Method for preparing high-titer recombinant influenza virus using pseudovirus model and its application

Abstract

The present invention provides methods and applications for preparing high-titer recombinant influenza viruses using pseudovirus models. High-titer influenza virus preparation for vaccines can effectively shorten vaccine preparation cycles and reduce vaccine production costs. Specifically, the present invention provides methods for transforming low-titer H5 subtype influenza viruses into high-titer H5 subtype influenza viruses. In an example, the method of the present invention significantly increased the titer of the low-titer A / blackbird / Hunan / 1 / 2004 virus after transformation, with minimal impact on immunogenicity.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a method for preparing high-titer recombinant influenza virus by using a pseudovirus model and application thereof. Background Art

[0002] Influenza viruses belong to the family Orthomyxoviridae. They primarily infect birds, humans, and mammals, causing seasonal influenza and avian influenza. Seasonal influenza causes casualties worldwide annually, and global influenza pandemics occur every few decades, significantly impacting both the economy and society. Influenza viruses are classified into four types: A, B, C, and D, based on the antigenicity of their membrane protein 1 (M1) and nucleoprotein (NP). Influenza A viruses are divided into 18 HA subtypes and 11 NA subtypes based on differences in their hemagglutinin (HA) and neuraminidase (NA) proteins. Influenza A viruses are spherical, with a phospholipid bilayer outer shell comprising HA, NA, and membrane protein (M2). The genome consists of eight segments of single-stranded negative-stranded RNA encoding 11 viral proteins.

[0003] Avian influenza viruses are categorized as highly pathogenic (HPAI) and low pathogenicity (LPAI) based on their pathogenicity. H5N1 is a HPAI. Poultry are the natural reservoir for HPAI, but studies have shown that the H5N1 virus can cross species barriers and spread between species, even between humans. Once an epidemic occurs, it poses a serious threat to public health. When H5N1 cases occur in a poultry population, all birds in the population are culled. According to statistics, in the first half of 2022 alone, 1,154 H5N1 influenza cases occurred in 25 countries and regions across the United States, Africa, Asia, and Europe, resulting in the deaths and culling of approximately 30 million animals and posing a serious threat to the global livestock industry.

[0004] Vaccines are currently the most effective method for preventing influenza pandemics. Inactivated vaccines are widely used due to their high safety, strong immunogenicity, the ability to produce polyvalent vaccines, and resistance to mutation. However, low titers of certain strains during inactivated influenza vaccine preparation are a common problem in large-scale inactivated vaccine production. Currently, commercially available inactivated vaccines for the highly pathogenic H5 subtype typically utilize the influenza virus's HA and NA as the viral envelope proteins. Six internal genes from the high-titer PR8 (or D7) are used as recombinant virus internal genes. The HA is modified to reduce the recombinant virus's pathogenicity in chicken embryos, resulting in seed virus. This method is then serially passaged in chicken embryos. However, the recombinant virus vaccine strains produced by this method have low titers and suffer from three drawbacks: ① Serial passage in chicken embryos is time-consuming; ② the results are unpredictable, and high-titer virus vaccine strains cannot always be obtained through serial passage in chicken embryos; and ③ the recombinant virus strains are susceptible to mutations during serial passage in chicken embryos.

[0005] Highly pathogenic influenza viruses are extremely contagious and pathogenic, requiring manipulation and study within a BSL-3 laboratory environment, which has always been a bottleneck for their operation. Influenza pseudoviruses, however, are produced by merging the influenza virus's envelope proteins HA and NA onto a backbone protein encoded by the retroviral genome. They possess the envelope characteristics of influenza viruses while retaining the inherent characteristics of retroviruses. Influenza pseudoviruses not only mimic the early stages of infection with live viruses, but are also replication-defective compared to wild-type viruses, possessing only a single round of infection. Because they lack the ability to replicate themselves, influenza pseudoviruses require a lower laboratory environment and are therefore far safer than live viruses.

[0006] Therefore, there is an urgent need in the art to develop a simple and rapid method and application for transforming low-titer influenza viruses into high-titer influenza viruses to solve the above problems. Summary of the Invention

[0007] The object of the present invention is to provide a method for simply and quickly transforming low-titer influenza virus into high-titer influenza virus by using a pseudovirus model.

[0008] The first aspect of the present invention provides a method for transforming a low-titer H5 subtype influenza virus into a high-titer influenza virus, comprising:

[0009] (a) providing a constructed H5 subtype high-titer influenza virus library and an H5 subtype low-titer influenza virus strain to be modified, and obtaining a genetic evolutionary tree based on the HA amino acid sequences of the H5 subtype high-titer influenza virus in the virus library and the H5 subtype low-titer influenza virus;

[0010] (b) obtaining, through the genetic evolutionary tree, an H5 subtype high-titer influenza virus strain that is closest in genetic distance to the H5 subtype low-titer influenza virus strain;

[0011] (c) exchanging the HA or NA protein of the high-titer H5 subtype influenza virus strain obtained in step (b) with the low-titer H5 subtype influenza virus to prepare a chimeric pseudovirus, and detecting the titer of the chimeric pseudovirus to determine whether the protein that affects the influenza virus titer is HA or NA;

[0012] (d) comparing the amino acid differences in HA (or NA) between the H5 subtype high-titer influenza virus strain obtained in step (b) and the H5 subtype low-titer influenza virus described in step (a);

[0013] (e) subjecting all amino acids in the HA (or NA) of the H5 subtype high-titer influenza virus obtained in step (b) that differ from those in the HA (or NA) of the H5 subtype low-titer influenza virus strain described in step (a) to single-point mutations, thereby preparing HA (or NA) mutant influenza pseudoviruses;

[0014] (f) respectively detecting the titer of the HA (or NA) mutant influenza pseudovirus obtained in step (e) and the pseudovirus of the H5 subtype high-titer influenza virus strain obtained in step (b), thereby determining the amino acid site information that may affect the H5 subtype low-titer influenza virus strain;

[0015] (g) performing single-site amino acid mutations on the key amino acids in the HA (or NA) of the H5 subtype low-titer influenza virus to be modified according to the amino acid sites obtained in step (f) to prepare pseudoviruses, and determining amino acid sites that may increase viral titer by titer detection; further performing double-site or multi-site amino acid mutations, and performing pseudovirus titer detection to determine the key amino acids that affect the H5 subtype low-titer influenza virus;

[0016] (h) constructing a plasmid based on the key amino acid sites determined in step (g), preparing an HA mutant influenza virus by reverse genetics, and verifying the confirmed key amino acid sites by titer detection, thereby obtaining an H5 subtype high-titer influenza virus.

[0017] In another preferred embodiment, the method further comprises step (i), detecting the adsorption capacity and fusion capacity of the H5 subtype high-titer influenza virus obtained in step (h).

[0018] In another preferred embodiment, the method further comprises step (j), detecting the immunogenicity of the H5 subtype high-titer influenza virus obtained in step (h), thereby obtaining an H5 subtype high-titer influenza virus with little or no immunogenicity.

[0019] In another preferred embodiment, the method further comprises the step of detecting the viral titer of a reference H5 subtype low-titer influenza virus.

[0020] In another preferred embodiment, the reference H5 subtype low-titer influenza virus is A / WSN / 33.

[0021] In another preferred example, the H5 subtype high-titer influenza virus library is constructed by the following method: based on the influenza virus HA sequence provided by GISAID, potential H5 subtype high-titer influenza virus strains are screened by bioinformatics methods, and influenza pseudoviruses are prepared by using a 4-plasmid expression system with the HA and NA of the influenza virus as envelope proteins and firefly luciferin as internal nucleic acid packaging, and high-titer H5 subtype influenza virus strains are screened by relative luciferase activity detection, thereby constructing a high-titer H5 subtype influenza virus strain library.

[0022] In another preferred embodiment, the bioinformatics method includes sequence comparison and analysis.

[0023] In another preferred embodiment, the amino acid after mutation in the H5 subtype high-titer virus strain is the amino acid at the same site in the low-titer H5 subtype influenza virus strain.

[0024] In another preferred embodiment, the amino acid mutated in the H5 subtype low-titer virus strain is an amino acid at the same position in the high-titer H5 subtype influenza virus strain that is closest to the low-titer H5 subtype influenza virus strain in genetic distance on the genetic evolutionary tree.

[0025] In another preferred embodiment, the H5 subtype high-titer influenza virus strain with the closest genetic distance refers to a virus strain that is located at the same node and the same evolutionary branch as the H5 subtype low-titer influenza virus strain on the genetic evolutionary tree.

[0026] In another preferred embodiment, the H5 subtype high-titer influenza virus refers to a pseudovirus whose relative luciferase activity (RLA) value is significantly higher than 1000.

[0027] In another preferred embodiment, the H5 subtype low-titer influenza virus refers to a pseudovirus whose relative luciferase activity (RLA) value is significantly lower than 500.

[0028] In another preferred embodiment, the H5 subtype high titer influenza virus strain is as follows:

[0029]

[0030] In another preferred embodiment, the H5 subtype low-titer influenza virus includes A / blackbird / Hunan / 1 / 2004.

[0031] In another preferred embodiment, the titer of HA and NA chimeric viruses, or viruses with single-point or multi-point mutations in HA (or NA) is detected by expressing a reporter gene (firefly luciferase) in the host cells after the virus infects the host cells. After the host cells are lysed, the virus titer is determined by reacting with a substrate containing firefly luciferase and a buffer solution, and detecting the luminescence value of the luciferase at 560 nm.

[0032] In another preferred embodiment, if the viral titer changes significantly (for example, the titer increases) after the HA of the low-titer virus strain is replaced with the HA of the high-titer virus strain, and the viral titer does not change significantly after the NA of the low-titer virus strain is replaced with the NA of the high-titer virus strain, it can be determined that the key amino acid affecting the low-titer virus strain is not located in NA but in HA; or

[0033] If the viral titer does not change after the HA of the low-titer virus strain is replaced with the HA of the high-titer virus strain, but the viral titer changes significantly (increases) after the NA of the low-titer virus strain is replaced with the NA of the high-titer virus strain, it can be determined that the key amino acid affecting the low-titer virus strain is not located in HA but in NA; or

[0034] If the viral titer changes significantly (e.g., the titer increases) after the HA of a low-titer virus strain is replaced with the HA of a high-titer virus strain, and the viral titer also changes significantly (e.g., the titer increases) after the NA of a low-titer virus strain is replaced with the NA of a high-titer virus strain, it can be determined that the key amino acids affecting the low-titer virus strain are located in both HA and NA.

