TWI620819B - Novel vaccines against pandemic influenza virus a/h1n1 - Google Patents

Abstract

The present invention relates to an influenza virus vaccine, and specifically to an influenza virus, which has at least its hemagglutinin gene containing a D222 mutation and an oseltamivir resistant neuraminidase gene. In some embodiments, the influenza virus vaccine further comprises a matrix protein that contains mutations that increase the specific immune response in humans. The virus can be used to produce vaccines against hypervirulence and oseltamivir-resistant influenza.

Description

Novel vaccine against pandemic influenza virus A / H1N1

The present invention relates to an anti-influenza virus vaccine, and specifically to a vaccine against a hypervirulent strain of pandemic A / H1N1 influenza virus. The present invention provides influenza virus strains that can be used to manufacture influenza vaccines carrying specific mutations in the HA and NA segments.

This application claims priority over US Provisional Patent Application No. 61,409 / 303 filed on November 2, 2010. The full text of this provisional application is incorporated herein by reference.

Influenza is caused by RNA viruses of the orthomyxoviridae family. There are three types of these viruses, and they cause three different types of influenza: type A, type B, and type C. Influenza A virus infects mammals (humans, pigs, ferrets, horses) and birds. This is extremely important for humans, because this type of virus has caused a pandemic worldwide. Influenza B virus (also referred to as influenza B) only infects humans. It sometimes causes local outbreaks of flu. Influenza C virus only infects humans. It has the largest number of early infections and rarely causes serious illness.

Influenza A virus infects many species of mammals, including humans, horses, pigs, ferrets and birds. The main human pathogens are related to epidemics and pandemics. There are at least 15 known hemagglutinin (H) serotypes and 9 known neuraminidase (N) serotypes. The industry believes that pigs and birds are particularly important reservoirs, which generate genetically and antigenically different pools of viruses that are transferred back to the population through close contact between humans and animals. Influenza B virus only infects mammals and causes disease, but is generally not as severe as type A. Unlike influenza A virus, influenza B virus does not have a discernable serotype. Influenza C virus only infects mammals, but rarely causes disease. It is genetically and morphologically different from type A and type B.

Unlike many other viruses, the influenza genome consists of RNA rather than DNA. The genome contains 8 segments and only the virus particles containing all 8 segments are viable. (Steinhauer DA, Skehel JJ., Genetics of influenza viruses. Ann Rev Genet. 2002 36: 305-332).

There are four kinds of antigens in influenza virus: hemagglutinin (HA), neuraminidase (NA), nucleocapsid (NA), matrix (M) and nucleocapsid protein (NP). NP is a type-specific antigen and exists in three forms A, B, and C, which provide the basis for classification of human influenza virus. The matrix protein (M protein) surrounds the core shell and accounts for 35-45% of the mass of the particles. On the surface, two surface glycoproteins were observed as rod-shaped protrusions. Hemagglutinin (HA) consists of two subunits HA1 and HA2. HA mediates virus attachment to cellular receptors. Neuraminidase (NA) molecules are present in lesser amounts in the mantle. Circulating human strains are widely known for accumulating mutations year after year and causing epidemics to recur.

Influenza virus is one of the very few genomes whose genomes are located in separate segments (8). The segmentation of the genome also makes it possible to exchange complete genes between different virus strains, thereby increasing the possibility of forming recombinants (if two different viruses infect the same cell, they can be formed by swapping gene segments). That is, if a cell is infected with two different influenza strains, each segment can be redistributed to produce a new virus with a segment from each original virus. In nature, this can help to quickly generate new influenza strains. A new genome can be assembled by mixing between the two viruses. This process can also be replicated in the laboratory (used to make vaccine strains). In the Far East, poultry and human strains recombined in pigs may allow the evolution of virulent human strains.

The genetic plasticity of influenza viruses is also likely related to vaccine design, pathogenicity, and the ability of new viruses to emerge from natural reservoirs and cause pandemic diseases worldwide.

The pandemic (H1N1) 2009 virus has evolved around the world, and it has become dominant during the pandemic process from the initial mixed branching pattern to a branch (branch 7). Therefore, the virus that constitutes this branch can be responsible for most of the pandemic outbreaks worldwide. The mutation S203T in the hemagglutinin (HA) protein sequence is unique to the branch 7 isolate. (Valli MB et al., Evolutionary pattern of pandemic influenza (H1N1) 2009 virus in the late phases of the 2009 pandemic. PLoS Curr Influenza. March 3, 2010: RRN1149).

In the branch 7 isolate containing the S203T mutation, the amino acid change corresponding to position 222 (position 225 in H3 numbering) in the hemagglutinin HA1 gene sequence (where aspartic acid (D) becomes glycine) (G)) is related to serious clinical outcomes. At different time points from April to November 2009, the D222G mutation (Chen GW, Shih SR (2009) Genomic signatures of influenza A) was recorded in the H1N1 pdm global isolate in the GenBank. pandemic (H1N1) 2009 virus. Emerg Infect Dis).

