-type WSN virus was used as a control.
  • the sequence of the M gene in the extracted RNA was qualitatively and quantitatively analyzed by the method of the above 3 to determine the presence of different M gene mutant viruses in each sample.
  • Figure 3 three different replication kinetics of the virus ( Figure 3).
  • the A cluster virus which has an effective replication ability, like wild-type viruses, may cause disease; the B-clustered virus may affect the structure and function of viral genes and proteins due to mutations.
  • W7-791 may be an ideal attenuated vaccine candidate strain;
  • Attenuated vaccine genetic stability test In order to ensure that the vaccine does not undergo back mutation, the attenuated vaccine returns to the ancestors, we will W7-791 The virus was subjected to a series of passages in MDCK cells and mice. The sequence of the gene sequence, especially the M gene, obtained from the cell or mouse lung homogenate was determined. We found that the W7-791 virus M gene was mutated. It can be stably inherited without the deletion or reversion of the insertion mutation. Moreover, as the number of passages increased, the titer of the W7-791 virus gradually decreased.
  • mice infected with w7-791 for 6 days were immunized with different titers of W7-791 virus, even when the virus inoculation amount was as high as 10 7 TCID50 per mouse, we did not find that the mice produced body weight. Drop and flu symptoms. Compared to wild-type WSN virus-infected mice of 10 3 TCID50, significant flu symptoms and weight loss occurred. The viral load of mice infected with w7-791 for 6 days was 100 times lower than that of wild-type WSN virus and H3 subtype virus-infected mice (Fig. 7A, B, C, D).