[0035] In another preferred embodiment, in step (f), if the viral titer of the HA (or NA) mutant influenza virus is significantly lower than that of the corresponding wild-type virus, it indicates that the corresponding mutation site in the HA mutant influenza virus is an amino acid that affects the titer of the H5 subtype low-titer influenza virus.

[0036] In another preferred embodiment, in step (f), if the virus titer of the NA mutant influenza virus is significantly lower than that of its corresponding wild-type virus, it indicates that the corresponding mutation site in the NA mutant influenza virus is an amino acid that affects the H5 subtype low-titer influenza virus.

[0037] The second aspect of the present invention provides an H5 subtype high-titer influenza virus, which is prepared using the method described in the first aspect of the present invention.

[0038] In another preferred embodiment, the virus is a mutant virus, and the mutation site corresponding to the HA protein in the genome of the virus and its corresponding mutant amino acid are the key amino acid sites and their corresponding key amino acids for improving the virus titer of the H5 subtype high-titer influenza virus strain with the closest genetic distance in the first aspect of the present invention.

[0039] In another preferred embodiment, the mutation is a single-site amino acid mutation or a double-site amino acid mutation, or a multi-site amino acid mutation.

[0040] In another preferred embodiment, the H5 subtype high-titer influenza virus is an HA chimera virus of the HA head zone and the stem zone.

[0041] The third aspect of the present invention provides a pharmaceutical composition, which contains the high-titer influenza pseudovirus described in the second aspect of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0042] In another preferred embodiment, the pharmaceutical composition is a vaccine composition.

[0043] In another preferred embodiment, the vaccine composition is monovalent or multivalent.

[0044] In another preferred embodiment, the pharmaceutical composition further contains an adjuvant, preferably various aluminum adjuvants.

[0045] In another preferred embodiment, the molar or weight ratio of the high-titer influenza pseudovirus to the adjuvant (such as aluminum) in the pharmaceutical composition is between 1:100, preferably between 1:40 and 1:60.

[0046] In another preferred embodiment, the pharmaceutical composition includes a single drug, a compound drug, or a synergistic drug.

[0047] In another preferred embodiment, the pharmaceutical composition is in the form of liquid, solid, or gel.

[0048] In another preferred embodiment, the pharmaceutical composition is administered in a manner selected from the group consisting of subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral and nasal spraying and aerosol inhalation.

[0049] The fourth aspect of the present invention provides a vaccine composition, which comprises the high-titer influenza pseudovirus described in the second aspect of the present invention, and an immunologically acceptable carrier and / or adjuvant.

[0050] In another preferred embodiment, the vaccine composition further contains an adjuvant.

[0051] In another preferred embodiment, the adjuvant includes: granular and non-granular adjuvants.

[0052] In another preferred embodiment, the particulate adjuvant is selected from the group consisting of aluminum salts, water-in-oil emulsions, oil-in-water emulsions, nanoparticles, microparticles, liposomes, immunostimulatory complexes, or combinations thereof.

[0053] In another preferred embodiment, the non-granular adjuvant is selected from the following group: muramyl dipeptide and its derivatives, saponin, lipid A, cytokines, derived polysaccharides, bacterial toxins, microorganisms and their products such as mycobacteria (tuberculosis, BCG), bacillus pumilus, Bordetella pertussis, propolis, or a combination thereof.

[0054] In another preferred embodiment, the adjuvant includes alumina, saponin, quil A, muramyl dipeptide, mineral oil or vegetable oil, vesicle-based adjuvant, non-ionic block copolymer or DEAE dextran, cytokines (including IL-1, IL-2, IFN-r, GM-CSF, IL-6, IL-12, and CpG).

[0055] In another preferred embodiment, the vaccine composition comprises an injectable dosage form.

[0056] The fifth aspect of the present invention provides the use of the high-titer influenza pseudovirus according to the second aspect of the present invention, the pharmaceutical composition according to the third aspect of the present invention, or the vaccine composition according to the fourth aspect of the present invention, (a) for preparing antibodies against HA or NA of H5 subtype influenza virus; and / or (b) for preparing drugs for preventing and / or treating influenza virus infection or related diseases.

[0057] In another preferred embodiment, the antibody includes antibodies against influenza virus HA or NA, or the HA head region, or the Ha rod region, or against key amino acid sites of HA that increase viral titer, such as the sites modified by the high-titer virus strain prepared as described in the second aspect of the present invention.

[0058] In another preferred embodiment, the antibodies include antibodies against HA or NA of H5 subtype influenza virus.

[0059] In another preferred embodiment, the antibodies include antibodies against H5 subtype influenza virus.

[0060] In another preferred embodiment, the influenza virus includes H5 subtype influenza virus and high-titer H5 subtype mutant influenza virus transformed as described in the method of the first aspect of the present invention.

[0061] In another preferred embodiment, the H5 subtype influenza virus includes an H5N1 subtype influenza virus.

[0062] In another preferred embodiment, the influenza virus infection or related diseases are selected from the group consisting of fever, headache, runny nose, sneezing, loss of smell, cough, chills, sore throat, body aches or muscle pain, or a combination thereof.

[0063] In another preferred embodiment, the treatment comprises treatment using gene therapy.

[0064] The sixth aspect of the present invention provides a method for generating an immune response against influenza virus, comprising the steps of administering to a subject in need thereof the high-titer influenza pseudovirus described in the second aspect of the present invention, the pharmaceutical composition described in the third aspect of the present invention, or the vaccine composition described in the fourth aspect of the present invention.

[0065] In another preferred embodiment, the subject includes humans or non-human mammals.

[0066] In another preferred embodiment, the non-human mammal includes a non-human primate (such as a monkey).

[0067] In another preferred embodiment, the method induces the production of neutralizing antibodies against H5 subtype influenza virus in the subject.

[0068] The seventh aspect of the present invention provides a treatment method, which comprises administering the high-titer influenza virus strain described in the second aspect of the present invention, the pharmaceutical composition described in the third aspect of the present invention, or the vaccine composition described in the fourth aspect of the present invention to a subject in need.

[0069] In another preferred embodiment, the treatment method includes gene therapy.

[0070] In another preferred embodiment, the treatment method includes in vitro transplantation of human DC cells transfected using electroporation technology and lymphocyte mRNA vaccine injection.

[0071] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 The effect of HA protein on virus titer.

[0073] Figure 2 Schematic diagram of the preparation of four plasmids for influenza pseudovirus.

[0074] Figure 3 Comparison of HA amino acids between A / Thailand / 1(KAN-1) / 2004 and A / blackbird / Hunan / 1 / 2004 and comparison of relative fluorescence intensities of different pseudoviruses.

[0075] Figure 4 Comparison of the infectivity of A / blackbird / Hunan / 1 / 2004 mutant pseudovirus and A / blackbird / Hunan / 1 / 2004 pseudovirus.

[0076] Figure 5 Comparison of hemagglutination titers of A / blackbird / Hunan / 1 / 2004 recombinant virus and mutant recombinant virus.

[0077] Figure 6 Comparison of immunogenicity of A / blackbird / Hunan / 1 / 2004 recombinant virus and mutant recombinant virus. DETAILED DESCRIPTION

[0078] Through extensive and in-depth research, the inventors unexpectedly discovered that by constructing a high-titer H5 subtype highly pathogenic avian influenza virus strain library, drawing a genetic evolutionary tree, and selecting virus strains with a close genetic distance to the low-titer virus strain to be modified from the evolutionary tree; constructing a chimeric pseudovirus with interchangeable HA and NA, and determining whether the key amino acid affecting the titer of the recombinant virus is located in HA or NA through changes in the pseudovirus titer; and comparing the amino acid sequence differences of HA (or NA) of the high-titer virus strain and the low-titer virus strain, further site-directed mutation of amino acids at different sites on HA (or NA); constructing mutant pseudoviruses, comparing the titers of the mutant pseudoviruses and the pseudoviruses, and finding the amino acids affecting the titer of the pseudovirus. According to the pseudovirus detection result, the recombinant virus of mutation is constructed, the titer of the mutant recombinant virus and the wild-type recombinant virus is compared, the amino acid that improves the replication ability of the virus on chicken embryo and MDCK cells is determined, the amino acid that the immunogenicity of the vaccine strain is less affected is selected to mutate, and the recombinant virus of high titer is constructed, thereby the influenza virus of low titer can be transformed into the influenza virus of high titer, further experimental confirmation is carried out the change of the adsorption capacity, fusion ability etc. of the recombinant virus strain, and the principle of the preparation of high titer virus strain is explained. With respect to the preparation of the influenza vaccine strain of low titer, the time and cost of vaccine preparation can be effectively shortened with the virus strain of high titer of the present invention, more effectively prevent and control. Concretely, the present invention is with the low titer influenza virus strain A / blackbird / Hunan / 1 / 2004 of H5N1 subtype as an example, and by the method of this patent, effectively transformed, by the amino acid mutation of hemagglutinin (HA) (NCBI sequence number AAS65615) the 42nd, 44th, 53rd position, can effectively improve the titer of virus, thereby can efficiently produce and prepare the H5 subtype influenza vaccine of high titer. On this basis, the inventors completed the present invention.

[0079] the term

[0080] For the convenience of description, the virus strain A / Thailand / 1(KAN-1) / 2004 is abbreviated as "TH" and the virus strain A / blackbird / Hunan / 1 / 2004 is abbreviated as "HN".

[0081] The term "AxxB" means that the amino acid A at position xx is changed to amino acid B, for example, "I42V" means that the amino acid I at position 42 is mutated to V, and so on.

[0082] The term "high-titer influenza (pseudo)virus" refers to a (pseudo)virus of the present invention whose relative luciferase activity (RLA) value is greater than 1000.