Influenza vaccine is an anniversary vaccine intended to protect against highly variable influenza viruses. Each seasonal influenza vaccine injected contains three influenza viruses: an A (H3N2) virus, the 2009 pandemic H1N1 virus, and an influenza B virus. (www.cdc.gov/vaccines/pubs/vis/downloads/vis-flu.pdf). However, the pandemic influenza vaccine is only weakly effective against influenza H1N1 virus carrying the D222G mutation in the HA protein.

Therefore, there is an urgent need in the industry for virus strains and virus-derived antigens that generate robust antibody titers against the D222G variant of H1N1.

The present invention relates to a method and composition for preparing a vaccine against a hypervirulent form of influenza A H1N1 virus and / or an oseltamivir resistant form. Vaccine compositions containing mutations in residues D222 of HA and S275 of NA are provided.

The present invention provides an influenza virus vaccine, which contains an influenza virus having at least the following: hemagglutinin (HA) gene segment, which contains a virus 1 isolated from influenza A H1 / N1 influenza virus Amino acid sequence SEQ ID NO: 34; and neuraminidase (NA) gene segment, which contains the amino acid sequence of the virus 1 isolate derived from influenza A H1 / N1 influenza virus SEQ ID NO: 39.

In one embodiment, the HA gene segment comprises the amino acid sequence encoded by the nucleotide sequence of segment 4 of virus 1 (SEQ ID NO: 4). In some forms, the HA gene segment contains the influenza A H1 / N1 influenza virus branching 7 specific HA sequence.

In one embodiment, the HA gene segment contains a mutation at residue D222 in the wild-type influenza A H1 / N1 influenza virus. In one embodiment, the HA gene segment contains the D222E mutation. In some embodiments, the HA gene segment contains the S203T mutation relative to the wild-type influenza A H1 / N1 influenza virus.

The present invention relates to a vaccine, wherein the HA gene segment further includes one or more of P83S and I321V mutations.

In one embodiment, the NA gene segment comprises the amino acid sequence encoded by the nucleotide sequence of segment 6 in virus 1 (SEQ ID NO: 6). The NA gene segment contains the Y275H mutation relative to the wild-type influenza A H1 / N1 influenza virus.

The present invention relates to a vaccine, wherein the NA gene segment further contains one or more of I106V mutation and D248N mutation.

The vaccine of the present invention may further comprise: c) a matrix (MA) gene segment encoding the amino acid sequence of SEQ ID NO: 50 comprising an isolate of virus 1 derived from influenza A H1 / N1 influenza virus M1 peptide.

In one embodiment, the MA gene segment comprises the amino acid sequence encoded by the nucleotide sequence of segment 7 in virus 1 (SEQ ID NO: 7).

In some embodiments, the matrix (MA) gene segment encodes an M2 peptide comprising the amino acid sequence SEQ ID NO: 51 of the virus 1 isolate derived from influenza A H1 / N1 influenza virus. In some embodiments, the M1 peptide comprises the A198P mutation.

The present invention relates to a method of immunizing a patient against influenza virus, which includes the step of administering the influenza vaccine disclosed herein to the patient.

The present invention relates to a method for immunizing a patient against a hypervirulent influenza virus, which includes the step of administering the influenza vaccine disclosed herein to the patient.

The present invention relates to a method for immunizing a patient against oseltamivir-resistant influenza virus, which includes the step of administering the influenza vaccine disclosed herein to the patient.

The present invention relates to the vaccine disclosed herein, wherein the vaccine is a redistribution vaccine. In some embodiments, redistribution influenza viruses are obtained by traditional redistribution.

The present invention relates to the vaccines disclosed herein, which are live attenuated vaccines or inactivated vaccines. In some embodiments, the vaccine is formulated for oral or intranasal administration.

The present invention relates to a method for preparing a vaccine for immunizing an individual against a hyperviral influenza virus strain, which includes the steps of mixing the influenza virus disclosed herein with a carrier and an optional adjuvant as needed.

These and other aspects can be understood from the following description of the preferred embodiments in conjunction with the following drawings, but changes and modifications can be implemented therein without departing from the spirit and scope of the novel concepts of the present disclosure.

The following drawings form part of this specification and are included to further demonstrate certain aspects of the disclosure. Refer to one or more of these drawings and the detailed description of specific embodiments described herein for a more thorough understanding this invention. This patent or application file contains at least one color scheme. After requesting and paying the necessary fees, the Patent Office (Office) will provide a copy of this patent or patent application publication with color drawings.

In the context of the present invention and in the specific context in which each term is used, the terms used in this specification generally have ordinary meanings in the industry. Some terms used to illustrate the present invention are discussed below or elsewhere in the specification, which provides additional guidance for practitioners concerned with the description of the present invention. For convenience, certain terms may be highlighted using italics and / or quotation marks, for example. The use of highlighting has no effect on the scope and meaning of the term; in the same context, regardless of whether it is highlighted, the scope and meaning of the term are the same. It should be understood that the same object can be described in more than one way.