  • the main contents are as follows: (1) Immunoprotective assay: Mice were immunized with W7-791, and mice were infected with wild-type WSN virus or PR8 virus of 4 times MLD50 one month after immunization. We found that the unimmunized mice had a severe weight loss and died during the experiment, while the W7-791 immunized mice maintained normal body weight and did not show any flu symptoms (Fig. 8A-E). (2) Detection of humoral immunity levels: Influenza-specific antibodies or virus-neutralizing antibodies in the serum of immunized mice can be determined by influenza virus hemagglutination inhibition assay or virus neutralization assay.

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Abstract

公开了一种流感病毒弱毒活疫苗毒株的筛选和鉴定方法，通过构建和筛选病毒基因由Mu噬菌体转座子介导的高密度随机插入突变体库，获得弱毒活疫苗候选毒株的方法，并开发了一套对所获得的弱毒活疫苗毒株进行系统性评价的技术体系。

Description

一种流感病毒弱毒活疫苗毒株的筛选和鉴定方法 技术领域
本发明涉及一种流感病毒弱毒活疫苗毒株的筛选和鉴定方法，属于生物制药技术领域。
背景技术
流感是一种严重威胁人类健康和公共卫生安全的呼吸道传染病。当前防治流感的主要方法是患者应用抗病毒药物进行治疗和对正常人群进行免疫接种。但是由于近些年来流行的流感病毒都具有普遍的耐药性，使得目前使用的抗流感病毒药物的疗效非常有限，流感的治疗面临无药可用的局面。因此疫苗接种已成为当前流感防治体系中最为基础和有效手段。目前使用的流感疫苗有两种，一种为流感病毒灭活疫苗，另一种为流感病毒减毒活疫苗。中国使用的是流感灭活疫苗。
传统的灭活疫苗都是利用鸡胚作为生产基质，这使得其应用面临诸多问题。首先，由鸡胚生产的流感疫苗中会残存一些鸡胚的成分，这些成分往往造成疫苗接种者出现比较严重的副反应，这使得某些人群根本无法接种流感疫苗；为了降低疫苗接种的副反应，现在要经过复杂的纯化过程去除鸡胚蛋白，而这又增加了疫苗生产的时间和经济成本。其次，目前所使用的灭活疫苗对于婴幼儿及老年人的免疫效果不是很好，而其主要原因可能是鸡胚疫苗中的异源性抗原分散了这些人群机体有限的免疫应答能力，另外也与禽类和人类细胞在抗原微修饰方面的差异有关。此外，当一种新型流感病毒出现后，利用当前疫苗的鸡胚生产体系一般需要将近6个月的时间才能生产出新的疫苗，这常使得我们无法在面临流感流行或大流行的时候及时生产和储备足够量的疫苗。而且某些流禽流感病毒由于其对鸡胚的高度致死性，可能根本就无法通过鸡胚来生产该类病毒的疫苗。流感减毒活疫苗由于其在介导细胞免疫和粘膜免疫方面的优势特点，它能给人体提供非常好的免疫保护效果。这些冷适应减毒疫苗是将野生型流感病毒在非病毒生理条件下进行持续的传代，使病毒在冷适应的过程中获得某种突变，进而从突变的病毒中筛选出能够作为弱毒活疫苗的毒株。这种方法非常的耗时，冷适应能获得的突变病毒量非常有限，导致在候选筛选过程中的可选择性很小。另外。冷适应获得的突变病毒，往往是在病毒的基因组发生一个或若干个点突变，这种突变在后续疫苗生产和免疫过程中非常容易发生回复性突变，可能造成疫苗的返祖，进而引 起疾病。尽管如此，现有的冷适应减毒疫苗都是由国外相关单位所研发，我国还没有自己独立知识产权的减毒疫苗毒株，这在一定程度上阻碍了流感病毒减毒活疫苗在中国的生产和应用。由此可见，发展新的流感减毒活疫苗筛选和评价体系，开发具有自主知识产权的减毒疫苗毒株，克服传统弱毒疫苗研制体系缺陷是应对当前严峻的流感防控形势迫切需求，也具有很高的经济价值。