[0083] The term "low-titer influenza (pseudo)virus" refers to a (pseudo)virus of the present invention whose relative luciferase activity (RLA) value is <500.

[0084] The term "genetic distance" refers to the unit length of the number of differences between sequences.

[0085] The term "close genetic distance" refers to the fact that the lengths of evolutionary branches at the same node on the evolutionary tree have the smallest numerical difference.

[0086] H5 subtype high titer influenza virus library

[0087] The H5 subtype high-titer influenza virus library is shown in the following table. Specifically, the present invention provides a high-titer influenza virus library, and the construction method is as follows: based on the HA sequence of the H5 subtype highly pathogenic avian influenza virus strain published by the WHO, representative virus strains of each subtype are screened through bioinformatics analysis, and plasmid synthesis is performed separately. Pseudoviruses are prepared by using a 4-plasmid expression system, using the HA protein and NA of the influenza virus as envelope proteins and firefly luciferin as the internal nucleic acid for packaging. High-titer virus strains are then selected by detecting relative luciferase activity, thereby constructing a high-titer H5 subtype highly pathogenic avian influenza virus strain library.

[0088] Serial number Virus strain Subclass 1 A / Hong Kong China / 156 / 1997 0 2 A / Cambodia / p0322095 / 05 1 3 A / Thailand / (KAN-1) / 2004 1 4 A / Indonesia / 5 / 2005 2.1.3.2 5 A / Turkey / 65596 / 2006 2.2.1 6 A / CommonMagpie / HongKong China / 5052 / 2007 2.3.2.1 7 A / duck / Guangdong / S1322 / 10(R6) 2.3.2.1 8 A / Shenzhen / 406H / 2006 2.3.4 9 A / chicken / Guizhou / 4 / 13(R8) 2.3.4.4 10 A / sichuan / 26221 / 2014(H5N6) 2.3.4.4 11 A / chicken / Netherlands / 14015526 / 2014(H5N8) 2.3.4.4 12 A / Chicken / Guangxi / 12 / 2004 2.4 13 A / Chicken / Korea / es / 2003 2.5 14 A / Silky Chicken / Hong Kong China / SF189 / 01 3 15 A / Goose / Guiyang / 337 / 2006 4 16 A / Duck / Guangxi / 1378 / 2004 5 17 A / blackbird / Hunan / 1 / 2004 6 18 A / Duck / Hubei / wg / 2002 6 19 A / Beijing / 01 / 2003 7.1 20 A / Chicken / Shanxi / 2 / 2006 7.2 21 A / Chicken / Henan / 16 / 2004 8 22 A / Goose / Shantou / 1621 / 05 9

[0089] avian influenza virus

[0090] Highly pathogenic avian influenza (H5) is a zoonotic disease caused by influenza A virus of the genus Influenzavirus in the family Orthomyxoviridae. Hemagglutinin (HA) is the primary target protein for broad-spectrum influenza A vaccines because it can induce neutralizing antibodies that can prevent viral infection, block viral invasion, and eliminate influenza viruses from the body.

[0091] Method for transforming low-titer influenza virus strains into high-titer virus strains

[0092] The present invention provides a method for transforming an H5 subtype low-titer highly pathogenic influenza virus into an H5 subtype high-titer influenza virus, comprising the steps of:

[0093] (a) providing a constructed H5 subtype high-titer influenza virus library, and constructing a genetic evolutionary tree based on the HA amino acid sequences of the high-titer influenza viruses and the low-titer influenza viruses in the virus library;

[0094] (b) obtaining a high-titer influenza virus strain that is closest in genetic distance to the low-titer influenza virus strain through a phylogenetic tree;

[0095] (c) exchanging the HA or NA protein of a high-titer influenza virus strain with that of a low-titer influenza virus to construct a chimeric influenza pseudovirus, and performing an RLA assay on the titer of the chimeric pseudovirus to determine whether the key amino acid affecting the influenza virus titer is located in the HA or NA;

[0096] (d) after determining that the key amino acid is located in HA (or NA), comparing the amino acid differences in HA (or NA) of the high-titer influenza virus obtained in step (b) and the low-titer influenza virus to be modified;

[0097] (e) using the HA (or NA) of the high-titer influenza virus obtained in step (b) as a backbone, performing single-point amino acid mutations on the different amino acid sites obtained in step (d), and constructing HA (or NA) mutant influenza pseudoviruses;

[0098] (f) respectively detecting the titer of the mutant pseudovirus obtained in step (e) and the pseudovirus of the high-titer virus strain obtained in step (b), and determining the key amino acid positions and quantities that may affect the low-titer virus strain;

[0099] (g) Based on the results of the key amino acids obtained in step (f), site-directed mutagenesis is performed on a single site or multiple sites in the above key amino acids in the HA (or NA) of the low-titer influenza pseudovirus to construct a mutant pseudovirus, and the key amino acid sites that increase the virus titer are determined by titer detection.

[0100] (h) constructing a plasmid based on the key amino acid sites determined in step (g), preparing HA mutant influenza viruses through reverse genetics, and verifying the key amino acid sites that can improve the low-titer virus strain confirmed by the pseudovirus model through titer detection, thereby obtaining a high-titer influenza virus strain.

[0101] The present invention takes the H5N1 subtype low-titer influenza virus strain A / blackbird / Hunan / 1 / 2004 as an example and identifies for the first time the amino acids 42, 44 and 53 in the HA protein (hemagglutinin), which are key amino acids affecting the virus titer. Simultaneously mutating the amino acids 42, 44 and 53 in the HA protein (hemagglutinin) (mutating V at position 42 to I, D at position 44 to E, and E at position 53 to D, respectively) will increase the virus titer, thereby obtaining a corresponding high-titer influenza virus.

[0102] According to the method provided by the present invention, a low-titer H5 subtype influenza virus strain was successfully transformed into a high-titer influenza virus strain. The specific implementation method is as follows: by constructing a genetic evolutionary tree (according to method step (a)), the high-titer virus strain A / Thailand / (KAN-1) / 2004 (according to method step (b)) with the closest genetic distance to A / blackbird / Hunan / 1 / 2004 was determined; the titer results of the prepared pseudovirus showed that there was a significant difference in the virus titer between the A / Thailand / (KAN-1) / 2004 virus strain and the A / blackbird / Hunan / 1 / 2004 virus strain: A / Thailand / (KAN-1) / 2004 was a high-titer virus strain, and A / blackbird / Hunan / 1 / 2004 was a low-titer virus strain; by using A / T The HA of the A / blackbird / Hunan / 1 / 2004 virus strain was exchanged with that of the A / blackbird / Hunan / 1 / 2004 virus strain to construct a chimeric pseudovirus. The results showed that when the HA of the A / blackbird / Hunan / 1 / 2004 virus strain was replaced with the HA of A / Thailand / (KAN-1) / 2004, the virus titer was significantly increased, indicating that the key amino acids affecting the titer of the A / blackbird / Hunan / 1 / 2004 virus strain are located in HA (according to method step (c)); by further comparing the amino acid sequences of the HA of the A / Thailand / (KAN-1) / 2004 virus strain and the HA of the A / blackbird / Hunan / 1 / 2004 virus strain, it was found that there were 17 amino acid differences (according to method step (d)).Using the HA of A / Thailand / (KAN-1) / 2004 as the backbone, 17 different amino acid sites were subjected to single-site mutation (the mutated amino acids were consistent with the amino acids at the same sites in A / blackbird / Hunan / 1 / 2004), HA mutant plasmids were synthesized, and HA single-site mutant pseudoviruses were prepared (according to method step (e)). Titer detection showed that mutations at three amino acid sites (positions 42, 44, and 53) significantly reduced the titer of the A / Thailand / (KAN-1) / 2004 pseudovirus, and the titer of the double-site mutant pseudovirus was also significantly reduced (according to method step (f)); In order to further verify the amino acid effects of the three amino acid sites on A / blackbird / Hunan To investigate the effect of mutations on the titer of the A / blackbird / Hunan / 1 / 2004 virus strain, researchers used the HA of the A / blackbird / Hunan / 1 / 2004 virus strain as the backbone and subjected it to single-site, double-site, and triple-site mutations (the mutated amino acids correspond to the amino acids at the same sites in the HA of A / Thailand / (KAN-1) / 2004). The results showed that simultaneous mutations at all three sites significantly increased the titer of the A / blackbird / Hunan / 1 / 2004 virus, confirming that amino acids 42, 44, and 53 of the A / blackbird / Hunan / 1 / 2004 HA sequence are key influencing the titer of the A / blackbird / Hunan / 1 / 2004 virus (according to step (g) of the method). Furthermore, the adsorption and fusion abilities of the A / blackbird / Hunan / 1 / 2004 mutant pseudoviruses were tested to determine the possible mechanism underlying the increased titer of the mutant pseudoviruses. Recombinant viruses were constructed and verified by reverse genetic methods, and the results were consistent: the titer of the A / blackbird / Hunan / 1 / 2004 recombinant virus with mutations at three amino acid sites was significantly improved, and the immunogenicity of the A / blackbird / Hunan / 1 / 2004 mutant recombinant virus was not significantly different from that of the A / blackbird / Hunan / 1 / 2004 recombinant virus (according to method step (h)).

[0103] Currently commercialized, highly pathogenic inactivated vaccines of the H5 subtype are typically prepared using influenza virus HA and NA as the viral envelope proteins, with six internal genes of the high-titer PR8 (or D7) used as internal genes of the recombinant virus to produce seed viruses. HA and NA are influenza virus envelope proteins and are the primary proteins that influence viral titer. Therefore, based on this, the present invention provides a method for increasing viral titer using a pseudovirus model. Furthermore, according to the method provided by the present invention, the originally low-titer virus strain A / blackbird / Hunan / 1 / 2004 was successfully transformed into a high-titer virus strain through steps (a) to (h). The successful transformation of the A / blackbird / Hunan / 1 / 2004 virus strain demonstrates the effectiveness of the method, which is not only applicable to A / blackbird / Hunan / 1 / 2004, but also to other low-titer H5 subtype influenza virus strains: first, a high-titer H5 subtype influenza virus strain with the closest genetic distance to the low-titer H5 subtype influenza virus to be transformed is selected, and a pseudovirus model is used to determine whether the key amino acids affecting the virus titer are derived from HA or NA, or both HA and NA. Further, by changing the key amino acid sites, the virus titer is improved. The present invention belongs to a platform technology and has universal applicability.