Therefore, alternative language and synonyms can be used for any one or more of the terms discussed herein, whether or not the terms are elaborated or discussed in this document in any way. Provide synonyms for certain terms. The listing of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification (including examples of any term discussed herein) is illustrative only, and in no way limits the scope and meaning of the invention or of any term exemplified. Likewise, the present invention is not limited to the various embodiments given in this specification.

Unless otherwise stated, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention belongs. In case of conflicts, this document (including definitions) shall prevail. The following references provide technicians with a general definition of many terms used in the present invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1993); The Cambridge Dictionary of Science and Technology (Edited by Walker, Cambridge University Press. 1990); The Glossary of Genetics, 5th Edition, R. Rieger et al. (Editor), Springer Verlag (1991); and Hale & Margham, The Harper Collins Dictionary of Biology (1991).

In general, the terms and techniques used in connection with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are well known and commonly used in the industry. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (eg, electroporation, lipofection). Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions or as commonly used in the industry or as described herein. Unless explicitly indicated to the contrary, the practice of the present invention will employ conventional methods of virology, immunology, microbiology, molecular biology, and recombinant DNA technology well known in the industry, many of which are described below for illustrative purposes. These techniques are fully explained in the literature. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual (Second Edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, Volume I and Volume II ( (Edited by D. Glover); Oligonucleotide Synthesis (Edited by N. Gait, 1984); Nucleic Acid Hybridization (Edited by B. Hames and S. Higgins, 1985); Transcription and Translation (Edited by B. Hames and S. Higgins, 1984); Animal Cell Culture (edited by R. Freshney, 1986); Perbal, A Practical Guide to Molecular Cloning (1984).

Influenza A virus contains a genome consisting of 8 individual segments of negative sense RNA as shown in Table 1 below. (Steinhauer DA, Skehel JJ., Genetics of influenza viruses. Ann Rev Genet. 2002 36: 305-332). Circulating human strains are widely known for accumulating mutations year after year and causing epidemics. However, the genome is segmented so that different virus strains can exchange complete genes. The genetic plasticity of influenza viruses is also likely related to vaccine design, pathogenicity, and the ability of new viruses to emerge from natural reservoirs and cause pandemic diseases worldwide.

A total of 950 viruses were isolated by cell culture, and 193 neuraminidase (NA) and 197 hemagglutinin (HA) partial gene sequences were obtained from these viruses via capillary sequencing. Because HA and NA are the most variable sequences in AH1N1, computer simulation analysis (genealogy analysis and BLAST) shows that all 386 (HA and NA sequences) can be divided into 20 clusters; In 454 Titanium Roche Pyrosequencing equipment (ROCHE), pyrosequencing was performed on the complete genome of a representative member in each cluster. A total of 20 complete AH1N1 genomes were obtained, each AH1N1 consisting of 8 genes.

Sequencing data showed four virus isolates with three different mutations: (i) pAH1N1 virus, which has mutations in NA (oseltamivir ^R ), HA (supervirulence) and matrix segments (virus 1); ii) pAH1N1 virus, with mutations in the NA and matrix segments (virus 2); (iii) pAH1N1 virus, with mutations in the matrix (MA) segment (virus 3); and (iv) pAH1N1 virus, which There is a mutation in the neuraminidase (NA) segment (virus 4).

Changes in hemagglutinin HA1 gene sequence at amino acids corresponding to position 222 (position 225 in H3 numbering) (where aspartic acid (D) becomes glycine (G)) are associated with serious clinical outcomes . At different time points between April and November 2009, the D222G mutation was recorded in the H1N1pdm global isolate in the gene pool (Chen GW, Shih SR (2009) Genomic signatures of influenza A pandemic (H1N1) 2009 virus. Emerg Infect Dis).

The preferential binding of influenza virus to sialic acid-α2,3-galactose (α2,3 receptor) or sialic acid-α2,6-galactose (α2,6 receptor) can determine its tropism, because α2,3 receptors and α2,6 receptors dominate the lower and upper respiratory tract cells (Shinya K, Ebina M, Yamada S, Ono M, Kasai N, Kawaoka Y. Avian flu: influenza receptors in the human airway. Nature. 2006; 440 (7083): 435-6).

The latest glycan microarray analysis shows that the replacement of hemagglutinin (HA) D222G may cause a change from α2,6 receptor specificity to mixed α2,3 / α2,6 receptor specificity, which can enhance the relationship with α2,3 Integration of the body and increase the severity of the disease. (Stevens J, Blixt O, Glaser L, Taubenberger JK, PaleseP, Paulson JC and others, Glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities. J Mol Biol. 2006; 355 (5): 1143- 55). This position seems to affect receptor binding specificity, and the D / G difference between the two 1918 virus variants (same as above) is related to the priority of linking sialic acid from α2-6 to dual α2-3 / α2-6 specific The change is related, that is, it may make the virus more susceptible to infection of the airways deeper (where the cells expressing α2-3 receptors are more abundant), thereby producing hypervirulence.

