WO2018157455A1 - 一种流感病毒株及其应用 - Google Patents

一种流感病毒株及其应用 Download PDF

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WO2018157455A1
WO2018157455A1 PCT/CN2017/080561 CN2017080561W WO2018157455A1 WO 2018157455 A1 WO2018157455 A1 WO 2018157455A1 CN 2017080561 W CN2017080561 W CN 2017080561W WO 2018157455 A1 WO2018157455 A1 WO 2018157455A1
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influenza
vaccine
virus
influenza virus
virus strain
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French (fr)
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程根宏
秦晓峰
王路岚
徐娟
刘舒扬
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Suzhou Institute of Systems Medicine
University of California Berkeley
University of California San Diego UCSD
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Suzhou Institute of Systems Medicine
University of California Berkeley
University of California San Diego UCSD
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—RNA viruses
    • C07K16/108—Orthomyxoviridae (F), e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K2039/525—Virus
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K2039/552—Veterinary vaccine
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    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to the field of viruses, and in particular to an influenza virus strain and an application thereof.
  • influenza virus The genome of influenza virus is composed of 8 single-stranded RNA fragments encoding 11 proteins belonging to the genus Influenzavirus of the Orthomyxoviridae family. Influenza viruses are classified into three types: A, B, and C. Types A and B are common types in the population and can cause a worldwide pandemic. However, influenza A virus infection is more widespread and more harmful.
  • the eight genomic fragments of influenza virus are PB2, PB1, PA, HA, P, NA, M and NS genes in descending order of molecular weight. Eight of the 11 viral proteins encoded by these gene fragments are viral structural proteins.
  • NS1 encodes NS1 and P NS2 are two non-structural proteins; two major surface glycoproteins: hemagglutinin (HA) and neuro-ammonium Acidase (NA) is the major surface antigen of influenza virus.
  • HA hemagglutinin
  • NA neuro-ammonium Acidase
  • influenza A virus is divided into different subtypes. Currently, 9 NA subtypes and 16 HA subtypes have been identified.
  • Influenza viruses can cause serious respiratory diseases, are highly contagious, and are prone to other serious complications. There have been many world pandemics that have caused great harm to human health. Due to the variability of the viral surface proteins HA and NA, two types of mutations, antigenic drift and antigenic shift, can be produced, especially in recent years, in the world and in China, the outbreaks of influenza viruses such as H1N1 and H7N9 And the epidemic has brought more difficulties to the prevention and control of diseases in the context of global integration.
  • influenza pandemics The widely used influenza vaccine is designed mainly for HA and NA proteins of viruses, and HA and NA are used as target antigens to induce immune protection.
  • the inactivated virus vaccines that have been approved for human use are two types that are harmful to humans.
  • a trivalent inactivated vaccine consisting of influenza A (H1N1 and H3N2) and a type B influenza virus.
  • the vaccine design method, research and development strategy and protection effect also have a direct impact.
  • the HA and NA proteins of influenza virus are prone to antigenic drift or transformation.
  • the production of new vaccines should be updated in time with the variation of the epidemic strains.
  • the breeding of virus vaccine strains is time-consuming and laborious, the production cycle is long, the cost is high, and it is difficult to adapt to the prevention.
  • Cellular immune response therefore, selecting a suitable target antigen for vaccine design, accelerating the screening of candidate vaccine strains, and fully improving the immunoprotective effect of vaccines are very important and urgent problems in current influenza vaccine research.
  • matrix protein M has been widely reported by many scholars in the selection of influenza virus vaccine target antigens.
  • M protein is encoded by viral RNA segment 7 and contains 1027 nucleotides, including non-sugar.
  • the M1 and M2 coding regions partially overlap, but have different open reading frames.
  • the M1 protein is encoded by 252 amino acids, and the M2 protein coding region includes nucleotides 26 ⁇ 51 and 740 ⁇ 1007, encoding 97 amino acids.
  • Ml forms a dimer that binds to viral RNA and envelope and plays a role in viral nucleocapsid assembly.
  • Ml has a low mutation rate and is specific in type, and the difference in antigen is one of the basis for virus typing.
  • M2 protein is one of the influenza virus membrane proteins, which is expressed at low density on the envelope of influenza A virus and is widely distributed on the cell membrane of infected cells. The M2 protein is present in the inner lipid membrane in the form of a homotetramer and has a proton pumping effect. The pH of the virus is regulated by controlling the proton channel activity, which affects the replication of influenza virus. Since the M2 protein is the third transmembrane protein in addition to HA and NA, it is highly conserved among human influenza A viruses. As a candidate target antigen for universal influenza vaccine with cross-protection ability, M2 protein has become a hot spot in the research of universal vaccine for influenza virus.
  • live attenuated influenza vaccine can stimulate humoral immunity and cellular immunity, which is one of the hotspots and main development directions of influenza vaccine research.
  • Influenza live attenuated vaccines have more advantages than inactivated vaccines.
  • the live attenuated vaccine is similar to the natural infection of the virus. Respiratory replication can induce an effective mucosal immune response, produce a large amount of secreted lgA, induce strong cellular and humoral immune responses, and effectively control the virus in the respiratory tract. Or nasal route, it is very convenient to avoid the problems caused by the injection route; cellular immunity induced by nasal attenuated vaccine and slgA antibody have a certain cross-protection effect on different subtypes of influenza virus.