发明内容
本发明是研究和开发了一种通过构建和筛选病毒基因由Mu噬菌体转座子介导的高密度随机插入突变体库，获得弱毒活疫苗候选毒株的方法，并开发了一套对所获得的弱毒活疫苗毒株进行系统性评价的技术体系。这些方法不但可以直接应用于流感病毒弱毒活疫苗毒株的筛选和评价，而且对于其他病毒弱毒活疫苗的研制和开发有广泛的借鉴意义。
为实现上述目的，本发明采用如下技术方案：
一种流感病毒弱毒活疫苗毒株的筛选和鉴定方法，其特征在于包括以下步骤：
(1)利用Mu噬菌体转座子介导的随机插入技术建立M基因的高密度突变库：
利用Finnzymes公司Mu噬菌体转座子介导的随机插入突变试剂盒向流感病毒A/WSN/1933M基因各个碱基间插入5’-NNNNNTGCGGCCGCA-3’这一15nt长的寡核苷酸序列，从而获得流感病毒M基因的高密度突变库；
(2)通过流感病毒反向遗传学技术获得病毒突变体库：
用电转化的方法将携有M基因突变体的质粒转化入大肠杆菌DH10B感受体细胞，从重组菌种提取M基因突变体库质粒，然后利用A/WSN/33H1N1流感病毒8质粒病毒反向遗传学操作系统获取病毒突变体库；
(3)通过第二代测序技术对病毒突变体库组分进行分析：
取步骤(2)获得的病毒突变体库病毒在MDCK细胞上进行传代，然后利用TRIzol试剂提取病毒RNA，并对各RNA按照反转录试剂盒iScriptTM cDNA Synthesis kit进行反转录产生相应的cDNA，以该cDNA为模板，分别使用3个M基因的特异性正向引物5’-AGCAAAAGCAGGTAGATATT-3’，5’-GGGGCCAAAGAAATAGCACT-3’，5’-TCCTAGCTCCAGTGCTGGTC-3’与Vic标记的插入序列特异性反向引物做PCR扩增， 由PCR获得的荧光标记PCR产物设置一次重复和Liz-500分子量标准利用96-毛细管3730xl DNA分析仪进行测序，所产生的数据应用ABI软件依照以下标准进行分析，去除PCR过程、引物及测序仪器产生的非特异性数据；
(4)小鼠体内筛选流感病毒弱毒活疫苗候选毒株：
首先通过超速离心的方法浓缩突变体库病毒，测定其病毒滴度后用于后续小鼠感染实验，病毒通过滴鼻的方法感染6-8周龄的C57/B6小鼠，分别在感染后第二天、第四天、第六天和第八天收取小鼠的肺脏组织并进行匀浆处理，从肺组织匀浆中用TRIzol试剂提取总RNA，依照上述步骤(3)的方法对样本中病毒M基因进行测序并进行定性和定量分析，根据不同M基因突变病毒在各样本中的存在情况，确定弱毒活疫苗候选毒株；
(5)弱毒活疫苗候选毒株遗传稳定性和安全性评价：
(a)弱毒活疫苗的分离和表型鉴定：首先对上述弱毒活疫苗候选毒株进行了单克隆化，对其中能在MDCK细胞中能够有效复制的W7-757、W7-791和W7-797三株病毒进行扩增，筛选出表现出更好的复制能力和较低的细胞毒性的W7-791病毒；
(b)弱毒活疫苗遗传稳定性检测：将W7-791病毒在MDCK细胞和小鼠体内进行传代，对从细胞或小鼠肺脏匀浆中获得病毒的基因序列中的M基因的序列进行测定，确定W7-791病毒M基因的突变能够被稳定地遗传下去；
(c)弱毒疫苗的安全性评估：用不同滴度的W7-791病毒免疫6-8周龄小鼠，没发现小鼠产生体重下降及流感症状；给15日龄的新生BALB/c小鼠滴鼻接种不同滴度的W7-791或104TCID50的野生型WSN病毒，对小鼠体重和肺脏病变进行检测，W7-791接种小鼠上未观察到像野生型WSN病毒感染小鼠那样的体重下降和肺部病变，由此确定W7-791病毒即为流感病毒弱毒活疫苗毒株。