However, a unique pedigree cluster of severe cases with D222G substitution has not been observed (Mak GC et al., Association of D222G substitution in hemagglutinin of 2009 pandemic influenza A (H1N1) with severe disease. Eurosurveillance, Vol. 15, Issue 14, (April 8, 2010).

All viruses found to contain changes at position 222 in HA1 belong to the subbranches of the pandemic H1N1 virus characterized by HA1 replacing S203T (branch 7 feature). The acid bag is exposed to the antigenic region unique to this hypervirulent strain and the remaining AH1N1 strains. This subbranch accounts for most of the pandemic viruses in Europe and the rest of the Northern Hemisphere. For the southern hemisphere, this subbranche is also widely distributed.

Based on the currently available data shared with WHO, the prevalence of D222G replacement is less than 1.8% (52 detected in more than 2755 HA sequences). Of the 364 lethal cases analyzed so far, the virus in 26 cases (7.1%) has been replaced by D222G. There is limited clinical information about the possible underlying medical conditions in these cases. The pandemic (H1N1) 2009 virus with D222G substitution is similar in antigenicity to the A / California / 7/2009 (H1N1) virus (a vaccine virus recommended by WHO).

In the same virus A (H1N1) 2009 above, (i) Gly222 mutation in hemagglutinin (HA) and (ii) Tyr275 in neuraminidase (NA) related to resistance to oseltamivir Mutation (McKimm-Breschkin JL. Resistance of influenza viruses to neuraminidase inhibitors-a review. Antiviral Res 2000; 47: 1-17; Collins PJ et al., Structural basis for oseltamivir resistance of influenza viruses. Vaccine 27 (2009) 6317-6323 ). Since both mutations are related to important phenotypes (supervirulence and oseltamivir resistance), and the antigenic regions are related to their mutations, the vaccine candidate of the present invention can effectively recognize the mutant strain and AH1N1 wild type II By.

The three D222G variant viruses contained H275Y substitutions related to oseltamivir resistance in neuraminidase (NA). WHO Report. Preliminary review of D222G amino acid substitution in the hemagglutinin of pandemic influenza A (H1N1) 2009 viruses, December 28, 2009 (www.who.int/csr/resources/publications/swineflu/cp165_2009_2812_review_d222g_amino_acid_substitution_in_ha_h1n1_viruses.pdf).

The currently available pandemic vaccine also recognizes hypervirulent and oseltamivir resistant mutants. However, although the mutation is located in the receptor binding cavity of H1 hemagglutinin rather than an antigenic site, it is controlled in infectious diseases in Sweden The preliminary antigen characterization carried out by the Swedish Institute for Infectious Disease Control and in our laboratory showed that the reactivity of the mutant virus with the antiserum against the current pandemic vaccine compared to the wild-type pandemic AH1N1 strain Significantly reduced. Therefore, the current vaccines are not as effective in identifying these new mutant strains that spread rapidly around the world as vaccine candidates of the present invention.

The mutation in the 3 'end of the viral genome segment 7 (matrix segment) showed similarity to the influenza BM segment (60%). It has been previously reported that this mutation can increase the specific immune response in humans, and it can also confer protection against influenza B. However, cell-mediated immunity that is primarily resistant to viral matrix and nuclear protein antigens cannot protect against infection, but it is the key to virus clearance and recovery from disease.

The amino acid sequences of the four virus isolates were deduced from nucleotide sequence determination. Virus 1 contains mutations of interest in HA, NA, and MA proteins. Virus 2 contains mutations of interest in NA and MA proteins. Virus 3 carries the mutation of interest in the NA protein. Virus 4 contains the mutation of interest in the MA protein.

The mutations of interest in the HA sequence were analyzed relative to the wild-type influenza A H1N1 sequence. All four isolates carry mutations P83S and I321V, and these mutations appear to be prevalent in branch 7 of the A / H1N1 pandemic strain.

Virus 1 also has (i) the S203T mutation characteristic of branch 7, and (ii) the D222E mutation at position 222 of the HA sequence. At positions 222-223, virus 1 has E-R amino acids instead of wild-type D-Q. In HA protein of influenza A virus (A / Novgorod / 01/2009 (H1N1)), an exact match to the HA sequence of virus 1 was found.

Viruses 1, 2 and 4 contained mutations at residues I106V, D248N and Y275H in the NA segment (6) relative to the wild-type A / California / 07/2009 sequence. The Y275H mutation is associated with oseltamivir resistance. The exact match of the NA sequences found in these three isolates was also found in other isolates.

Current wild-type vaccines are less effective against virus 1 carrying both HA and NA mutations at these specific sites. (Puzelli et al., Transmission of Hemagglutinin D222G mutant strain of pandemic (H1N1) 2009 virus. Emerging Infectious Diseases 16 (5): 863-865 (2010)). Vaccines generated from viruses (e.g. virus 1) containing both hypervirulence (D222G / E in the HA area) and oseltamivir resistance (Y275H in the NA area) Inefficient and provides protection against both hypervirulence and oseltamivir resistant viruses.