  • the traditional design method of live attenuated vaccines is to select candidate live vaccines by positive genetic methods with multiple selective mutations under non-physiological conditions, and only a small number of candidate vaccine strains can be produced.
  • the preparation process of influenza virus cold adaptation and attenuated vaccine is complicated, time consuming, laborious and technically demanding.
  • six genes from cold-adapted virus strains and two HA and NA genes derived from the current epidemic strains were cloned into eight plasmids by reverse genetics, which can be used to transfect mammalian cells, which can simplify cold adaptation.
  • the live vaccine preparation process accelerates vaccine development.
  • reverse genetics still cannot perform large-scale rapid screening of live attenuated vaccine candidates, and it is difficult to adapt to the current influenza prevention and control needs.
  • new technical methods are combined with reverse genetic manipulation to accelerate and large-scale screening of candidate vaccine strains, it can provide new research directions for the design of live attenuated influenza vaccines, and can also be used for other viruses with reverse genetic manipulation platforms. Provide a reference for vaccine design and development.
  • the object of the present invention is to obtain an influenza virus strain and apply it to the production of a vaccine. [0009]
  • the present invention adopts the following technical solutions:
  • M gene sequence of the influenza virus strain is as shown in SEQ ID No. 1.
  • influenza virus strain has a 15 nt sequence inserted after the 78th nt of the M2 gene coding region of the wild type influenza virus strain, as shown in SEQ ID No. 2.
  • the drug is selected from the group consisting of a live attenuated influenza vaccine, an influenza inactivated vaccine, an influenza polypeptide vaccine, or a influenza genetically engineered vaccine.
  • influenza vaccine comprising the influenza virus strain described above.
  • the vaccine is a live attenuated vaccine of influenza virus.
  • a method for preparing an antibody, a hybridoma cell or an antiserum according to the above influenza virus strain, a lytic component of the virus strain, a genetic engineering protein of the virus strain or a polypeptide of the virus strain is The immunogen was prepared.
  • the diagnostic preparation includes an antigen detection kit, an antibody detection kit, and a nucleic acid detection reagent.
  • the antigen in the antigen detection kit is selected from the group consisting of the influenza virus strain, the lysing component of the influenza virus strain, the genetic engineering protein of the influenza virus strain or the polypeptide of the influenza virus strain;
  • the antibody in the detection kit is selected from the group consisting of the influenza virus strain, the lysing component of the influenza virus strain, the genetic engineering protein of the influenza virus strain, or the monoclonal antibody or polyclonal antibody prepared from the polypeptide of the influenza virus strain.
  • influenza virus strain is safe and stable, and can provide an efficient and broad-spectrum immune protection to the body.
  • the further development and use of influenza strains not only overcomes the problems faced by traditional influenza inactivated vaccines, but also outperforms conventional attenuated live attenuated vaccines currently available in many aspects.
  • the development and utilization of influenza virus strains can also provide reference for the development of other attenuated live vaccines.
  • FIG. 1 Comparison of wild-type WSN virus and W7-791 mutant virus ⁇ 2 protein nucleic acid and protein sequences.
  • Figure 3 Virus titer determination. (Infected MDCK with 0.25 ⁇ wild-type WSN virus and W7-791 virus Cells to detect viral titers at different time points).
  • FIG. 5 (A, C) was tested for body weight after inoculation of 10 7 , 10 7 or 10 8 TCID50 of W7-791 or wild-type WSN virus; (B, D) fourth and sixth days after inoculation Virus titer determination; (E) Mice weight monitoring after inoculation of newborn BALB/c mice with W7-791, WSN or PBS.
  • FIG. 6 (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 4 times after one month of immunization. MLD50 WSN virus, the body weight and survival of mice were detected periodically 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 PR8 after one month of immunization Virus, virus infection, timed detection of mouse weight and survival. *** Representative! >-value ⁇ 0.001.
  • Figure 7 W7-791 single immunization activates strong cross-protection against lethal doses of influenza infection.
  • mice were immunized with 10 6 pfu of W7-791 or PBS by intranasal immunization. After three weeks of immunization, the mice were inoculated with 2MLD50 Cam/H5. Mouse body weight and survival were measured at the indicated time points.
  • Figure 8 W7-791 is better able to protect mice from heterotypic H3 virus infection.
  • FIG. 9 W7-791 is capable of activating humoral and cell-mediated immune responses.
  • 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) trace amounts And experimentally determined the neutralizing antibody titer in the serum of W7-791 immunized mice; (EF) passed the serum of W7-791 immunized mice to unimmunized mice, and inoculated with lethal doses of WSN and HK68/H3 virus 24 hours later.
  • FIG. 10 (A) changes in body temperature of the ferrets were observed after intranasal inoculation of 10 6 , 10 7 or 10 8 TCID50 W7-791 or PBS. (B) Clinical scores were obtained after the ferrets were infected with W7-791 or 10 6 TCID50 WSN. (C) HAI analysis shows W7-
  • the 791 immunized ferrets increased the anti-W7-791 antibody titer in serum.
  • D HAI analysis showed elevated anti-H1HA or H3HA antibodies in serum after 21 days of infection.
  • Immune or unimmunized ferrets were inoculated with 10 6 TCID50 WSN or HK68/H, and evaluated (EF) virus titer and (GH) ) Clinical scoring.
  • influenza virus strain of the present invention is hereinafter referred to as W7-791.