[0104] Inactivated or attenuated vaccines

[0105] The present invention also provides an inactivated vaccine for preventing H5 subtype avian influenza virus.

[0106] Inactivated vaccines are vaccines made by culturing viruses or bacteria and then inactivating them using physical (such as heating) or chemical agents (such as β-propiolactone), rendering them ineffective in terms of infectivity or toxicity while retaining their immunogenicity. Inactivated vaccines can consist of whole viruses or bacteria, or split fragments of these fragments, which are then further purified to contain only the desired antigenic components. Attenuated vaccines, on the other hand, involve various treatments that weaken the pathogenic microorganism's toxicity while still retaining its immunogenicity.

[0107] Typically, the method for producing an inactivated vaccine and an attenuated vaccine comprises obtaining the avian influenza virus by co-transfecting cells with a plasmid based on the nucleotide sequence of the H5 subtype avian influenza virus using reverse genetics, and further amplifying the virus in cells or chicken embryos before inactivating or treating the virus to lose or weaken its infectivity (or toxicity). It should be understood that in a preferred embodiment of the present invention, the hemagglutinin protein of the influenza virus obtained using reverse genetics is a mutant protein, wherein one or more amino acid mutations (preferably two or three) are respectively performed at amino acids 42, 44, and 53 of the hemagglutinin protein of a wild-type influenza virus (e.g., the H5N1 subtype low-titer influenza virus strain A / blackbird / Hunan / 1 / 2004) (V at position 42 is mutated to I, D at position 44 is mutated to E, and E at position 53 is mutated to D).

[0108] Preparation of vaccine compositions

[0109] The present invention also provides a method for preparing a vaccine composition, specifically comprising the steps of:

[0110] The high-titer influenza pseudovirus prepared by the present invention is mixed with a pharmaceutically acceptable vaccine adjuvant to form a vaccine composition.

[0111] In another preferred embodiment, the adjuvant is an aluminum adjuvant or a GLA adjuvant, preferably an aluminum adjuvant.

[0112] Compositions and methods of administration

[0113] The present invention also provides a composition comprising: (i) a high-titer influenza pseudovirus prepared using the method of the present invention, and (ii) a pharmaceutically or immunologically acceptable excipient or adjuvant. As used herein, the term "comprising" indicates that various components can be used or present together in the composition of the present invention. Therefore, the terms "consisting essentially of" and "consisting of" are encompassed by the term "comprising."

[0114] The compositions of the present invention include pharmaceutical compositions and vaccine compositions. The compositions of the present invention can be monovalent or multivalent.

[0115] The pharmaceutical composition or vaccine composition of the present invention can be prepared into various conventional dosage forms, including (but not limited to): injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays, etc.

[0116] (i) Pharmaceutical Compositions

[0117] The pharmaceutical composition of the present invention comprises an effective amount of a recombinant protein or vaccine polypeptide prepared by the method of the present invention. The recombinant protein or vaccine polypeptide may be monovalent or multivalent.

[0118] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or preventive effect. This effect can be detected, for example, by antigen levels. A therapeutic effect also includes a reduction in physiological symptoms. The precise effective amount for a given subject depends on the subject's size and health, the nature and extent of the condition, and the therapeutic agent and / or combination of therapeutic agents selected for administration. Therefore, it is not useful to specify an exact effective amount in advance. However, for a given condition, the effective amount can be determined by routine experimentation.

[0119] For the purposes of the present invention, an effective dosage is about 0.2 μg / kg to 2 μg / kg administered to a subject.

[0120] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier used for administering a therapeutic agent (e.g., a recombinant protein or other therapeutic agent). The term refers to pharmaceutical carriers that do not themselves induce the production of antibodies harmful to the individual receiving the composition and that are not excessively toxic after administration. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, etc. These carriers are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0121] Pharmaceutically acceptable carriers in the compositions may include liquids such as water, saline, glycerol, and ethanol. These carriers may also contain auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like. Typically, the compositions are formulated as injectables, such as liquid solutions or suspensions. They may also be formulated as solid forms suitable for incorporation into solutions, suspensions, or liquid excipients prior to injection. Liposomes are also included within the definition of pharmaceutically acceptable carriers.

[0122] (ii) Vaccine Composition

[0123] The vaccine composition of the present invention can be prophylactic (i.e., to prevent infection) or therapeutic. The vaccine composition comprises an immunogenic antigen (including a high-titer influenza pseudovirus of the present invention) and is typically combined with a "pharmaceutically acceptable carrier," which includes any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), etc. These carriers are well known to those of ordinary skill in the art. In addition, these carriers can act as immunostimulants ("adjuvants"). In addition, antigens can also be coupled to bacterial toxins (such as toxins of pathogens such as diphtheria, tetanus, cholera, and Helicobacter pylori).

[0124] Preferred adjuvants for enhancing the effect of the immunocomposition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations, for example, (a) MF59 (see WO90 / 14837), (b) SAF, and (c) Ribi TM Adjuvant system (RAS) (Ribi Immunochem, Hamilton, MT), (3) saponin adjuvant; (4) Freund's complete adjuvant (CFA) and Freund's incomplete adjuvant (IFA); (5) cytokines, such as interleukins (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (such as gamma interferon), macrophage colony stimulating factor (M-CFS), tumor necrosis factor (TNF), etc.; (6) detoxified variants of bacterial ADP-ribosylating toxins (such as cholera toxin CT, pertussis toxin PT or Escherichia coli heat-labile toxin LT), see for example WO93 / 13302 and WO92 / 19265; and (7) other substances that act as immunostimulants to enhance the effect of the composition.

[0125] Vaccine compositions, including immunogenic compositions (e.g., may include an antigen, a pharmaceutically acceptable carrier, and an adjuvant), typically contain a diluent such as water, saline, glycerol, ethanol, etc. In addition, auxiliary substances, such as wetting agents or emulsifiers, pH buffering substances, etc., may be present in such carriers.

[0126] More specifically, vaccines, including immunogenic compositions, contain an immunogenic polypeptide in an immunologically effective amount, as well as the other required components described above. An "immunologically effective amount" refers to an amount that is effective for treatment or prevention when administered to an individual as a single dose or as part of a continuous dose. This amount can be determined based on the health and physiological condition of the individual being treated, the type of individual being treated (e.g., human), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating physician's assessment of the medical condition, and other relevant factors. It is expected that this amount will be within a relatively wide range and can be determined through routine experimentation.

[0127] Typically, vaccine compositions or immunogenic compositions are formulated as injectable formulations, such as liquid solutions or suspensions. They can also be formulated in solid form suitable for incorporation into solutions, suspensions, or liquid excipients prior to injection. Such formulations can also be emulsified or encapsulated in liposomes to enhance the adjuvant effect.

[0128] (iii) Route of administration and dosage

[0129] The composition can be administered directly to a subject. The subject can be a human or non-human mammal, preferably a human. When used as a vaccine, the high-titer influenza virus-like particles of the present invention can be administered directly to an individual using known methods. These vaccines are typically administered using the same routes of administration as conventional vaccines and / or by simulating pathogen infection pathways.

[0130] Routes of administration for the pharmaceutical or vaccine compositions of the present invention include, but are not limited to, intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, intravaginal, oral, or other parenteral routes. If desired, routes of administration may be combined or adjusted based on the disease. Vaccine compositions may be administered in single or multiple doses, and may include booster doses to elicit and / or maintain immunity.

[0131] The virus-like particle vaccine should be administered in an "effective amount", that is, the amount of virus-like particles is sufficient to induce an immune response in the selected administration route and can effectively protect the host against infection with avian influenza virus.

[0132] The amount of virus-like particles selected in each vaccine dose is determined based on the amount that can induce an immune protective response without significant side effects. Generally, after infecting the host cells, each dose of vaccine is sufficient to contain about 1μg-1000μg, preferably 1μg-100μg, and more preferably 10μg-50μg of protein or VLP. The optimal dosage of a specific vaccine can be determined by standard research methods including observing antibody titers and other reactions in the subjects. The need for a booster dose can be determined by monitoring the level of immunity provided by the vaccine. After evaluating the antibody titer in the serum, it may be necessary to select a booster dose for immunization. The administration of adjuvants and / or immunostimulants can enhance the immune response to the protein of the present invention. The preferred method is to administer the immunogenic composition by injection from a parenteral (subcutaneous or intramuscular) route.

[0133] The main advantages of the present invention are:

[0134] (1) The present invention utilizes the influenza pseudovirus model for the first time and develops a simple and rapid method for transforming low-titer influenza viruses into high-titer influenza viruses;

[0135] (2) The present invention provides for the first time a high-titer H5 subtype highly pathogenic avian influenza virus library;

[0136] (3) The present invention first discovered that a genetic evolutionary tree was drawn using the HA amino acid sequence of the high-titer H5 subtype highly pathogenic avian influenza virus library constructed by the present invention and the low-titer H5 subtype. Virus strains with a close genetic distance to the low-titer virus strains on the evolutionary tree were selected, and chimeric pseudoviruses with interchangeable HA and NA were constructed to determine whether the key amino acids affecting the titer of the recombinant virus are located in HA or NA. The HA (or NA) amino acid sequences of the high-titer virus strains and the low-titer virus strains were compared, and further amino acids at different sites on HA (or NA) were site-directed mutated to construct mutant pseudoviruses to find out the amino acids affecting the titer of the pseudoviruses. Based on the pseudovirus detection results, mutant recombinant viruses were constructed, and the titers of the mutant recombinant viruses and wild-type recombinant viruses were compared to determine the amino acids that improve the virus's ability to replicate in chicken embryos and MDCK cells. Amino acids with less impact on the immunogenicity of the vaccine strain were selected for mutation to construct high-titer recombinant viruses, thereby transforming low-titer influenza viruses into high-titer influenza viruses. Vaccines prepared with high-titer influenza viruses can effectively improve vaccine preparation time and save preparation costs.