进一步地，步骤(2)中的电转化的条件是2.0kV、200Ω、25μF。
进一步地，步骤(2)中获取病毒突变体库的具体方法是：培养的HEK293T细胞转至6孔培养板中，待细胞汇合度达到80～90％时，按照转染试剂操作说明，将含插入突变M基因的质粒和含有流感病毒其他7个基因片段的质粒等量混合，与转染试剂按比例混匀，室温孵育15min，逐滴加入HEK293T细胞培养液中，37℃，5％CO2培养箱中培养48h，收集转染细胞上清，并将病毒接种于 MDCK细胞上进行扩增，感染48小时后收集病毒，将部分病毒冻存以备后用。
进一步地，步骤(3)中PCR使用Novagen的PCR酶KOD Hot-Start polymerase，PCR的反应条件是预变性95℃，10min；变性95℃，45s；退火52℃，30s；延伸72℃，90s；运行30个循环；最后72℃延伸10min。
进一步地，步骤(3)中去除非特异性数据的方法为：(a)所有数据都满足标准的默认检测水平；(b)由于序列的初始70bp具有较强的非特异性背景，所以被去除；(c)所有的序列都与流感病毒M基因对应的DNA序列进行联配；(d)对测序数据分别做相对于野生型A/WSN/1933病毒感染细胞、未感染病毒细胞、及不同基因文库对照的归一化处理。
进一步地，步骤(4)中确定弱毒疫苗候选毒株的标准为：病毒在感染后能够有效地复制，且在6-8天时被机体清除。
进一步地，步骤(5)中的滴度为106-108TCID50。
通过本发明方法筛选得到的流感病毒弱毒活疫苗毒株在中国微生物菌种保藏管理委员会普通微生物菌种保藏中心进行了保藏，保藏号为CGMCC No.13784。其M基因具有如SEQ ID No.1所示的序列。
本发明的优点和有益效果
Mu噬菌体转座子介导的随机插入技术能够快速、高通量获得任何基因的高密度突变体库，结合流感病毒反向遗传学操作系统，能获得很大库容的流感病毒突变体库，该病毒突变体库为筛选流感病毒弱毒疫苗毒株提供了物质基础。这优于传统的通过冷适应突变的办法构建弱毒疫苗毒株的方法。另外，该发明中综合运用新兴第二代高通量测序技术和小鼠体内对病毒突变体库进行筛选进而获得弱毒活疫苗候选毒株的疫苗筛选技术，该技术也具有高通量、速度快和结果可靠等特点，可以大大缩短弱毒疫苗的研制周期。最后，本发明发展了一套系统全面的疫苗免疫效果评价体系，不但能够科学评价疫苗为机体所能提供的免疫保护力，而且也能在一定程度上阐明疫苗提供免疫保护的具体机制，为疫苗的进一步改良和优化提供帮助。
附图说明
图1转座子介导的M基因片段突变流感病毒库的建立流程示意图；
图2M基因突变体库的基因分析图；
图3.用M基因突变体库滴鼻感染小鼠后在不同时间所做的基因分型结果；
图4.通过初次突变分离出来的不同突变克隆在M基因上的分布概况(A)；上述M基因突变质粒被转染到293T细胞中病毒滴度变化示意图(B)；
图5.流感病毒对MDCK细胞活力的影响示意图；
图6.感染MDCK细胞的病毒滴度变化示意图；
图7.本发明的流感病毒株(W7-791)与野生流感病毒株(WT-WSN)对小鼠的影响示意图；
图8.小鼠接种本发明的流感病毒株(W7-791)后，小鼠对同亚型流感病毒抵御作用示意图；
图9.小鼠对本发明的流感病毒株(W7-791)的免疫应答示意图。
其中，图1中(A)在流感病毒(A/WSN/1933)M基因片段上利用转座子介导的突变随机插入15个碱基，获得高密度突变的M基因质粒库。将获得的M基因突变质粒库和其他七个野生型流感病毒基因反向遗传学操作质粒共转染来建立突变病毒库。含有突变病毒库的细胞上清被继续用来感染MDCK细胞或者直接感染小鼠。在不同时间点收集被感染的小鼠的肺和淋巴器官来分离病毒。导致病毒生长减缓的突变用基因分型鉴定出来并进一步评价体内和体外感染效果；(B)用基因特异性引物和识别15nt插入片段的引物进行PCR扩增，并对获得的PCR产物测序，就可获得库里所有突变点的位置。(C)流感突变库可在体内或体外做筛选。通过比较未被筛选和被筛选过的库基因分型结果便可确定基因组中必需的和非必需的区域。