  • Mu phage transposon-mediated mutagenesis random insertion high-density mutation technique is a method capable of randomly inserting a 15 bp ( 5 ' -NNNNNTGCGGCCGCA-3 ' in DNA, N represents the target sequence
  • a short nucleotide sequence of 5 repeat bases on DNA thereby producing a high-capacity gene insertion mutant library.
  • AFLP fragment length polymorphism analysis
  • the present invention establishes a high-density mutation library containing M gene by using a Mu-phage transposon-mediated random insertion technique using influenza virus (A/WSN/1933 (H1N1)) matrix protein M as a target gene (mutation efficiency is greater than 10 5 ), and a library of influenza viruses with high-density mutations of the M gene was obtained by reverse genetic manipulation techniques.
  • a weaker mutant influenza virus strain W7-791 was screened from the virus mutant library by comprehensive in vivo screening method and second generation sequencing technology. After comprehensive detection and evaluation of the virulence and immunoprotection of W7-791, it was found that W7-791 is an ideal attenuated influenza virus vaccine strain with a vaccine strain that can be used for influenza virus control. .
  • Embodiment 2 Basic information of W7-791
  • W7-791 is a live attenuated influenza vaccine strain that has been screened from a pool of influenza virus mutants with high frequency mutations by combining the emerging second-generation high-throughput sequencing technology and vaccine in vivo screening technology.
  • Analysis of the specific genetic material of the virus revealed that W7-791 inserted the 15nt sequence of GTCATTGCGGCCGCA (SEQ ID No. 2) after the 78nt (the corresponding cDNA of the viral genome) in the coding region of the M2 gene.
  • the peptide of RHCGRI was inserted after the 26th amino acid of the W7-791 virus M2 protein. From the overall structure of the M2 protein, this insert peptide is located in the cytoplasmic segment of the M2 protein ion channel (as shown in Figures 1 and 2).
  • W7-791 When we infected MDCK cells with wild-type WSN (WT-WSN) and W7-791 at an MOI of 0.25, and then tested the titer of the virus in the infected cell supernatant at different time points, the results showed that the replication of W7-791 was It is slower than WT-WSN, but it also shows good replication ability in MDCK cells. At the peak of replication, W7-791 can achieve the virus titer which is basically consistent with WT-WSN (as shown in Figure 3).
  • the W7-791 virus can effectively replicate in mice in the first six days after infection, the virus can detect higher titers in the lungs, but is cleared by the body 6-8 days after infection. The presence of these viruses was not detected. However, the mice did not develop any flu-related symptoms throughout the infection.
  • Attenuated live vaccine needs to be absolutely safe, and its phenotype and genotype need to be able to be stably inherited between generations. Therefore, we conducted a systematic and comprehensive safety and genetic stability evaluation of the attenuated vaccine candidate strain W7-791.
  • Toxicity testing of W7-791 infection on cells We examined the cell viability of W7-791-infected MDCK cells at different time points. We found that W7-791 was significantly less toxic to cells than WT-WSN virus (Fig. 4);
  • Attenuated vaccine genetic stability test In order to ensure that the vaccine does not undergo back mutation, and the attenuated vaccine returns to the ancestors, we carried out a series of passages of W7-791 virus in MDCK cells and mice.
  • the sequence of the gene sequence of the virus obtained in the mouse lung homogenate, especially the M gene was determined.
  • the titer of the W7-791 virus gradually decreased. This indicates that the mutations and phenotypes of the W7-791 virus can be stably inherited.
  • mice infected with W7-791 for 6 days were 100 times lower than that of wild-type WSN virus and H3 subtype virus-infected mice (Fig. 5A, B, C, D). If the lungs of the mice were observed 4 days after infection, we found that the lungs of the PBS group and the W7-791-infected mice did not show significant lesions, while the wild-type WSN virus-infected mice showed severe lung tissue damage.
  • mice were immunized with W7-791, and mice were infected with 4 times MLD50 parental wild-type WSN virus or the same subtype PR8 virus one month after immunization. We found that the unimmunized mice lost weight and died during the experiment, while the W7-791 immunized mice maintained normal body weight and did not show any flu symptoms ( Figure 6A-E). This suggests that an immunization of W7-791 can effectively protect mice against lethal doses of subtype influenza virus infection.
  • One-time immunization can provide cross-protection to mice against infection by different subtypes of influenza virus
  • influenza viruses can be divided into different subtypes, and there is no cross protection between each subtype of virus, traditional inactivated vaccines need to be continuously updated according to different subtypes of viruses that are prevalent in different time periods. So we further studied whether W7-791 can provide the body with cross-protection ability against different subtypes of influenza virus infection.
  • W7-791 can provide the body with cross-protection ability against different subtypes of influenza virus infection.
  • BALB/c mice with W6-791 at a dose of 10 6 pfii, 3 weeks after immunization, with 2 MLD50 of H5N1 subtype highly pathogenic avian influenza virus A/Cambodia/P0322095/05 (Cam/H5) The mice in the immunized group and the control group were challenged.
  • W7-791 can provide cross-protection to newborn mice to protect against lethal doses of parental WSN virus or other different subtypes of lethal doses of influenza virus.
  • 15 day old BALB/c mice were immunized with 10 6 TCID50 of W7-791 virus, followed by a lethal dose of WSN virus (10 5 or 10 6 TCID50/mice) or A/Hong Kong/68 H3N1 (HK68/H3) (10 6 or 10 7 TCID50/mice)
  • the virus was challenged in mice. Similar to adult mice, all immunized mice were protected and the virus was cleared from the body (Fig. 7E, F).