Viruses 1, 2 and 3 carry the mutation A198P in the M1 region (segment 7) relative to the wild-type A / California / 07/2009 sequence. In addition, these three isolates lack residues corresponding to D34 in the M2 region.

This mutation has been reported to increase cell-mediated immunity (but does not protect against infection). The mutation region has 60% sequence similarity to the influenza B virus MA region and a vaccine generated against a virus isolate carrying this mutation can develop immunity against influenza B.

In addition, M1 mutations can be used as therapeutic targets for virus clearance and recovery.

The method of the present invention uses the influenza virus antigen disclosed herein to immunize against influenza virus infection. The specific virus from which the antigen is derived may be the same as or different from the specific virus that provides protection against it, because cross-protection is known to exist between different isolates within influenza virus, especially the same virus subtype.

The currently used influenza vaccines are named whole virus (WV) vaccines or subviral particles (SV) (also known as "lysed" or "purified surface antigens"). The WV vaccine contains intact non-activated virus, while the SV vaccine contains purified virus split by a cleaning agent that dissolves the lipid-containing virus coat and then chemically deactivates the remaining virus. Attenuated virus vaccines against influenza are also under development. For a discussion of the preparation methods of conventional vaccines, see Wright, PF and Webster, RG., FIELDS VIROLOGY, 4th edition (edited by Knipe, DM, etc.), 1464-65 (2001).

If the vaccine includes more than one influenza strain, the different strains are usually grown separately and mixed after the virus has been harvested and the antigen has been prepared. Alternatively, different segments of different isolates of influenza virus can be combined to produce a pluripotent vaccine. The influenza virus used in the method of the present invention can be a redistributive strain, and / or can be obtained by reverse genetics techniques. The virus can be an attenuated virus. The virus can be a temperature-sensitive virus. The virus can be a cold virus. Redistributive strains including HA and / or NA virus segments from pathogenic strains and the remaining 6 or 7 segments from non-pathogenic strains can be used.

The influenza virus antigen used in the immunogenic composition of the present invention may be in the form of live virus or preferably inactivated virus. Viral inactivation usually involves treatment with chemicals such as formalin or β-propiolactone. If inactivated virus is used, the antigen can be whole virus, split virus or virus subunit. Virus lysis is achieved by using detergents (e.g. diethyl ether, polysorbate 80, deoxycholate, tri-N-butyl phosphate, Triton X-100, Triton N101, cetyltrimethyl bromide (Ammonium, etc.) obtained by treating viral particles to produce a subviral particle preparation. The subunit vaccine contains one or both of the influenza surface antigen hemagglutinin and neuraminidase. Influenza antigens can also be presented in the form of virions.

If the antigen is prepared from influenza virus (ie not produced in a recombinant or synthetic system that does not involve influenza virus growth), the virus can grow on eggs or in cell culture. Growing in embryonated eggs without specific pathogens is a traditional way to grow influenza virus for vaccine production, and cell culture is a new development method. If cell culture is used, the influenza virus vaccine will usually grow on mammalian cells (such as MDCK cells, VERO cells, or PER.C6 cells). Such cell lines are available everywhere, for example from the American Type Cell Culture (ATCC) collection, or from Coriell Cell Repositories. For example, ATCC supplies various VERO cells under catalog numbers CCL-81, CCL-81.2, CRL-1586 and CRL-1587, and it supplies MDCK cells under catalog numbers CCL-34. It can also be grown on avian cell lines, including cell lines derived from hens, such as chicken embryo fibroblasts (CEF).

The immunogenicity and pharmaceutical composition of the present invention are suitable for administration to patients. This can be achieved in a variety of ways, including (but not limited to): intradermal injection; transdermal administration; and local administration. These methods can be used in conjunction with, for example, skin abrasions by emery paper or by using micro-abrasives.

The immunogenicity and pharmaceutical composition of the present invention preferably exist in the form of a vaccine.

The composition of the present invention may include an adjuvant. Adjuvants used in influenza vaccines include aluminum salts, chitosan, CpG oligodeoxynucleotides (e.g. CpG 7909), oil-in-water emulsions (e.g. MF59), water-in-oil emulsions, E. coli ( E.coli ) Heat-labile toxin and its detoxified mutant, monophosphoryl lipid A and its 3- O -deamidated derivative, pertussis toxin mutant, cell wall acetyl dipeptide, etc.

Hemagglutinin (HA) is the main immunogen in inactivated influenza vaccine, and the vaccine dose is standardized with reference to HA content, usually measured by one-way radiation immunodiffusion (SRID) analysis. Vaccines for intramuscular injection usually contain about 15 μg HA / strain, but lower doses can also be used (eg for children, or in a pandemic situation) and divided doses have been used, such as 1/2 (ie 7.5 μg HA / strain), 1/4 and 1/8 doses. The composition of the present invention will generally comprise between 0.1 μg and 8 μg of HA per influenza strain, preferably for example about 7.5, about 5, about 3, about 2.5, about 2, about 1.5, about 1, about 0.75 , About 0.5, about 0.4, about 0.2 μg, etc.