图2中，流感病毒A WSN(H1N1)M1和M2蛋白总体突变库的基因分析结果概要(上部分)。柱状表示基因中插入位点。所有突变库被转染到293T细胞中，并在MDCK细胞中扩增几轮，标记成1-4代。峰值代表的荧光强度反应了病毒RNA的量(下部分)。
图3中，用M基因片段突变病毒库滴鼻感染8只6-8周龄C57/B6小鼠.在不同时间点收集肺脏并做基因分型。各峰值表示插入位置的病毒RNA量。PBS处理的和野生型WSN感染的肺匀浆作为阳性对照。
图4中，从M基因突变库里筛选出的的单个病毒克隆(A)通过初次突变分离出来的67个不同突变克隆在M基因上的分布概况。(B)上述M基因突变质粒 被转染到293T细胞中，细胞上清被收集并测定病毒滴度。
图6中，用0.25MOI的野生型WSN病毒和W7-791病毒感染MDCK细胞来检测不同时间点的病毒滴度。
图7中，(A，C)接种106、107或者108TCID50的W7-791或野生型WSN病毒后小鼠体重检测；(B，D)接种后第四和第六天病毒滴度测定；(E)W7-791、WSN或PBS接种新生BALB/c小鼠后小鼠体重监测。
图8中，(A)小鼠免疫和病毒感染流程示意图；(B-C)每组5只小鼠滴鼻免疫105PFU的W7-791或者同体积PBS，免疫一个月后接种4倍MLD50的WSN病毒，在病毒感染后定时检测小鼠体重和存活状况；(D-E)每组5只小鼠滴鼻免疫105PFU的W7-791或者PBS，免疫一个月后接种4倍MLD50的PR8病毒，病毒感染后定时检测小鼠体重和存活状况。***代表P-值<0.001。
图9中，(A)小鼠肺匀浆液中病毒滴度检测；(B)免疫小鼠血清HAI活性检测；(C)免疫小鼠血清抗流感病毒抗体检测；(D)微量中和实验测定W7-791免疫小鼠血清中的中和抗体滴度；(E-F)过继W7-791免疫小鼠的血清到未免疫小鼠体内，24小时后接种致死量的WSN和HK68/H3病毒，观察并记录各时间点小鼠存活率；(G-H)过继W7-791免疫小鼠的T细胞到未免疫小鼠体内，24小时后接种致死量的WSN和HK68/H3病毒，观察并记录各时间点小鼠的存活率。
具体实施方式
下面结合附图，通过实施例对本发明进行具体描述和说明：
本发明所建立的新型流感病毒弱毒活疫苗筛选和评价方法的具体技术方案如图1所示，具体描述如下：
1.利用Mu噬菌体转座子介导的随机插入技术建立M基因的高密度突变库
首先，根据Finnzymes公司Mu噬菌体转座子介导的随机插入突变试剂盒(MGS kit,Finnzymes)说明书的操作步骤，向流感病毒A/WSN/1933M基因个各碱基间插入5’-NNNNNTGCGGCCGCA-3’这一15nt长的寡核苷酸序列，从而获得流感病毒M基因的高密度突变库(如图1A；图2)。
2.通过流感病毒反向遗传学技术获得病毒突变体库
用电转化的方法将携有M基因突变体的质粒转化入大肠杆菌(E.coli)DH10B感受体细胞，电转化的条件是2.0kV、200Ω、25μF(ElectroMax TM DH10B, Invitrogen)。从重组菌种提取M基因突变体库质粒，然后利用Hoffmann等(PNAS，2000)建立的A/WSN/33(H1N1)流感病毒8质粒病毒反向遗传学操作系统获取病毒突变体库。具体方法是：培养的HEK293T细胞转至6孔培养板中，待细胞汇合度达到80～90％时，按照转染试剂操作说明，将含插入突变M基因的质粒和含有流感病毒其他7个基因片段的质粒等量混合，与转染试剂按比例混匀，室温孵育15min，逐滴加入HEK293T细胞培养液中，37℃，5％CO2培养箱中培养48h，收集转染细胞上清，并将病毒接种于MDCK细胞上进行扩增。感染48小时后收集病毒，将部分病毒冻存以备后用(如图1A)。