  • FluMist® is composed of four attenuated strains of influenza, including two attenuated strains of influenza B virus, one H3N2 (Switzerland/9715293/2013) and one attenuated strain of H1N1 (California/7/2009 pandemic virus). Immunization of mice with the same amount of two attenuated vaccines and challenge with the same amount of HK68/H3 virus showed that the immune protection of W7-791 was superior to that of FluMist® (Fig. 8). It can be seen from the above studies that one-time immunization of W7-791 can provide very effective cross-immunization protection for the body.
  • Example 6 W7-791 is capable of simultaneously eliciting effective humoral and cellular immune responses
  • Influenza-specific antibodies or viruses in the serum of immunized mice can be determined by influenza virus hemagglutination inhibition assay or virus neutralization assay Neutralizing antibodies.
  • the results of antibody immunization of mice showed that W7-791 immunized mice only produced WSN virus-specific antibodies, but no antibodies against PR8 virus, HK68 (H3N1), Wis (H3N2) virus (Fig. 9A-C).
  • the serum of W7-791 immunized mice was adoptively transferred to non-immunized mice. When these mice were infected with various viruses, the blood clearance of the immunized mice could provide partial protection against WSN itself, and could not protect other Infection of the virus in mice (Fig. 9D-F). This suggests that humoral immunity is not the only source of immunity provided by the W7-791 strain.
  • T lymphocytes of W7-791 immunized mice were adoptively transferred to non-immunized mice, and then the mice were infected with different wild-type influenza viruses to observe the possible immunity of the adoptive T lymphocytes.
  • T cell immunity To determine the role of T cell immunity in vaccine protection. We found that when T cells from W7-791-immunized mice were adoptively transferred to non-immunized mice, they were able to obtain partial broad-spectrum protection, thus reducing the mice's infection by various influenza viruses to some extent. The extent of the disease and the symptoms of the disease (Figure 9D-F). This shows that W7-791 can effectively induce the body to produce a protective T cell immune response, which is also consistent with the characteristics of influenza virus attenuated live vaccine immunization.
  • Example 7 W7-791-immunization can effectively protect ferrets from infection by different influenza viruses
  • the snow scorpion immunized by W7-791 was basically unable to detect the virus in the ferrets two days after the attack (Fig. 10E-F). Moreover, the flu-related symptoms exhibited by the immunized ferrets after the challenge were also significantly lighter (Fig. 10 GH).