[0137] (4) The present invention discovered for the first time that a hemagglutinin mutant (e.g., mutations at amino acids 42, 44, and 53) derived from the low-titer H5N1 subtype influenza virus strain A / blackbird / Hunan / 1 / 2004 can effectively increase the virus titer, thereby enabling the efficient production and preparation of a high-titer H5 subtype influenza vaccine.

[0138] (5) The present invention discovered for the first time that, through the method provided by the present invention, three key amino acid sites were found, and the viral titer of the constructed mutant recombinant virus strain was significantly improved, and the immunogenicity was not affected, thereby enabling the preparation of high-titer recombinant viruses.

[0139] The present invention is further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental methods in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0140] Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial channels.

[0141] The HA pseudovirus and recombinant pseudovirus used in this study were obtained from the Shanghai Pasteur Institute, Chinese Academy of Sciences. Wild-type influenza virus was obtained from Shenzhen Third People's Hospital. Experiments with wild-type and recombinant viruses were performed at the Department of Microbiology, Shenzhen Center for Disease Control and Prevention, Shenzhen Third People's Hospital. Plasmids pHR-Luc and pCMV / RΔ8.2 were gifts from Professor Luigi Naldini of the University of Torino Medical School, Torino, Italy.

[0142] The hemagglutinin amino acid sites involved in the present invention all use the H3 numbering method.

[0143] 1. Experimental Materials

[0144] 1.1 The main consumables and reagents used in the experiment are shown in Table 1

[0145] Table 1. Information on reagents and consumables used in the experiment

[0146] name brand Item No. 6-well cell culture plates Thermo fisher 140675 T75 cell culture flask Thermo fisher 156499 Plasmid extraction kit Macherey-Nagel 740412.50 96-well flat-bottom cell culture plates Corning 3599 96-well U-bottom cell culture plate Corning 3799 Luciferase Assay Kit Promega E1500 Mycoplasma detection kit Yeasen 40612 DMEM high glucose medium Gibco 12800-017 Penicillin-Streptomycin (double antibody) Gibco 15140-122 Gluta MAX Gibco 35050061 Fetal bovine serum Gibco 16000-044 0.25% trypsin-EDTA Gibco 25200-072 BSA Sigma 9048-46-8 Lipo2000 Thermo fisher 11668-019 HIV p24 Protein Detection Kit Zeptometrix 0801111 TRIzol reagent Thermo 15596018 Reverse transcription kit Takara RR037Q

[0147] 1.2 Cells

[0148] HEK293T cells (Human embryonic kidney, ATCC, CRL-3216);

[0149] MDCK.1 cells (Madin-Darby canine kidney, ATCC, CRL-2935);

[0150] MDCK-NBL-2 cells (Madin-Darby canine kidney, ATCC, CCL-34);

[0151] The cell culture medium was DMEM high-glucose complete medium (containing 10% fetal bovine serum, 100 units / mL penicillin, and 100 μg / mL streptomycin);

[0152] Cell culture conditions were 37°C, 5% CO2;

[0153] All cells used were tested for mycoplasma and the test results were negative.

[0154] 1.3 Plasmids

[0155] 1) The six gene segments PB1, PB2, PA, NP, M, and NS of the A / WSN / 1933 virus were inserted into the pHW2000 vector to construct the vectors pHW2000-PB1, pHW2000-PB2, pHW2000-PA, pHW2000-NP, pHW2000-M, and pHW2000-NS, respectively;

[0156] 2) The alkaline cleavage site PERERRRKKRG of the HA protein of the H5 subtype avian influenza recombinant virus A / blackbird / Hunan / 1 / 2004 (HN) was mutated to PEIETRG, and amino acids at positions 42, 44, and 53 were mutated to V42I, D44E, and E53D. The mutated amino acids were identical to amino acids 42, 44, and 53 of the HA amino acid sequence of TH. The HA nucleotide sequence was synthesized using this method to construct the vector pHW2000-HNHA (424453);

[0157] 3) Using the HA and NA genes of TH and HN as templates, after human-preferred codon optimization, the company synthesized the nucleotide sequences to construct the vectors pCMV / R-THHA, pCMV / R-HNHA, pCMV / R-THNA, and pCMV / R-HNNA;

[0158] 4) There are 17 amino acids different in the HA amino acid sequences of TH and NA. Using the HA amino acid sequence of TH as a template, mutations were performed on 17 amino acid sites. The amino acids after mutation were the same as the amino acids at the HN phase point. The 17 different HA nucleotide sequences after mutation were used as templates. After human-preferred codon optimization, the sequences were handed over to the company for sequence synthesis. The vectors pCMV / R-THHA(I42V), pCMV / R-THHA(E44D), pCMV / R-THHA(D53E), pCMV / R-THHA(N92aS), pCMV / R-THHA(V93A), and pCMV / R-T were constructed. HHA(S129D), pCMV / R-THHA(L133bS), pCMV / R-THHA(R143G), pCMV / R-THHA(K144N), pCMV / R-THHA(S145P), pCMV / R-THHA(T160A), pCM V / R-THHA(E174A), pCMV / R-THHA(R216K), pCMV / R-THHA(T266A), pCMV / R-THHA(D449E), pCMV / R-THHA(K498N) and pCMV / R-THHA(I511T).

[0159] 5) The amino acids at positions 42, 44, and 53 of the HA amino acid sequence of TH were subjected to double-site mutation and triple-site mutation, respectively. The mutated amino acid sequence was used as a template and the human-preferred codons were optimized and then handed over to the company for sequence synthesis to construct the vectors pCMV / R-THHA (4244), pCMV / R-THHA (4253), pCMV / R-THHA (4453), and pCMV / R-THHA (424453).

[0160] All plasmid sequences were confirmed by Sanger sequencing.

[0161] 1.4 Others

[0162] SPF eggs were purchased from Shandong Haotai Experimental Animal Breeding Co., Ltd.

[0163] 2. Experimental Methods

[0164] The specific methods are shown in 2.1 to 2.10 below.

[0165] 2.1 Recombinant virus preparation

[0166] The present invention utilizes a reverse genetics system to package and prepare the A / blackbird / Hunan / 1 / 2004 recombinant virus using PB1, PB2, PA, NP, M, and NS from the A / WSN / 33 virus (using a low-titer H5 subtype influenza virus as a reference) as internal genes and hemagglutinin HA and ceramide NA from the A / blackbird / Hunan / 1 / 2004 virus as external genes. The A / WSN / 33 recombinant virus is also packaged using PB1, PB2, PA, NP, M, NS, HA, and NA from the A / WSN / 33 virus. The amino acid sequence of the A / blackbird / Hunan / 1 / 2004 hemagglutinin is SEQ ID NO.: 1, and the amino acid sequence number is EPI25072.

[0167] A / blackbird / Hunan / 1 / 2004 (an example of a low-titer influenza pseudovirus of the H5 subtype) hemagglutinin amino acid sequence (NCBI sequence number AAW19638, available by downloading from the NCBI website https: / / www.ncbi.nlm.nih.gov / protein / 56672737):

[0168] MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDVLDKTHNGKLCELDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKASPANDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGNPSFFRNVVWLIKKNSAYPTIKRSYNNTNQADLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYEKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLNREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICI

[0169] The amino acid sequence of the hemagglutinin of A / Thailand / 1(KAN-1) / 2004 (an example of a high-titer influenza pseudovirus of the H5 subtype) (NCBI sequence number AAS65615, available for download at the NCBI website: https: / / www.ncbi.nlm.nih.gov / protein / AAS65615.2) is: MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQRKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTS TLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDG VTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICI

[0170] The specific method is:

[0171] 1) Prepare 10 mL of DMEM complete medium for a total of 9 x 10 6 A mixed single cell suspension of HEK293T and MDCK-NBL-2 (HEK293T:MDCK-NBL-2=6:1) was inoculated into a T75 culture flask and cultured at 37°C, 5% CO2 for 20 h;

[0172] 2) According to the instructions of the lipofectamine lipo2000 transfection reagent, six plasmids containing the internal gene segments of the WSN virus, pHW2000-PB1, pHW2000-PB2, pHW2000-PA, pHW2000-M, pHW2000-NP, and pHW2000-NS, were co-transfected with pHW2000-HN HA and pHW2000-HNNA (or pHW2000-WSN HA and pHW2000-WSN NA) into a mixture of HEK293T and MDCK-NBL-2 cells to prepare recombinant virus HN (or WSN);

[0173] 3) 72 h after transfection, centrifuge at 3000 rpm and 4°C for 20 min to collect the supernatant containing the virus.

[0174] 2.2 Recombinant virus amplification

[0175] 2.2.1 MDCK.1 cell expansion

[0176] 1) Prepare 10 mL of DMEM complete medium for a total of 9 x 10 6 The MDCK.1 single cell suspension was inoculated into a T75 culture flask and cultured at 37°C and 5% CO2;

[0177] 2) After 24 hours, the original cell culture medium was discarded and the recombinant viruses WSN and HN were added from 10 -1 Dilute to 10 -10 times, added to the cells, incubated at 37°C, 5% CO2 for 1 h, then added with 10 mL of serum-free medium, and continued to culture at 37°C, 5% CO2;

[0178] 3) After culturing for 48 to 72 hours, when CPE reaches 80%, collect the cell culture medium into a 50 mL centrifuge tube and centrifuge at 3000 rpm and 4°C for 20 minutes. Discard the cell debris and collect the supernatant virus liquid. Take an appropriate amount for hemagglutination titer detection. Aliquot the remaining virus and store at -80°C until use.

[0179] The results of hemagglutination assay showed that the titer of recombinant virus A / WSN / 33 after amplification in MDCK.1 cells was significantly higher than that of recombinant virus A / blackbird / Hunan / 1 / 2004, and the difference was significant (e.g. Figure 1 (as shown in A in the figure).

[0180] 2.2.2 SPF chicken embryo expansion

[0181] The recombinant viruses WSN and HN were isolated from 10 -1 Dilute to 10 -10The virus supernatant was collected after centrifugation at 3000 rpm and 4°C for 20 min. An appropriate amount of virus was used for hemagglutination titer detection. The remaining virus was packaged and stored at -80°C for later use.