Vaccines for intramuscular injection usually have a volume of 0.5 ml. The composition may include a preservative, such as thiomersal or 2-phenoxyethanol. However, the composition should preferably be substantially free (ie, less than 1 μg / ml) of mercury-containing materials, such as thimerosal. A mercury-free vaccine is better.

If influenza virus has grown on cell culture, the composition of the present invention preferably contains less than 100 pg residual host cell DNA / dose, but trace amounts of host cell DNA may be present. Standard purification procedures such as chromatography can be used to remove contaminating DNA during vaccine preparation. Residual host cell DNA can be removed by nuclease treatment (for example by using Benzonase DNase) to enhance. Vaccines containing <100 pg host cell DNA / 10 μg hemagglutinin are preferred, and vaccines containing <100 pg host cell DNA / 0.25 ml volume are also preferred. Vaccines containing <100 pg host cell DNA / 50 μg hemagglutinin are better, and vaccines containing <100 pg host cell DNA / ml volume are also better.

The vaccine of the present invention can be delivered according to a single dose immunization schedule. Alternatively, it can be delivered as the priming element of the priming-boosting regimen (meaning that after the first immunization, a second injection of similar antigenicity within weeks or months).

Methods for testing the immunogenicity of influenza vaccines are well known in the industry. One method involves the following procedures: (a) Immediately before vaccination, a 10 ml venous blood sample is taken from the patient (usually from the arm) for baseline titration of circulating anti-HA antibodies; (b) After that, the patient immediately receives a dose of vaccine If the vaccine is administered to the arm, it should be given to the opposite arm of the blood-drawing arm; (c) A blood sample of 10 ml should be taken from the patient approximately 3 weeks after vaccination. Separate the serum from the blood sample and store (if necessary) at -20 ° C. The anti-hemagglutinin antibodies against the relevant strains of the serum were analyzed by hemagglutination inhibition (HI) or unidirectional radiation hemolysis (SRH). Positive and negative sera and reference preparations can be obtained from public reference laboratories. Antibody titration was performed in duplicate, and the pre-vaccination serum and post-vaccination serum were titrated simultaneously. The titration value assigned to each sample is the geometric mean of two independent determinations (but for calculation purposes, under standard conditions, any HI results <10 (= undetectable) are expressed as 5 and any negative SRH results are expressed as 4 mm ² ).

In the HI test, the serum conversion rate corresponds to The pre-immunization and post-immunization titer ratios of 40 and the antibody titer increased significantly (eg at least 4 times). In the SRH test, the seroconversion rate corresponds to The area of 25 MM ² after vaccination and at least 50% of the area before vaccination. Examples

Without wishing to limit the scope of the present invention, the following gives example instruments, devices, methods and related results of embodiments of the present invention. Note that for the convenience of the reader, the title or subtitle can be used in the examples, which should in no way limit the scope of the present invention. In addition, certain theories are proposed and disclosed herein; however, regardless of whether they are correct or incorrect, they should in no way limit the scope of the present invention, as long as the present invention is practiced according to the present invention, without considering any specific theory or scheme of action.

Example 1: Cell line

The VERO cell line is a well-studied cell line that was first isolated from the African green monkey kidney ( Cercopithecus aethiops ) and has been widely used in microbiology, cell biology, and virology research. It has been approved for the production of viral vaccines (rotavirus and polivirus) in the United States, it has also been tested and in some cases approved for the fight against rabies (Rabies), Rio Production of vaccines for reovirus and Japanese encephalitis. This cell line was chosen because it is susceptible to co-infection and it can grow on a commercial scale in a fermentor or bioreactor (this is a desirable feature of vaccine production).

The VERO cell line used was purchased from ATCC, and maintained at -70 ° C with dimethyl sulfoxide (DMSO). Thaw cells according to CORNING instructions.

Example 2: Aseptic conditions

All of our facilities at BSLIII are carefully handled and monitored to avoid contamination, and room, material, reagent, and equipment sterility tests are conducted regularly. The cell culture area is cleaned and disinfected regularly every month, and benzyl, 70% ethanol, and 5% chlorine are used for several rounds of disinfection every 24, 48, and 72 hours. At the end of these sterilizations, a sterility test was carried out, and all reagents were also tested for contamination by culturing aliquots in thioglycollate liquid medium.