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Abstract

提供了一种流感病毒株，其保藏号为CGMCC No.13784。所述流感病毒株在野生型流感病毒株的M2基因编码区的第78nt后面插入了15nt的序列，如SEQ ID No.2所示。所述流感病毒株的M基因序列，如SEQ ID No.1所示。还提供了上述流感病毒株在制备用于预防和/或治疗流感的疫苗中的应用。

Description

一种流感病毒株及其应用 技术领域
[0001] 本发明涉及病毒领域， 具体涉及一种流感病毒株及其应用。
背景技术
[0002] 流感病毒 （influenza virus) 的基因组由 8个单链 RNA片断组成， 编码 11种蛋白， 属于正粘病毒科流感病毒属。 流感病毒分为 A、 B、 C 三型， A、 B 两型为人群中流行的常 见类型， 并能引起世界性大流行， 但 A型流感病毒感染范围更广， 危害更大。 流感病毒的 8个基因组片断按分子量由大到小顺序依次为 PB2、 PB1、 PA、 HA、 P、 NA、 M和 NS基 因， 这些基因片段编码的 11 种病毒蛋白中有 8 个是病毒结构蛋白 （HA、 NA、 P、 Ml、 M2、 PB1、 PB2禾 P PA); NS 基因编码的 NS1 禾 P NS2为两个非结构蛋白； 两种主要表面 糖蛋白： 血凝素 （HA) 和神经氨酸酶 （NA) 为流感病毒主要表面抗原。 根据 HA和 NA 的不同， A型流感病毒又分为不同亚型， 目前己鉴定出 9种 NA亚型和 16种 HA亚型。
[0003] 流感病毒能够引起严重的呼吸道疾病， 传染性很强， 容易诱发其他严重并发症。 已 发生多次世界性大流行， 给人类生命健康带来了极大危害。 由于病毒表面蛋白 HA和 NA 易发生变异， 能够产生抗原漂移 （antigenic drift) 和抗原转换 （antigenic shift) 两种突变形 式， 尤其近年来在世界及中国出现的 H1N1和 H7N9等流感病毒突变株的爆发和流行在全球 一体化背景下给疾病的防控带来了更多困难， 随着交流越来越频繁， 病毒发生重组或重配的 频率也随之增加， 对于新型突变株的预测也会更加困难， 不仅给各国、 各地区造成巨大经济 损失， 给人们健康和生命安全带来了极大威胁， 更是加大了对疾病的防控难度。
[0004] 接种疫苗是当前预防流感病毒大流行的最有效手段。 现在广泛使用的流感疫苗设计 主要针对病毒的 HA和 NA蛋白， 以 HA和 NA作为靶抗原诱导机体产生免疫保护， 目前已 批准应用于人体的灭活病毒疫苗是由对人类危害较大的两种甲型流感病毒 （H1N1 和 H3N2) 和一种乙型流感病毒组成的三价灭活疫苗。 虽然针对 HA和 NA设计的灭活全病毒 疫苗安全性较高、 抗原组分齐全、 免疫原性强， 能够抵抗同亚型流感病毒的攻击， 提供良好 的免疫保护。 但当前疫苗在流感病毒流行和爆发期间的防治效果并不非常理想， 一方面和病 毒本身的特点具有重要的关系， 同时疫苗设计方法、 研发策略和保护效果等也有着直接的影 响。 首先流感病毒的 HA和 NA蛋白易发生抗原漂移或转变， 新疫苗的生产要随着流行毒株 的变异而及时更新， 病毒疫苗株的选育费时费力， 生产周期长、 成本高， 难以适应防控流感 大流行的需要。 其次灭活疫苗难以对病毒的感染产生完全充分的免疫保护， 无法有效的剌激 细胞免疫反应， 因此选择疫苗设计的合适靶抗原、 加快候选疫苗株的高效筛选及充分提高疫 苗的免疫保护效果是当前流感疫苗研究中非常关键和急需解决的问题。
[0005] 流感病毒疫苗靶抗原选择中， 除了病毒表面 HA和 NA蛋白外， 基质蛋白 M也被众 多学者广泛报道， M蛋白由病毒 RNA节段 7编码， 含 1027个核苷酸， 包括非糖基化结构 蛋白 Ml和 M2。 Ml和 M2编码区部分重叠， 但具有不同的开放读码框架， Ml蛋白由 252 个氨基酸编码， M2蛋白编码区包括核苷酸 26〜51和 740〜1007， 编码 97个氨基酸。 Ml形 成二聚体， 结合病毒 RNA和囊膜， 在病毒核衣壳组装时发挥作用。 Ml 变异率低、 具有型 特异性， 抗原的差异是病毒分型的依据之一。 M2 蛋白是流感病毒膜蛋白之一， 甲型流感病 毒包膜上低密度表达， 在感染细胞胞膜上分布广泛。 M2 蛋白以同源四聚体形式存在内脂膜 上， 具有质子泵作用， 通过控制质子通道活性调节病毒内的 pH值， 影响流感病毒的复制。 由于 M2 蛋白是除 HA、 NA外的第三种跨膜蛋白， 在人类 A型流感病毒中高度保守。 M2 蛋白作为具有交叉保护能力的 "通用流感疫苗"（universal influenza vaccine) 候选靶抗原， 已成为目前流感病毒通用疫苗研究的热点。
[0006] 疫苗保护效果的提高上， 减毒活疫苗 （live attenuated influenza vaccine, LAIV) 能够 剌激体液免疫和细胞免疫， 是目前流感疫苗研究的热点和开发的主要方向之一。 流感病毒减 毒活疫苗与灭活疫苗相比有较多优势。 减毒活疫苗的免疫途径与病毒自然感染相似， 呼吸道 复制能够诱导有效的黏膜免疫应答， 产生大量分泌型 lgA， 诱导较强的细胞和体液免疫应 答， 有效控制病毒在呼吸道繁殖； 可以通过滴鼻或喷鼻途径给药， 非常方便， 避免了注射途 径带来的问题； 经鼻免疫减毒疫苗所诱导细胞免疫及 slgA抗体对不同亚型流感病毒具有一 定的交叉保护作用。
[0007] 减毒活疫苗传统的设计方法是在非生理条件下多次选择性突变的正向遗传学方法进 行候选活疫苗筛选， 仅能产生少量候选疫苗株。 流感病毒冷适应减毒疫苗制备过程复杂耗时 费力， 技术要求高。 当前利用反向遗传技术将 6个来自冷适应病毒株的基因和 2个来源于当 年流行病毒株的 HA和 NA基因分别克隆到 8个质粒中， 共转染哺乳动物细胞， 能够简化冷 适应减毒活疫苗制备过程， 加快疫苗开发。 然而反向遗传技术仍不能进行减毒活疫苗候选株 的大规模快速筛选， 难以适应当前流感流行的防控需要。 如果使用新的技术方法结合反向遗 传操作加快并大规模进行候选疫苗株筛选， 能够为当前流感病毒减毒活疫苗的设计提供新的 研究方向， 也能够为其他具有反向遗传操作平台的病毒的疫苗设计和开发提供参考。