[0182] The results of the hemagglutination test showed that the hemagglutination titer of the recombinant virus A / WSN / 33 after chicken embryo amplification was significantly higher than that of the recombinant virus A / blackbird / Hunan / 1 / 2004, and the difference was significant (e.g. Figure 1 (as shown in B in the figure).

[0183] 2.3 Virus titer detection

[0184] 2.3.1 Virus hemagglutination titer detection

[0185] 1) Add 50 μL of 1× PBS to wells 2-12 of a 96-well U-bottom cell culture plate. Add 100 μL of virus solution to well 1. Pipette 50 μL of virus solution from well 1 and add it to well 2, mixing five times. Perform a two-fold serial dilution of the virus starting from well 2 through well 11, discarding 50 μL from well 11. Well 12 serves as a negative control.

[0186] 2) Add 50 μL of 0.5% chicken red blood cells to each well 1-12, mix gently three times, and let stand at room temperature for 30-60 minutes, or until the red blood cells in the negative control wells sink to the bottom of the well.

[0187] 3) The highest virus dilution factor corresponding to 100% agglutination of red blood cells is the virus hemagglutination titer.

[0188] 2.3.2 Detection of Pseudovirus p24 Protein Content

[0189] 1) Take out an appropriate amount of microporous strips from the HIV-1 P24 antigen ELISA kit, equilibrate at room temperature, wash, lyse the sample to be tested, and then serially dilute the lysed sample and P24 protein standard. Add the diluted sample to the microporous strips and incubate at 4°C overnight.

[0190] 2) Block at room temperature, add detection antibody, and incubate at 37°C.

[0191] 3) Add chemically labeled secondary antibody and incubate at 37°C.

[0192] 4) Add substrate and incubate at room temperature in the dark for 30 minutes.

[0193] 5) Add stop solution to terminate the reaction and place the sample in a microplate reader to read the sample absorbance (OD 450nm).

[0194] 6) Calculate the P24 protein content corresponding to the sample to be tested based on the standard curve drawn with the standard.

[0195] 2.3.3 Pseudovirus reporter gene copy number detection

[0196] The experiment used reverse transcription and real-time fluorescence quantitative PCR to detect the copy number of pseudovirus reporter genes. The experimental steps refer to the "Invitrogen TRIzol reagent" reagent instructions. The specific steps are as follows:

[0197] 1) Use Trizol to lyse the pseudovirus and release the viral RNA.

[0198] 2) Purify and concentrate viral RNA, and reverse transcribe the RNA into cDNA using universal primers according to the instructions of Takara "PrimeScript™ RT Master Mix (Perfect Real Time)".

[0199] 3) cDNA and standards were amplified by real-time fluorescence quantitative PCR, and viral particles were quantified according to the CT value.

[0200] 2.3.4 Pseudovirus relative fluorescence intensity detection

[0201] After influenza virus infects MDCK.1 cells, the internal firefly luciferase reporter gene will be expressed in the host MDCK.1 cells. After the MDCK.1 cells infected with influenza virus are lysed, luciferase is released from the cells. The fluorescence signal (i.e., relative fluorescence intensity) of luciferase at a wavelength of 560nm can be detected using a firefly luciferase detection kit. In the present invention, the titer of influenza virus or pseudovirus is determined by detecting the relative fluorescence intensity. The specific method is as follows:

[0202] 1) Prepare 5×10 MDCK.1 single cell suspensions in DMEM complete medium. 4 cell / mL, 100 μL per well was inoculated into a 96-well flat-bottom cell culture plate and cultured at 37°C, 5% CO2 for 20 h;

[0203] 2) Add 50 μL of DMEM complete medium to each well of a 96-well round-bottom cell culture plate, add 100 μL of pseudovirus solution to well 2, pipette 50 μL of the mixture from well 2 into well 3, pipette and pipette five times to mix thoroughly, and perform a 2-fold serial dilution of the virus starting from well 2 to well 11, discarding 50 μL of the solution.

[0204] 3) Incubate the diluted pseudovirus and culture medium mixture at 37°C in 5% CO2;

[0205] 4) After 1 hour, the incubated pseudoviruses were added to MDCK.1 cells and cultured at 37°C and 5% CO2;

[0206] 5) After 60 h, the cell status was observed and the relative fluorescence intensity was measured according to the instructions of the luciferase assay kit.

[0207] 2.4 Pseudovirus Preparation

[0208] The present invention uses HEK-293T cells as host cells, a 4-plasmid expression system, and a calcium phosphate-mediated plasmid DNA transfection method to package and prepare pseudoviruses. The experimental procedures are based on the fourth edition of the Molecular Cloning Laboratory Manual.

[0209] The 4 plasmid systems are:

[0210] 1) pCMV / R is used to express the envelope protein HA of influenza pseudovirus;

[0211] 2) pCMV / R is used to express the envelope protein NA of influenza pseudovirus (or the G protein of vesicular stomatitis virus (VSV-G), a control pseudovirus);

[0212] 3) pCMV / RΔ8.2 is used to express the pseudovirus capsid protein;

[0213] 4) pHR'CMV-luc is used to express the pseudoviral reporter protein - firefly luciferase.

[0214] Plasmid schematic diagram Figure 2 shown.

[0215] The specific method is:

[0216] 1) HEK293T cells were cultured at 9×10 6 / 10mL was inoculated into a T75 cell culture flask and cultured at 37°C, 5% CO2 for 20 hours. The original culture medium was replaced with 10mL of DMEM complete medium containing 10μmol / L chloroquine diphosphate and cultured at 37°C, 5% CO2 for 2 hours;

[0217] 2) Prepare a mixture of plasmid, transfection reagent, and buffer according to Table 2, let it stand at room temperature for 20 minutes, and then add the mixture to the culture flask prepared in step 1);

[0218] 3) After culturing at 37°C and 5% CO2 for 15 h, replace with 15 mL of fresh DMEM complete medium and continue culturing;

[0219] 4) After 48 h, the cell culture medium was collected and centrifuged at 3000 rpm and 4°C for 20 min. The viral supernatant was collected and an appropriate amount was used for relative fluorescence intensity detection. The remaining virus was aliquoted and stored at -80°C for later use.

[0220] Table 2. Pseudovirus preparation mixed system

[0221] Plasmids / reagents volume <![CDATA[ddH2O]]> 529.2μL pCMV / R-HA (100 ng / μL) 27.0μL pCMV / R-NA (50 ng / μL) 13.5μL pHR-Luc (1000 ng / μL) 18.9μL pCMVΔ8.9 (1000 ng / μL) 18.9μL <![CDATA[CaCl2(2.5M)]]> 67.5μL 2×HEPES (pH 7.1) 675.0μL

[0222] The titer of A / Thailand(KAN-1) / 2004 pseudovirus and A / blackbird / Hunan / 1 / 2004 pseudovirus were determined as described in 2.3.4. The hemagglutination test results showed that there was no significant difference in the hemagglutination titer between A / Thailand(KAN-1) / 2004 pseudovirus and A / blackbird / Hunan / 1 / 2004 pseudovirus (e.g. Figure 1 The results of p24 protein detection showed that there was no significant difference in the p24 protein content between the A / Thailand(KAN-1) / 2004 pseudovirus and the A / blackbird / Hunan / 1 / 2004 pseudovirus (as shown in C). Figure 1 The results of reporter gene copy number showed that there was no significant difference in the copy number of reporter gene between A / Thailand(KAN-1) / 2004 pseudovirus and A / blackbird / Hunan / 1 / 2004 pseudovirus (as shown in D). Figure 1 However, the relative fluorescence intensity results showed that the relative fluorescence intensity of the A / Thailand(KAN-1) / 2004 pseudovirus was stronger, and there was a significant difference between the relative fluorescence intensity of the A / blackbird / Hunan / 1 / 2004 pseudovirus (as shown in E). Figure 1 F in the figure).

[0223] 2.5 Preparation of Chimeric Pseudovirus

[0224] By exchanging the NA of A / Thailand(KAN-1) / 2004 and A / blackbird / Hunan / 1 / 2004, chimeric pseudoviruses THHAHNNA and HNHATHNA (e.g. Figure 1 The pseudovirus preparation method is as described in 2.4.

[0225] The titer of the chimeric pseudovirus was detected as described in 2.3.4. The relative fluorescence intensity of luciferase showed that the fluorescence intensity of the THHAHNNA chimeric pseudovirus was significantly higher than that of the HNHATHNA chimeric pseudovirus (e.g. Figure 1 (as shown in H in ).

[0226] The experimental results showed that the titer of the pseudovirus did not change much after the NA of A / Thailand(KAN-1) / 2004 was replaced, but the titer of the pseudovirus did change significantly after the HA of A / Thailand(KAN-1) / 2004 was replaced. Furthermore, the titer of the pseudovirus did not change much after the NA of A / blackbird / Hunan / 1 / 2004 was replaced, but the titer of the pseudovirus did change significantly after the HA of A / blackbird / Hunan / 1 / 2004 was replaced. This shows that the envelope protein HA affects and changes the titer and infectivity of the pseudovirus.

[0227] 2.6 HA amino acid differences between A / Thailand(KAN-1) / 2004 and A / blackbird / Hunan / 1 / 2004

[0228] The present invention compared the HA amino acid sequences of A / Thailand(KAN-1) / 2004 and A / blackbird / Hunan / 1 / 2004 and found that there were 17 amino acid differences (as shown in Table 3), and these 17 amino acids were located in the head region and the stem region of the HA protein (as shown in Table 3). Figure 3 (as shown in A).