Example 3: Cell proliferation

Vero cell proliferation is performed under suitable physical and chemical conditions (for example, storage in polystyrene cell culture flasks (25 cm ² , 50 cm ² and 75 cm ² )). These flasks were also irradiated with gamma to reduce hydrophobicity. Such containers allow effective cell adhesion and thus monolayer formation. To promote cell reproduction, use growth medium M199 or DMEM (Dulbecco's Modified Medium) supplemented with 10% fetal bovine serum (BFS) (SIGMA-Aldrich); monitor the maintenance of physiological pH by using Phenol Red . Antimicrobial mixtures (penicillin, streptomycin and amphotericin B) are added, and anti-PPLO is also added to suppress fungal contamination.

The cell culture was incubated at 35 ° C / 5% CO ₂ /5% humidity. Cell quality and growth are monitored by phase contrast microscopy, which makes it possible to discard their cultures showing bacterial or fungal contamination.

The subculture of the completely filled flask is performed as follows:

1. Remove and discard the medium.

2. Rinse the cell layer briefly with 0.25% (w / v) trypsin-0.53 mM EDTA solution to remove all traces of serum containing trypsin inhibitor.

3. Add 2.0 ml to 3.0 ml of trypsin-EDTA solution to the flask and observe the cells under an inverted microscope until the cell layer is dispersed (usually within 5 to 15 minutes).

4. Add 6.0 ml to 8.0 ml of complete growth medium and aspirate the cells by slowly pipetting.

5. Add a suitable aliquot of cell suspension to the new culture vessel.

6. Cultivate the culture at 37 ° C.

When 80% confluence was observed, cells were inoculated with 1x10 ⁶ p AHN1 virus. This degree of coverage is most suitable for observing the cytopathic effect caused by influenza virus A / H1N1.

Example 4: Virus isolation

Virus isolation was performed in a 25 cm ² flask seeded with VERO cells (80% confluent). In the virus isolation, the growth medium was removed, the cells were centrifuged and resuspended three times to discard cell debris. A 50 μl swab sample from a patient clinically diagnosed with influenza AH1N1 and confirmed by real-time PCR was inoculated in cell culture and incubated for 30 minutes to allow virus to attach to the cell surface. Thereafter, M199 was supplemented with 1% BFS and incubated at 40 ° C / 40 min.

The presence of cytopathic effects in the inoculated culture was monitored by phase contrast microscopy. At the same time, the viral load was measured by real-time PCR. Upon reaching 1x10 ^6, the cultures were frozen at -80 ℃ (DMSO 10%) and stored for further study.

Example 5: Confirm and quantify virus infection by real-time RT-PCR.

Viral RNA was extracted from VERO cell culture by using a commercial kit (ROCHE) as described previously, and thereafter, RNA was quantified by spectrophotometry using a NanoDrop device. The CDC protocol for AH1N1 detection was implemented by real-time PCR in the Light Cycler 480 temperature cycler (ROCHE), which included influenza influenza for quantification of the standard (101 to 106 H1 genes selected in the plasmid) copy). The viral load measurement is based on the calculation of the crosspoint format for each sample. Figure 1 shows an example of quantitative viral load.

Example 6: Sample selection

A total of 193 samples for sequencing from 3760 pAH1N1 positive samples were based on clinical and epidemiological data. The clinical selection criteria are: severity of infection, drug resistance and atypical clinical environment. The epidemiological criteria are based on location, gender and age. Selected samples were collected during the four peak bursts reported from March to November 2009. The samples were resuspended in 2.5 mL of virus delivery medium, 500 μl aliquots were dispensed and stored at -70 ° C.

Example 7: Nucleic acid extraction

According to the manufacturer's instructions, use the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche) to separate the total nucleic acid of the clinical sample by the External Lysis protocol on the MagNA Pure LC instrument (Roche) and resuspend it in deionization In water (100 μ), in short: place 500 μL of sample on a MagnaPure nucleic acid column and add a volume of lysis buffer. After that, set all parameters and start separation. The extracted RNA was placed at -70 ° C for further analysis.

Example 8: Influenza p / AH1N1 real-time PCR detection

The detection of pandemic influenza AH1N1 / 09 is carried out by two methods: real-time influenza A / H1N1 detection group (Roche), and real-time RNA virus master set (Roche). Thermal cycling was performed on the Light Cycler 2.0 instrument (Roche) under the following conditions: 30 min at 50 ° C; 15 min at 95 ° C; 45 cycles of 15 s at 94 ° C; and 30 s at 60 ° C.

The second method is for the real-time RT-PCR CDC protocol for influenza A (H1N1), which uses the primers and probes described in the above protocol and the one-step RT-PCR Invitrogen SuperScript ^TM III Platinum One-step quantitative kit. Thermal cycling was performed on the Light Cycler 2.0 instrument (Roche) under the following conditions: 50 min at 50 ° C; 2 min at 95 ° C; 45 cycles of 15 s at 95 ° C; and 30 s at 55 ° C.

All reactions in both methods were performed using 5 μL of total RNA in a total reaction volume of 20 μL according to the manufacturer's instructions. Among the two detection methods, there are internal controls (human myostatin gene for Roche method and RnaseP for CDC protocol).