发明内容
[0008] 本发明的目的在于得到一种流感病毒株， 并将其应用于疫苗的生产。 [0009] 本发明采用如下技术方案：
一种流感病毒株， 其保藏号为 CGMCC No. l3784。 保藏日期为 2017年 2月 21 日。 分类命名 为 Influenza A virus (A/WSN/1933(H1N1))0
[0010] 其中， 所述流感病毒株的 M基因序列， 如 SEQ ID No. l所示。
[0011] 其中， 所述流感病毒株在野生型流感病毒株的 M2基因编码区的第 78nt后面插入了 15nt的序列， 如 SEQ ID No.2所示。
[0012] 上述流感病毒株在制备用于预防和 /或治疗流感的药物中的应用。
[0013] 特别的， 所述的药物选自流感弱毒活疫苗、 流感灭活疫苗、 流感多肽疫苗或流感基 因工程疫苗。
[0014] 一种流感疫苗， 所述的疫苗中含有上述流感病毒株。
[0015] 进一步的， 所述疫苗为流感病毒弱毒活疫苗。
[0016] 一种用于制备抗体、 杂交瘤细胞或抗血清的方法， 根据上述的流感病毒株、 所述病 毒株的裂解成份、 所述病毒株的基因工程蛋白或所述病毒株的多肽为免疫原进行制备。
[0017] 由上述制备方法得到的制备抗体、 杂交瘤细胞或抗血清。
[0018] 上述流感病毒株在制备流感诊断制剂中的应用。
[0019] 进一步的， 所述的诊断制剂包括抗原检测试剂盒、 抗体检测试剂盒和核酸检测试剂
[0020] 进一步的， 所述的抗原检测试剂盒中的抗原选自上述流感病毒株、 上述流感病毒株 的裂解成份、 上述流感病毒株的基因工程蛋白或上述流感病毒株的多肽； 所述抗体检测试剂 盒中的抗体选自所述流感病毒株、 所述流感病毒株的裂解成份、 所述流感病毒株的基因工程 蛋白或流感病毒株的多肽制备的单抗或多抗。
[0021] 本发明的有益效果在于： 所述流感病毒株安全稳定， 能够给机体提供高效广谱的免 疫保护。 流感病毒株的进一步开发使用， 不但能够克服传统流感灭活疫苗所面临的各种问 题， 而且其很多方面都优于目前市售的常规弱毒活疫苗。 此外， 流感病毒株的开发利用， 也 能为其他病毒弱毒活疫苗研制提供参考。
附图说明
[0022] 图 1 野生型 WSN病毒和 W7-791突变型病毒 Μ2蛋白核酸和蛋白序列比较。
[0023] 图 1中， （Α) 核酸比较； （Β) 蛋白质序列比较。
[0024] 图 2在 Μ2蛋白晶体结构上标记 W7-791插入位置。
[0025] 图 3病毒滴度测定。 （用 0.25 ΜΟΙ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）。
[0026] 图 4 W7-791感染对 MDCK细胞存活的影响。
[0027] 图 5 W7-791病毒免疫效果评价。
[0028] 图 5中， （A， C) 接种 106、 107或者 108 TCID50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。
[0029] 图 6 W7-791单次免疫能够激活对致死剂量流感病毒感染的保护。
[0030] 图 6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 105 PFU 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 105 PFU 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！>-值< 0.001。
[0031] 图 7 W7-791单次免疫能够激活针对致死剂量异型流感感染的强大交叉保护作用。
[0032] 图 7 中， （A-B) 6 只小鼠被滴鼻免疫了 106 pfu W7-791 或 PBS. 三个星期免疫以 后， 小鼠被接种 2MLD50 Cam/H5。 在标明的时间点检测小鼠体重和存活情况。 （C-D) 小鼠 被滴鼻免疫 105 pfu W7-791 (n=9) 或 PBS (n=6)免疫一个月以后， 小鼠被接种 2MLD50 Vic/H3。 在标明的时间点检测小鼠体重和存活情况。 （E-F)新生小鼠被 W7-791 免疫三周以后 接种 WSN (105 or 106 TCID50) 禾 P HK68/H3 (106 or 107 TCID50), 观察小鼠体重变化。 *** 代表？-值< 0.001。
[0033] 图 8 W7-791能够更好的保护小鼠被异型 H3病毒感染。
[0034] 图 8中， C57BL/6小鼠被免疫了 106 TCID50 FluMist (2016年） 或者 W7-791。 一个 月以后， 这些小鼠被感染 2MLD50 HK68 H3N1。 两图分别显示感染以后小鼠体重变化和存 活情况。
[0035] 图 9 W7-791能够激活体液和细胞介导的免疫应答。
[0036] 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。 [0037] 图 10 单次 W7-791免疫能够在雪貂体内激抗活异型病毒保护作用。
[0038] 图 10中， （A) 滴鼻接种 106, 107 或 108 TCID50 W7-791 或 PBS后观察雪貂体温变 化。 （B) 雪貂感染 W7-791 或 106 TCID50 WSN后进行临床打分。 （C) HAI分析显示 W7-
791免疫过的雪貂提高了血清中抗 W7-791抗体滴度。 （D) HAI分析 显示感染 21天以后， 血清中抗 H1HA或者 H3HA抗体升高.免疫或者没有免疫过的雪貂接种 106 TCID50 WSN 或 HK68/H后， 评价 (E-F)病毒滴度和 (G-H)临床打分。