3.通过第二代测序技术对病毒突变体库组分进行分析
取上述所获得的病毒突变体库病毒在MDCK细胞上进行传代，然后利用TRIzol试剂(Invitrogen)提取病毒RNA，并对各RNA按照反转录试剂盒iScriptTM cDNA Synthesis kit(Bio-Rad)操作说明的要求进行反转录产生相应的cDNA。以该cDNA为模板，使用分别3个M基因的特异性正向引物(5’-AGCAAAAGCAGGTAGATATT-3’，5’-GGGGCCAAAGAAATAGCACT-3’，5’-TCCTAGCTCCAGTGCTGGTC-3’)与Vic标记的插入序列特异性反向引物做PCR，PCR使用Novagen的PCR酶KOD Hot-Start polymerase。PCR的反应条件是预变性95℃10min(1循环)；变性95℃，45s；退火52℃，30s；延伸72℃，90s；运行30个循环；最后72℃延伸10min(1循环)。由PCR获得的荧光标记PCR产物(设置一次重复)和Liz-500分子量标准(Applied Biosystem)利用96-毛细管3730xl DNA分析仪(3730xl DNA Analyzer,Applied Biosystems)进行测序(如图1B)。所产生的数据应用ABI软件依照以下标准进行分析，(1)所有数据都满足标准的默认检测水平；(2)由于序列的初始70bp具有较强的非特异性背景，所以被去除；(3)所有的序列都与流感病毒M基因对应的DNA序列进行联配；(4)对测序数据分别做相对于野生型A/WSN/1933病毒感染细胞、未感染病毒细胞、及不同基因文库对照的归一化处理，这样就去除了PCR过程、引物及测序仪器产生的非特异性数据。
4.小鼠体内筛选流感病毒弱毒活疫苗候选毒株
在本发明中，我们应用小鼠模型和上述第二代测序技术鉴定病毒突变体库组 分的方法从M基因突变病毒库中筛选流感病毒弱毒活疫苗候选毒株(如图1A)。首先通过超速离心的方法浓缩突变体库病毒，测定其病毒滴度后用于后续小鼠感染实验。病毒通过滴鼻的方法感染6-8周龄的C57/B6小鼠，每组8只，分别在感染后第二天、第四天、第六天和第八天收取小鼠的肺脏组织并进行匀浆处理，从肺组织匀浆中用TRIzol试剂提取总RNA，依照上述3的方法对样本中病毒M基因进行测序并进行定量分析。在实验中，以PBS处理和野生型WSN病毒感染小鼠肺组织中提取的总RNA作为对照。利用上述3的方法对所提取RNA中M基因的序列进行定性和定量分析，以确定不同M基因突变病毒在各样本中的存在情况。我们观察到三种不同复制动力学的病毒(如图3)。如图3所示，A簇病毒，这种病毒具有有效的复制能力，它们与野生型病毒一样，可能会导致疾病；B簇突变病毒可能由于突变影响了病毒基因和蛋白的结构与功能或造成病毒逃逸宿主免疫应答功能的丧失而被严重致弱，生长缓慢，这些病毒由于在机体内几乎不能存活，所以不能有效刺激机体产生免疫应答，所以不是理想的疫苗候选毒株。相比之下，C簇病毒虽然在感染后前六天能够有效地复制，但是在6-8天时则被机体清除，此时几乎检测不到这些病毒的存在。这样的病毒就能刺激机体产生较强的免疫应答，但由于其不能持续复制而不能引起疾病，所以就可以作为弱毒活疫苗候选毒株。
5.候选弱毒活疫苗毒株遗传稳定性和安全性评价
良好的弱毒活疫苗需要具有绝对的安全性，而且其表型和基因型需要能够在代际之间稳定遗传。所以，我们对上述所筛选获得的弱毒疫苗候选毒株进行系统全面的安全性和遗传稳定性评价是本技术体系非常重要的组成部分。为此，我们进行了如下实验：(1)弱毒毒的分离和表型鉴定：我们首先对上述C簇病毒进行了单克隆化，一共获得了67个病毒克隆。并对其中能在MDCK细胞中能够有效复制的W7-757、W7-791和W7-797三株病毒进行了扩增(图4)。而其中W7-791表现出更好的复制能力和较低的细胞毒性(图5，图6)。