[0229] Table 3. 17 amino acid differences in HA between A / Thailand(KAN-1) / 2004 and A / blackbird / Hunan / 1 / 2004

[0230] H3 Numbering 42 44 53 92a 93 129 Amino acids(TH) I E D N V S Amino acids(HN) V D E S A D H3 Numbering 133b 143 144 145 160 174 Amino acids(TH) L R K S T E Amino acids(HN) S G N P A A H3 Numbering 216 266 449 498 511 / Amino acids(TH) R T D K I / Amino acids(HN) K A E N T /

[0231] 2.7 Construction of A / Thailand (KAN-1) / 2004HA mutant pseudovirus

[0232] 2.7.1 Construction of pseudoviruses with single-point amino acid mutations

[0233] The present invention mutated 17 amino acids in the hemagglutinin amino acid residues of TH that differed from those of HN. The mutated amino acids were the amino acids at the corresponding amino acid sites of HN. According to the method for preparing pseudoviruses in 2.4, 17 mutant pseudoviruses were obtained (as shown in Table 4). The titers of the mutant pseudoviruses were tested according to the method described in 2.3.4. The experimental results showed (as shown in Figure 3 (as shown in B):

[0234] 1) The relative fluorescence intensities of the pseudoviruses TH(I42V), TH(E44D), and TH(D53E) were all weaker than those of the unmutated TH pseudovirus, and the differences were significant;

[0235] 2) Except for TH(I42V), TH(E44D), and TH(D53E), the relative fluorescence intensity of the pseudoviruses with the remaining 14 amino acid mutations was no different from that of the TH pseudovirus.

[0236] It can be seen from this that changes in amino acids at positions 42, 44, and 53 of the hemagglutinin of TH may affect the viral titer of TH.

[0237] Table 4.17 strains of A / Thailand (KAN-1) / 2004 single-site amino acid mutation pseudoviruses

[0238]

[0239]

[0240] 2.7.2 Construction of double-site amino acid mutation pseudovirus

[0241] Furthermore, the amino acids at positions 42, 44, and 53 of TH hemagglutinin were mutated at two sites to construct pseudoviruses (as shown in Table 5), and the titer of the pseudoviruses was tested. The results showed:

[0242] The relative fluorescence intensity of MDCK.1 cells infected with pseudoviruses TH(I42V,E44D), TH(I42V,D53E), and TH(E44D,D53E) was weaker than that of MDCK.1 cells infected with non-mutated TH pseudovirus, and the numerical differences were significant. However, the differences among pseudoviruses TH(I42V,E44D), TH(I42V,D53E), and TH(E44D,D53E) were not significant (e.g. Figure 3 (as shown in C in the figure).

[0243] Table 5.3 A / Thailand (KAN-1) / 2004 double-site amino acid mutation pseudovirus

[0244] Double mutant pseudovirus 1 Double mutant pseudovirus 2 Double mutant pseudovirus 3 TH(I42V,E44D) TH(I42V,D53E) TH(E44D,D53E)

[0245] 2.8 Construction of A / blackbird / Hunan / 1 / 2004 mutant pseudovirus

[0246] 2.8.1 A / blackbird / Hunan / 1 / 2004 mutant pseudovirus titer detection

[0247] The amino acids at positions 42, 44, and 53 of the HN hemagglutinin were subjected to single-point, double-point, and triple-point mutations, respectively, to construct a total of 7 mutant pseudoviruses. The mutated amino acids were consistent with the amino acids at positions 42, 44, and 53 corresponding to TH, as shown in Table 6.

[0248] Table 6. Construction of 7 mutant pseudoviruses of strain A / blackbird / Hunan / 1 / 2004

[0249]

[0250] The titer detection method of 7 mutant pseudovirus strains was as described in 2.3.4. The results showed that ( Figure 4 A in the figure):

[0251] 1) The relative fluorescence intensities of the single-point mutated pseudoviruses HN(V42I), HN(D44E), and HN(E53D) were not significantly different from those of the unmutated HN pseudovirus, indicating that single-point mutations at amino acid positions 42, 44, and 53 of the HN hemagglutinin do not affect the titer of the HN pseudovirus.

[0252] 2) The relative fluorescence intensity values ​​of the double-point mutant pseudoviruses HN(V42I,D44E), HN(V42I,E53D), and HN(D44E,E53D) were significantly higher than those of the unmutated HN pseudovirus, and the values ​​were significantly different, indicating that pairwise mutations at amino acid positions 42, 44, and 53 in the HN hemagglutinin can significantly increase the titer of the HN pseudovirus;

[0253] 3) The relative fluorescence intensity values ​​of the triple-point mutated pseudovirus HN (V42I, D44E, E53D) were also significantly higher than those of the unmutated HN pseudovirus, and the values ​​were extremely significantly different. The relative fluorescence intensity values ​​of HN (V42I, D44E, E53D) were also significantly higher than those of the three groups of double-point mutated pseudoviruses HN (V42I, D44E), HN (V42I, E53D), and HN (D44E, E53D), and the values ​​were extremely significantly different. This indicates that amino acids at positions 42, 44, and 53 are key amino acids affecting the titer of the HN pseudovirus. When the three sites are mutated simultaneously, the titer of the HN pseudovirus can be significantly increased.

[0254] To further determine whether the increase in viral titer of the pseudovirus HN (V42I, D44E, E53D) was due to the enhanced adsorption ability of the pseudovirus to host cells or the improved fusion ability with host cells, the present invention continued to conduct the following experiments.

[0255] 2.8.2A / blackbird / Hunan / 1 / 2004 Hemagglutinin Mutant Pseudovirus Adsorption Capacity Test

[0256] 1) Prepare MDCK.1 single cell suspension (4×10) in DMEM complete medium 4 / mL, 100 μL / well was inoculated into a 96-well flat-bottom cell culture plate, and cultured at 37°C, 5% CO2 for 20 h.

[0257] 2) Mix the pseudovirus and DMEM (1% BSA) culture medium and incubate at 4°C overnight.

[0258] 3) The original culture medium of the 96-well flat-bottom cell culture plate was discarded, and DMEM (1% BSA) culture medium was added at 100 μL / well and incubated at 4° C. for 1 h.

[0259] 4) Replace the cell culture medium in step 3 with the pseudovirus and serum mixture in step 2 and incubate at 4°C for 2 h.

[0260] 5) Wash the 96-well flat-bottom cell culture plate 4 times with sterile PBS (1% BSA) to remove unbound virus, add 100 μL of luciferase lysis buffer, and lyse the cells at room temperature for 30 minutes.

[0261] 6) According to the instructions of the HIV-p24 detection kit, read the absorbance at 450 nm and calculate the number of viruses bound to the cells.

[0262] The experimental results show that (such as Figure 4 B in the figure):

[0263] 1) Among the pseudoviruses with single-site amino acid mutations HN(V42I), HN(D44E), and HN(E53D), only the pseudovirus with amino acid mutation at D44E showed a change in adsorption capacity, and the adsorption capacity was weaker than that of HN, with different values;

[0264] 2) Among the pseudoviruses HN(V42I and D44E), HN(V42I, E53D), and HN(D44E, E53D) with double amino acid mutations, only the pseudovirus with HN(V42I, E53D) mutation showed a difference in adsorption capacity compared with HN, and the adsorption capacity of both pseudoviruses was weaker than that of HN. The adsorption capacity of the pseudoviruses HN(V42I and D44E) and HN(D44E, E53D) showed no difference compared with HN.

[0265] 3) The adsorption capacity of the pseudovirus HN with amino acid mutations at three sites (V42I, D44E, E53D) is no different from that of HN.

[0266] It can be seen that the amino acid at position 44 can affect the adsorption capacity of HN pseudovirus alone, and the amino acids at positions 42 and 53 can jointly affect the adsorption capacity of HN pseudovirus.

[0267] 2.8.3A / blackbird / Hunan / 1 / 2004 Hemagglutinin Three-Point Mutation Pseudovirus Fusion Capacity Test

[0268] 1) Incubate pseudovirus and 2% red blood cells on ice for 10 min.

[0269] 2) Sodium citrate buffer was mixed with the mixture in step 1, the final pH was adjusted to 5.0, and incubated at room temperature for 30 minutes.

[0270] 3) Centrifuge at 3000 rpm for 3 min and take the supernatant for Elisa assay to detect the hemoglobin content in the supernatant.

[0271] Figure 4 The experimental results of C show that:

[0272] 1) The fusion abilities of pseudoviruses with single-site amino acid mutations HN(V42I), HN(D44E), and HN(E53D) were no different from those of pseudovirus HN;

[0273] 2) The fusion abilities of the double-site amino acid mutation pseudoviruses HN (V42I and D44E), HN (V42I, E53D), and HN (D44E, E53D) were all enhanced compared to the pseudovirus HN, and the differences in experimental values ​​were very significant;

[0274] 3) The fusion ability of the pseudovirus HN with three-point amino acid mutations (V42I, D44E, E53D) was enhanced compared with that of the pseudovirus HN, and the numerical difference was extremely significant; and the fusion ability of the pseudovirus HN with three-point mutations (V42I, D44E, E53D) was stronger than that of the pseudovirus HN with two-site amino acids (V42I and D44E), HN (V42I, E53D) and HN (D44E, E53D).

[0275] It can be seen from this that the simultaneous mutation of the three amino acids at positions 42, 44 and 53 can significantly improve the pseudovirus fusion ability, which may also be the reason for the significant increase in the titer of HN (V42I, D44E, E53D).

[0276] comprehensive Figure 3 From the experimental data results of A, B, and C, it can be seen that when the amino acids at positions 42, 44, and 53 are mutated simultaneously, the adsorption ability of the pseudovirus HN (V42I, D44E, E53D) is not affected, and the fusion ability with the host cells is significantly improved, thereby significantly increasing the viral titer of HN (V42I, D44E, E53D).

[0277] Based on the experimental results of 2.6-2.8, it can be determined that amino acids 42, 44, and 53 in the HN hemagglutinin HA are critical sites. By mutating the amino acids at these three sites, high-titer HN virus can be obtained. Therefore, the present invention constructed a mutant recombinant virus HN (V42I, D44E, E53D) and tested the virus titer to verify the results.

[0278] 2.9 Construction of A / blackbird / Hunan / 1 / 2004 mutant recombinant virus

[0279] According to the methods described in 2.1-2.4, a mutant recombinant virus HN-424453 was constructed. Based on the hemagglutinin amino acid sequence of the A / blackbird / Hunan / 1 / 2004 virus strain, the amino acids at positions 42, 44, and 53 of the hemagglutinin amino acid sequence of the A / blackbird / Hunan / 1 / 2004 virus strain were mutated as follows: V42I, D44E, and E53D, respectively. This constructed the hemagglutinin amino acid sequence of the recombinant virus HN-424453, and a plasmid was synthesized to prepare the recombinant virus. The hemagglutinin amino acid sequence of the recombinant virus HN-424453 is shown below:

[0280] .