具体实施方式
[0039] 为了更加清楚明了的叙述， 下文中， 本发明的流感病毒株采用 W7-791指代。
[0040] 实施例 1 W7-791的制备方法
Mu 噬菌体转座子介导 (transposon-mediated mutagenesis) 的随机插入高密度突变技术是一 种能够在 DNA 中随机性插入一段 15bp ( 5 ' -NNNNNTGCGGCCGCA-3 ' , N代表靶序列
DNA上 5 个重复碱基） 的短核苷酸序列， 从而产生高库容的基因插入突变体文库的方法。 将其与病毒方向遗传学操作技术结合就能获得相应的病毒突变体文库， 再结合 PCR扩增、 毛细管电泳、 荧光标记 DNA测序技术和片段长度多态性分析 （AFLP) 等技术就能够准确鉴 定突变数量和插入位点。 这一技术已被广泛用于各种病毒基因组功能及病毒与宿主相互作用 的研究。
[0041] 本发明以流感病毒 （A/WSN/1933(H1N1)) 基质蛋白 M为靶基因通过 Mu噬菌体转座 子介导的随机插入技术建立了含 M基因的高密度突变库 （突变效率大于 105)， 并通过反向 遗传操作技术获得了 M基因高密度突变的流感病毒库。 在此基础上综合应用体内筛选的方 法和第二代测序技术从病毒突变体库中筛选到一株致弱性突变流感病毒株 W7-791。 在对 W7-791 的致病力及免疫保护性进行全面检测和评价后， 发现 W7-791 是一株非常理想的流 感病毒弱毒活疫苗毒株， 具有能够用于流感病毒防治的候选疫苗毒株。
[0042] 实施例 2 W7-791的基本信息
W7-791是通过结合新兴第二代高通量测序技术和疫苗体内筛选技术， 从病毒 M基因被高频 突变的流感病毒突变体库中筛选得到的流感弱毒活疫苗毒株。 对病毒具体遗传物质 （病毒基 因组 RNA) 的分析显示， W7-791 在其 M2 基因编码区的第 78nt (指病毒基因组对应的 cDNA) 后面插入了 GTCATTGCGGCCGCA这一 15nt的序列 （SEQ ID No.2)。 对应到蛋白 质水平， W7-791病毒 M2蛋白的第 26个氨基酸的后面插入了 RHCGRI的肽段。 从 M2蛋白 的整体结构来看， 这段插入肽段是位于 M2蛋白离子通道的胞浆段 （如图 1和图 2所示）。
[0043] 实施例 3 W7-791在体外细胞培养中和小鼠体内的复制动力学 ( 1 ) W7-791在细胞培养中的复制
当我们用野生型 WSN (WT-WSN) 和 W7-791 以 MOI为 0.25感染 MDCK细胞， 然后在不 同的时间点检测感染细胞上清中病毒的滴度， 结果显示， W7-791 的复制虽然要比 WT-WSN 要慢， 但其在 MDCK细胞中也表现出良好的复制能力， 在复制的高峰点， W7-791能够达到 与 WT-WSN基本一致的病毒滴度 （如图 3所示）。
[0044] ( 2 ) W7-791在小鼠体内的复制
W7-791 病毒虽然在感染小鼠后前六天能够在小鼠体内有效地复制， 在肺脏能够检测到较高 滴度的病毒， 但是在感染后 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。 但 在整个感染过程中小鼠不会出现任何流感相关症状。
[0045] 实施例 4 W7-791的安全性和遗传稳定性
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， 我们对弱毒疫苗候选毒株 W7-791 进行了系统全面的安全性和遗传稳定性评价。 ( 1 ) W7-791 感染对细胞的毒性检测： 我们检测了 W7-791 感染 MDCK细胞在不同时间点 的细胞活力， 我们发现 W7-791对细胞的毒性明显小于 WT-WSN病毒 （图 4); (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， 103 TCID50 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 5A， 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, 107 or 108 TCID50)的 W7-791或 104 TCID50 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 5E)。 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。 [0046] 实施例 5 W7-791的免疫保护能力
( 1 ) 一次免疫能够有效保护小鼠抵御致死剂量同亚型流感病毒的感染
用 W7-791免疫小鼠， 在免疫后一月， 用 4倍 MLD50的亲本野生型 WSN病毒或同亚型的 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 6A-E)。 这 就说明一次免疫 W7-791就能够有效保护小鼠抵抗致死剂量同亚型流感病毒的感染。
[0047] (2) 一次免疫能够为小鼠提供交叉保护抵御不同亚型流感病毒的感染
由于流感病毒可以分为不同的亚型， 各亚型病毒的之间缺乏交叉保护， 传统的灭活疫苗需要 根据不同时间段流行的不同亚型病毒进行不断的更新。 所以我们进一步研究了 W7-791 能否 为机体提供抗不同亚型流感病毒感染的交叉保护能力。 为此， 我们用先用 106 pfii 剂量的 W7-791免疫 BALB/c小鼠， 在免疫后 3周， 用 2 MLD50量的 H5N1亚型高致病性禽流感病 毒 A/Cambodia/P0322095/05(Cam/H5)对免疫组和对照组小鼠进行攻毒。 结果显示， 未免疫的 小鼠表型出各种流感症状， 体重严重下降并死亡； 而免疫组小鼠未出现显著的体重下降， 表 现出对 Cam/H5 良好的抵御能力 （图 7A， B)。 另外， 我们也检测了 W7-791 对一株不同遗 传谱系流感病毒 A/Victoria/3/75 H3N2 (Vic/H3)的免疫保护作用， 小鼠用 105 pfu 的 W7-791 免疫后 4周用 2 MLD50的 Vic/H3进行感染。 结果显示， W7-791免疫小鼠在攻毒后 3-5天 的时间段内体重只下降了约 10%后逐渐恢复， 而免疫对照组的小鼠都死亡 （图 7C， D)。 另 外， 我们也检测了 W7-791 能否为新生小鼠提供交叉保护， 从而抵御致死剂量的亲本 WSN 病毒或其他不同亚型致死剂量流感病毒的感染。 用 106 TCID50 的 W7-791病毒免疫 15 日龄 的 BALB/c小鼠， 然后用致死剂量的 WSN病毒（105 or 106 TCID50/mice)或 A/Hong Kong/68 H3N1 (HK68/H3) (106 or 107 TCID50/mice)病毒对小鼠进行攻毒。 与成年小鼠类似， 所有被免 疫的小鼠都得到了保护， 并从体内清除了病毒 （图 7E， F)。