所以我们初步认为W7-791可能是比较理想的弱毒疫苗候选毒株；(2)弱毒疫苗遗传稳定性检测：为了确保疫苗不会发生回复突变，发生弱毒疫苗返祖的现象，我们将W7-791病毒在MDCK细胞和小鼠体内进行了一系列的传代，对从细胞或小鼠肺脏匀浆中获得病毒的基因序列特别是M基因的序列进行了测定，我们发现W7-791病毒M 基因的突变能够被稳定地遗传下去，并不会发生插入突变的删除或回复突变的现象。而且随着传代次数的增多，W7-791病毒的滴度也逐渐降低。这说明w7-791病毒所具有的突变和表型能够稳定的遗传下去。(3)疫苗的安全性评估：用不同滴度的W7-791病毒免疫6-8周龄小鼠，甚至是当每只小鼠病毒接种量高达107TCID50，我们也没发现小鼠产生体重下降及流感症状。与相比，103TCID50的野生型WSN病毒感染的小鼠则出现明显的流感症状并出现体重下降。w7-791感染小鼠6天病毒载量要比野生型WSN病毒及H3亚型病毒感染小鼠肺内病毒滴度低100倍(图7A，B，C，D)。如果观察感染后4天小鼠的肺脏，我们发现PBS组和W7-791感染小鼠的肺脏没有发生明显病变，而野生型WSN病毒感染的小鼠则呈现严重的肺组织损伤。为了进一步确认W7-791的安全性，我们给15日龄的新生BALB/c小鼠滴鼻接种不同量(106,107or 108TCID50)的W7-791或104TCID50的野生型WSN病毒，小鼠体重和肺脏病变检测结果表明，W7-791接种小鼠上未观察到像野生型WSN病毒感染小鼠那样的体重下降和肺部病变(图7E)。这些结果都表明，我们筛选获得的流感病毒突变株W7-791是只能在体外和体内呈限制性复制，对成年和新生小鼠都具有较高安全新的弱毒株。
6.候选疫苗毒株保护效果的系统性评价
确定候选疫苗株后，就需要对其为机体所能提供的免疫保护力进行评价。其中主要涉及一下内容：(1)免疫保护性检测：用W7-791免疫小鼠，在免疫后一月，用4倍MLD50的野生型WSN病毒或PR8病毒对小鼠进行感染。我们发现未免疫组小鼠在实验过程中体重严重下降并死亡，而W7-791免疫小鼠一直保持了正常的体重，而且也未表现出任何流感症状(如图8A-E)。(2)体液免疫水平检测：通过流感病毒血凝抑制实验或病毒中和实验可以测定免疫小鼠血清中流感特异性抗体或病毒中和抗体。免疫小鼠抗体检测结果表明，W7-791免疫的小鼠只产生了WSN病毒特异性的抗体，而没有针对PR8病毒、HK68(H3N1)、Wis(H3N2)病毒的抗体(如图9A-C)。而将W7-791免疫小鼠的血清过继转移给未免疫小鼠，在用各种病毒对这些小鼠感染时，免疫小鼠血清除能提供部分针对WSN本身的保护了外，并不能保护其他病毒对小鼠的感染(图9D-F)。这就说明体液免疫并不是W7-791病毒株所提供免疫力的唯一来源。(3)细胞免疫应答水平检测：将W7-791免疫小鼠的T淋巴细胞过继转移给未免疫小鼠，然后用不同的野生型流 感病毒感染小鼠，观察所过继T淋巴细胞可能为小鼠所能提供的免疫力，从而确定T细胞免疫在疫苗保护中所发挥的作用。我们发现，当W7-791免疫小鼠的T细胞过继转移给未免疫小鼠后，能够使小鼠获得部分广谱的保护力，从而在一定程度上降低小鼠在受到各种流感病毒感染时的发病程度和疾病症状(图9D-F)。由此说明W7-791能有效诱导机体产生保护性T细胞免疫应答，这也符合流感病毒弱毒活疫苗免疫的特点。

Claims (5)

  1. 一种流感病毒弱毒活疫苗毒株的筛选和鉴定方法，其特征在于包括以下步骤：
    (1)利用Mu噬菌体转座子介导的随机插入技术建立M基因的高密度突变库：
    利用Finnzymes公司Mu噬菌体转座子介导的随机插入突变试剂盒向流感病毒A/WSN/1933的M基因各个碱基间插入5’-NNNNNTGCGGCCGCA-3’这一15nt长的寡核苷酸序列，从而获得流感病毒M基因的高密度突变库；
    (2)通过流感病毒反向遗传学技术获得病毒突变体库：
    用电转化的方法将携有M基因突变体的质粒转化入大肠杆菌DH10B感受体细胞，从重组菌种提取M基因突变体库质粒，然后利用A/WSN/33H1N1流感病毒8质粒病毒反向遗传学操作系统获取病毒突变体库；