[0281] The results of hemagglutination titer test on the mutant recombinant virus showed that ( Figure 5 shown):

[0282] 1) The hemagglutination titer of the mutant recombinant virus (HN-424453) obtained by amplification in MDCK.1 cells was higher than that of the recombinant virus HN, and the difference in values ​​was significant (e.g. Figure 5 (as shown in A in the figure).

[0283] 2) The hemagglutination titer of the recombinant virus (HN-424453) obtained by chicken embryo amplification was higher than that of the recombinant virus HA, and the difference in values ​​was significant (e.g. Figure 5 (as shown in B in the figure).

[0284] In summary, the present invention increases the titer of A / blackbird / Hunan / 1 / 2004 virus by mutating the amino acids at three key sites of A / blackbird / Hunan / 1 / 2004 hemagglutinin.

[0285] 2.10 Comparison of immunogenicity of A / blackbird / Hunan / 1 / 2004 recombinant virus and mutant recombinant virus

[0286] Recombinant virus HN and mutant recombinant virus HN-424453 were amplified in MDCK.1 cells, and the cell supernatant was collected and concentrated by ultracentrifugation at 25,000 rpm. The supernatant was purified by sucrose gradient centrifugation, inactivated with β-propiolactone, and then dialyzed. The inactivated virus HA protein was quantified by ELISA kit. The quantified inactivated virus was emulsified with MF59 adjuvant and stored at 4°C until use.

[0287] Twelve 6-8 week old Balb / c mice were randomly divided into two groups of six. One group received the inactivated HN vaccine, while the other group received the inactivated HN-M vaccine. Both groups received two immunizations, each receiving 10 μg of the emulsified immunogen via intramuscular injection. Fourteen days after the second immunization, the immune serum was collected, inactivated at 56°C, centrifuged, aliquoted, and stored at -80°C until further use.

[0288] MDCK.1 cells were plated one day in advance, and the immune serum was diluted doubly and incubated with pseudovirus at 37°C. After 60 minutes, it was added to the MDCK cells and cultured. After 60 hours, RLA was detected and fitted using Graphpad software to draw a sigma curve and calculate the neutralization titer of the immune serum.

[0289] The neutralization titer of immune serum against HN and HN mutant pseudovirus was tested by PN experiment, and VSV pseudovirus was used as negative control. Figure 6 As shown in the figure, the immune serum had no neutralizing activity against VSVG pseudovirus, but had good neutralizing activity against HN and HN mutant viruses; there was no significant difference in the PN titer of HN inactivated virus immune serum against HN and HN mutant pseudoviruses ( Figure 5A). Similarly, there was no significant difference in the PN titer of immune serum between HN mutant inactivated virus and HN and HN mutant pseudovirus ( Figure 5 B) This indicates that the immunogenicity of the HN and HN mutant recombinant viruses did not change significantly.

[0290] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A method for transforming a low-titer H5 subtype influenza virus into a high-titer influenza virus, characterized in that: include: (a) providing a constructed H5 subtype high-titer influenza virus library and an H5 subtype low-titer influenza virus strain to be modified, and obtaining a genetic evolutionary tree based on the HA amino acid sequences of the H5 subtype high-titer influenza virus in the virus library and the H5 subtype low-titer influenza virus; (b) obtaining, through the genetic evolutionary tree, an H5 subtype high-titer influenza virus strain that is closest in genetic distance to the H5 subtype low-titer influenza virus strain; (c) exchanging the HA or NA protein of the H5 subtype high-titer influenza virus strain and the H5 subtype low-titer influenza virus obtained in step (b) to prepare a chimeric pseudovirus, and detecting the titer of the chimeric pseudovirus to determine whether the protein affecting the influenza virus titer is HA or NA; Among them, if the viral titer is significantly increased after the HA of the low-titer virus strain is replaced with the HA of the high-titer virus strain, and the viral titer does not change significantly after the NA of the low-titer virus strain is replaced with the NA of the high-titer virus strain, it can be determined that the key amino acid affecting the low-titer virus strain is not located in NA but in HA; or If the viral titer does not change after the HA of the low-titer virus strain is replaced with the HA of the high-titer virus strain, but the viral titer increases significantly after the NA of the low-titer virus strain is replaced with the NA of the high-titer virus strain, it can be determined that the key amino acid affecting the low-titer virus strain is not located in HA but in NA; or If the viral titer is significantly increased after the HA of the low-titer virus strain is replaced with the HA of the high-titer virus strain, and the viral titer is also significantly increased after the NA of the low-titer virus strain is replaced with the NA of the high-titer virus strain, it can be determined that the key amino acids affecting the low-titer virus strain are located in both HA and NA; (d) comparing the amino acid differences in HA or NA between the H5 subtype high-titer influenza virus strain obtained in step (b) and the H5 subtype low-titer influenza virus of step (a); (e) based on the results of steps (c) and (d), single-point mutations are performed on all amino acids in the HA or NA of the H5 subtype high-titer influenza virus obtained in step (b) that differ from those in the HA or NA of the H5 subtype low-titer influenza virus strain described in step (a), thereby preparing HA or NA mutant influenza pseudoviruses; (f) respectively detecting the titer of the HA or NA mutant influenza pseudovirus obtained in step (e) and the pseudovirus of the H5 subtype high-titer influenza virus strain obtained in step (b), thereby determining the amino acid position information that may affect the H5 subtype low-titer virus strain; (g) performing single-site amino acid mutations on the key amino acids in the HA or NA of the H5 subtype low-titer influenza virus to be modified according to the amino acid sites obtained in step (f) to prepare pseudoviruses, and determining amino acid sites that may increase viral titer by titer detection; further performing double-site or multi-site amino acid mutations, and performing pseudovirus titer detection to determine the key amino acids that affect the H5 subtype low-titer influenza virus; (h) constructing a plasmid based on the key amino acid sites determined in step (g), preparing an HA mutant influenza virus by reverse genetics, and verifying the confirmed key amino acid sites by titer detection, thereby obtaining an H5 subtype high-titer influenza virus; as well as (i) detecting the adsorption capacity and fusion capacity of the H5 subtype high-titer influenza virus obtained in step (h); and / or (j) testing the immunogenicity of the H5 subtype high-titer influenza virus obtained in step (h), thereby obtaining an H5 subtype high-titer influenza virus with little or no immunogenicity.

2. The method according to claim 1, wherein The H5 subtype high-titer influenza virus library is constructed using the following method: based on the influenza virus HA sequence provided by GISAID, potential H5 subtype high-titer influenza virus strains are screened using bioinformatics methods, and influenza pseudoviruses are prepared using a 4-plasmid expression system with the HA and NA of the influenza virus as envelope proteins and firefly luciferin as internal nucleic acid packaging. High-titer H5 subtype influenza virus strains are screened by relative luciferase activity detection, thereby constructing a high-titer H5 subtype influenza virus strain library.

3. The method according to claim 2, wherein The bioinformatics method includes sequence comparison and analysis.

4. The method according to claim 1, wherein The method further comprises a step of detecting the virus titer of the reference H5 subtype low-titer influenza virus.

5. The method according to claim 4, wherein The reference H5 subtype low-titer influenza virus is A / WSN / 33.

6. The method according to claim 1, wherein The amino acid mutated in the H5 subtype low-titer influenza virus strain is an amino acid at the same position in the high-titer H5 subtype influenza virus strain that is closest to the low-titer H5 subtype influenza virus strain in genetic evolution tree.

7. The method according to claim 1, wherein The H5 subtype high-titer influenza virus strain with the closest genetic distance refers to a virus strain that is located at the same node and the same evolutionary branch as the H5 subtype low-titer influenza virus strain on the genetic evolutionary tree.

8. The method according to claim 2, wherein The H5 subtype high-titer influenza virus refers to a pseudovirus whose relative luciferase activity value is significantly higher than 1000.

9. The method according to claim 2, wherein The H5 subtype low-titer influenza virus refers to a pseudovirus whose relative luciferase activity value is significantly lower than 500.

10. The method according to claim 1, wherein The H5 subtype high titer influenza virus strains are as follows: 。 11. The method according to claim 1, wherein The H5 subtype low-titer influenza virus includes A / blackbird / Hunan / 1 / 2004.

12. The method according to claim 1, wherein The titer of HA and NA chimeric viruses, or viruses with single-point or multi-point mutations in HA or NA, is determined by expressing the reporter gene firefly luciferase in the host cells after the virus infects the host cells. After the host cells are lysed, the virus titer is determined by reacting with a firefly luciferase substrate and buffer solution, and detecting the luminescence value of the luciferase at 560 nm.

13. An H5 subtype high titer influenza virus, characterized in that The method according to claim 1 is used to prepare the H5 subtype high-titer influenza virus, and the H5 subtype high-titer influenza virus is a hemagglutinin mutant of the H5N1 subtype low-titer influenza virus strain A / blackbird / Hunan / 1 / 2004, wherein the mutation is a three-point amino acid mutation at positions 42, 44 and 53.

14. The H5 subtype high-titer influenza virus according to claim 13, wherein The mutations are three-site amino acid mutations of V42I, D44E and E53D.

15. A pharmaceutical composition, characterized in that The composition contains the H5 subtype high-titer influenza virus according to claim 13, and a pharmaceutically acceptable carrier and / or excipient.

16. A vaccine composition, characterized in that The composition contains the H5 subtype high-titer influenza virus according to claim 13, and an immunologically acceptable carrier and / or adjuvant.

17. Use of the high-titer influenza virus according to claim 13, the pharmaceutical composition according to claim 15, or the vaccine composition according to claim 16, characterized in that: (a) for preparing antibodies against HA or NA of H5 subtype influenza virus; and / or (b) for preparing drugs for preventing and / or treating H5 subtype influenza virus infection or related diseases.

18. The use according to claim 17, characterized in that The H5 subtype influenza virus infection or related diseases thereof are selected from the group consisting of fever, headache, runny nose, sneezing, loss of smell, cough, chills, sore throat, body aches or muscle pains, or a combination thereof.

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