[0048] 最后， 我们比较了 W7-791与商品化的在 2015-2016期间推荐使用的流感病毒弱毒活 疫苗 FluMist®的免疫效果。 FluMist®是由四种流感弱毒株组成， 包括两个 B 型流感病毒弱 毒株、 一个 H3N2(Switzerland/9715293/2013)和一个 H1N1 (California/7/2009 pandemic virus) 弱毒株。 用同样量的两种弱毒疫苗免疫小鼠并用同样量的 HK68/H3 病毒攻毒， 结果显示， W7-791 的免疫保护效果要优于 FluMist®的免疫效果 （图 8 )。 从上述研究可以看出， W7- 791的一次接种免疫， 能够为机体提供非常有效的交叉免疫保护。
[0049] 实施例 6 W7-791能够同时激发有效的体液免疫和细胞免疫应答
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。
[0050] 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。
[0051] 实施例 7 W7-791—次免疫能够有效保护雪貂免受不同流感病毒的感染
雪貂目前认为是更好的流感病毒感染模型。 为了进一步研究和确认 W7-791 作为流感病毒弱 毒活疫苗的有效性， 我们测试了 W7-791 对雪貂的免疫保护作用。 首先， 为了评估 W7-791 对雪貂的感染和致病力， 我们分别用 106, 107 and 108 TCID50的 W7-791疫苗株病毒感染雪 貂， 然后观察病毒所引起的流感症状。 我们发现， 108 TCID50剂量的 W7-791并未导致雪貂 出现体温升高和其他流感症状， 这就说明 W7-791 对于雪貂有着与小鼠相同的安全性 （图 10A, B)。 然后， 我们检测了 W7-791 免疫雪貂体内的抗体水平， 发现雪貂体内疫苗特异性 抗体明显增加 （图 10C)， 但是血凝抑制实验结果表明， 这些抗体只能结合 WSN的 HA， 而 不能结合 HK68/H3 或 H5N1病毒的 HA (图 10D)。 在雪貂用 W7-791免疫后 4周， 我们分 别用 106 TCID50的 WSN、 106 TCID50的 HK68/H3病毒进行攻毒， 结果显示， 与未免疫动 物相比， 用 103和 104.7 TCID50的 W7-791免疫的雪貂， 攻毒后两天雪貂体内基本检测不到 病毒 (图 10E-F)。 而且免疫雪貂在攻毒后所表现出的流感相关症状也明显较轻 (图 10 G-H)。
[0052] 根据上述的实施例对本发明作了详细描述。 需说明的是， 以上的实施例仅为了举例 说明发明而已。 在不偏离本发明的精神和实质的前提下， 本领域技术人员可以设计出本发明 的多种替换方案和改进方案， 其均应被理解为在本发明的保护范围之内。 打印件 (原件为电子形式)
Figure imgf000011_0001
下面的说明与本申请说明书中此处提到的
保藏的微生物或其他生物材料相关：
-1 页码 第【0009】段
-2 行号： 第 2~3行
-3 保藏事项
-3-1 保藏单位名称 中国微生物菌种保藏管理委员会普通微生物中心
-3-2 保藏单位地址 中国微生物菌种保藏委员会， 中国北京市 2714信箱， 邮 政编码：100080， Be i j i ng (CN)。
-3-3 保藏曰期 2017年 2月 21日 （21 . 02. 2017)
-3-4 保藏号 CGMCC 13784
-4 补充说明
-5 本说明是对下列指定国 所有指定国
-6 单独提交的说明
这些说明将随后提交给国际局 由受理局填写 -4 本表格与国际申请一起收到：
(是或否）
-4-1 受权官员 由国际局填写 -5 国际局收到本表格日期： -5-1 受权官员

Claims

权 利 要 求 书
1. 一种流感病毒株， 其特征在于， 其保藏号为 CGMCC No. l3784。
2. 根据权利要求 1所述的一种流感病毒株， 其特征在于， 所述流感病毒株的 M基因序列， 如 SEQ ID No. l所示。
3. 根据权利要求 1 所述的一种流感病毒株， 其特征在于， 所述流感病毒株在野生型流感病 毒株的 M2基因编码区的第 78nt后面插入了 15nt的序列， 所述 15nt的序列如 SEQ ID No.2 所示。
4. 如权利要求 1所述的流感病毒株在制备用于预防和 /或治疗流感的疫苗中的应用。
5. 根据权利要求 4 所述的应用， 其特征在于， 所述的疫苗选自流感弱毒活疫苗、 流感灭活 疫苗、 流感多肽疫苗或流感基因工程疫苗。
6. 一种流感疫苗， 其特征在于， 所述的疫苗中含有如权利要求 1所述的流感病毒株。
7. 根据权利要求 6所述的一种流感疫苗， 其特征在于， 所述疫苗为流感病毒弱毒活疫苗。
8. 一种用于制备抗体、 杂交瘤细胞或抗血清的方法， 其特征在于， 根据权利要求 1 所述的 流感病毒株、 所述病毒株的裂解成份、 所述病毒株的基因工程蛋白或所述病毒株的多肽为免 疫原进行制备。
9. 根据权利要求 8所述的方法得到的制备抗体、 杂交瘤细胞或抗血清。
10. 如权利要求 1所述的流感病毒株在制备流感诊断制剂中的应用。
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