    (3)通过第二代测序技术对病毒突变体库组分进行分析：
    取步骤(2)获得的病毒突变体库病毒在MDCK细胞上进行传代，然后利用TRIzol试剂提取病毒RNA，并对各RNA按照反转录试剂盒iScriptTM cDNA Synthesis kit进行反转录产生相应的cDNA，以该cDNA为模板，分别使用3个M基因的特异性正向引物，其序列分别为SEQ ID No.2，SEQ ID No.3，SEQ ID No.4，与Vic标记的插入序列特异性反向引物做PCR扩增，由PCR获得的荧光标记PCR产物设置一次重复和Liz-500分子量标准利用96-毛细管3730xl DNA分析仪进行测序，所产生的数据应用ABI软件进行分析，去除PCR过程、引物及测序仪器产生的非特异性数据；
    (4)小鼠体内筛选流感病毒弱毒活疫苗候选毒株：
    首先通过超速离心的方法浓缩突变体库病毒，测定其病毒滴度后用于后续小鼠感染实验，病毒通过滴鼻的方法感染6-8周龄的C57/B6小鼠，分别在感染后第二天、第四天、第六天和第八天收取小鼠的肺脏组织并进行匀浆处理，从肺组织匀浆中用TRIzol试剂提取总RNA，依照上述步骤(3)中方法对样本中病毒M基因进行测序并进行定性和定量分析，根据不同M基因突变病毒在各样本中的存在情况，确定弱毒活疫苗候选毒株；
    (5)弱毒活疫苗候选毒株遗传稳定性和安全性评价：
    (a)弱毒活疫苗的分离和表型鉴定：首先对上述弱毒活疫苗候选毒株进行了单克隆化，对其中能在MDCK细胞中能够有效复制的W7-757、W7-791和W7-797三株病毒进行扩增，初步筛选出表现出更好的复制能力和较低的细胞毒性的W7-791病毒；
    (b)弱毒活疫苗遗传稳定性检测：将W7-791病毒在MDCK细胞和小鼠体内进行传代，对从细胞或小鼠肺脏匀浆中获得病毒的基因序列中的M基因的序列进行测定，确定W7-791病毒M基因的突变能够被稳定地遗传下去；
    (c)弱毒疫苗的安全性评估：用不同滴度的W7-791病毒免疫6-8周龄小鼠，没发现小鼠产生体重下降及流感症状；给15日龄的新生BALB/c小鼠滴鼻接种不同滴度的W7-791或104TCID50的野生型WSN病毒，对小鼠体重和肺脏病变进行检测，W7-791接种小鼠上未观察到像野生型WSN病毒感染小鼠那样的体重下降和肺部病变，由此确定W7-791病毒即为流感病毒弱毒活疫苗毒株。
  2. 根据权利要求1所述的流感病毒弱毒活疫苗毒株的筛选和鉴定方法，其特征在于：步骤(2)中的电转化的条件是2.0kV、200Ω、25μF。
  3. 根据权利要求1所述的流感病毒弱毒活疫苗毒株的筛选和鉴定方法，其特征在于：步骤(2)中获取病毒突变体库的具体方法是：培养的HEK293T细胞转至6孔培养板中，待细胞汇合度达到80～90％时，按照转染试剂操作说明，将含插入突变M基因的质粒和含有流感病毒其他7个基因片段的质粒等量混合，与转染试剂按比例混匀，室温孵育15min，逐滴加入HEK293T细胞培养液中，37℃，5％CO2培养箱中培养48h，收集转染细胞上清，并将病毒接种于MDCK细胞上进行扩增，感染48小时后收集病毒，将部分病毒冻存以备后用。
  4. 根据权利要求1所述的流感病毒弱毒活疫苗毒株的筛选和鉴定方法，其特征在于：步骤(3)中PCR使用Novagen的PCR酶KOD Hot-Start polymerase，PCR的反应条件是预变性95℃，10min；变性95℃，45s；退火52℃，30s；延伸72℃，90s；运行30个循环；最后72℃延伸10min。
  5. 根据权利要求1所述的流感病毒弱毒活疫苗毒株的筛选和鉴定方法，其特征在于：步骤(4)中确定弱毒活疫苗候选毒株的标准为：病毒在感染后能够在小鼠肺部有效地复制，但在感染后6-8天时被机体清除。
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