Each sample was tested and the following probes were used separately for real-time PCR: Influenza A (InfA), Universal Swine (SwFlu A), H1 Swine (SwH1) and internal controls for human nucleic acid RNaseP (RP). Introduce positive and negative controls.

The reaction mixture contained 0.8 μM each primer, 0.2 μM each probe, 1X PCR buffer, and 0.5 U of SuperScript III / Platinum Taq mix (Invitrogen).

An example of sample configuration is shown in Figure 2.

The amplification conditions are as follows:

Reverse transcription -50 ℃ / 30 min

Inactive -15 ℃ / 2 min

Extend -95 ℃ / 15 cycles and 55 ℃ / 30 cycles.

Example 9: Data analysis

Analyze the results according to the WHO standard as follows:

1.- There should be no fluorescence in the negative control.

2.- The fluorescence of the RP probe should be present in the experimental sample.

3.- The human nucleic acid control should not emit the fluorescence of InfA, SwFluA and SwH1 probes.

4.- The positive control (A / H1N1 nucleic acid) should emit the fluorescence of all primers and probes.

5.-If all criteria are met, the sample should be considered positive.

Figures 3A and 3B show the graphical window of the Light Cycler 2.0 real-time temperature cycler interface, which shows the fluorescence curve. Figure 3A) Clinical samples. Figure 3B) RNAseP (RP) detection.

Example 10: Sequencing

A total of 950 viruses were isolated by cell culture, from which 193 neuraminidase (NA) and 197 hemagglutinin (HA) partial gene sequences were obtained by capillary sequencing in an ABI 3100 sequencer (Applied Biosystems). Because HA and NA are the most variable sequences in AH1N1, computer simulation analysis (genealogy analysis and BLAST) shows that all 386 (HA and NA sequences) can be divided into 20 clusters; In 454 Titanium Roche Pyrosequencing equipment (ROCHE), pyrosequencing was performed on the complete genome of a representative member in each cluster.

Example 11: Proliferation of influenza virus in chicken embryos

There have been several attempts in the industry to propagate proposed candidates for influenza vaccines in VERO cells. However, these attempts initially failed because the amount of recovered virus was extremely low even after 3 passages. Taking this event into consideration, one or two subcultures were performed in SPF (Specific Pathogen Free) chicken embryos. Each candidate was inoculated into amniotic fluid with 0.1 mL of each candidate. It also includes 40 other viruses previously isolated during the influenza pandemic, and each virus is vaccinated using two eggs.

After three days at 35 ° C, amniotic fluid was harvested from each infected embryo, the collected liquid was centrifuged at 3000 x g to remove cell debris, and the supernatant was analyzed by qRT-PCR and stored at -80 ° C. All procedures are implemented in BSL III in accordance with the required standards to avoid biosecurity risks.

After subsequent generations, there are three embryos with very high viral load, one of which is a virus candidate. Ten other isolates were also tested by RT-PCR test, but showed low viral load. Therefore, it was inoculated again in another SPF chicken embryo, but in this case inoculated into the allantoic fluid. After 72 hr of inoculation following the same conditions as above, the virus was harvested by collecting only allantoic fluid. The smaller volume was analyzed by using RT-PCR, and the remaining portion was stored at -80 ° C. The results show that 6 out of 10 virus isolates can replicate with high efficiency, thus realizing a high virus load.

Proof of the sequence from the Karolinska Institute

Before the chicken embryos proliferated, the virus strain cDNA was sent to Karolinska for research so the sequence was verified by using the 454 pyrosequencing system ROCHE. Bioinformatics analysis confirmed the sequence previously determined in our laboratory.

Master seed identification

The virus candidate for influenza vaccine was named HZFLUJAL.

Influenza virus adaptation to growth on VERO cell line

The virus candidate (HZFLUJAL) harvested from the embryo and the other two viruses were inoculated on the VERO cell line. To achieve adaptation, several passages were performed and the culture supernatant was collected 7 to 10 days after infection. Each supernatant was used to infect new VERO cell passages. Quantify the viral load of each subculture. Figure 4A shows the difference between the viral load of the same virus in embryos and various subcultures. The figure shows that until four passages, the virus has achieved adaptation to this tissue cell line.

Adaptation of influenza virus to growth on MDCK cell line

Not only is the VERO cell line evaluated as a substrate for influenza virus, but the MDCK cell line is also used to propagate the virus. The same procedure used to achieve adaptation to VERO cells was performed in MDCK cells. Preliminary results show a better adaptation to this cell line because it seems that only three passages are needed to obtain a viral load larger than that of chicken embryos (Figure 4B).

Multiply the virus to obtain the main species HZFLUJAL

For the proliferation of viruses that had previously been adapted to grow on VERO cell lines, the fourth passage on VERO cells in 1 cryotube was thawed and used to infect VERO cells on 175 cm ² flasks. At 33 ℃ CO ₂ incubator at 10 days, to obtain 120 mL of the main species, showing viral load of greater than 10 ⁷ viral particle count / mL ^(7.7 to about 10). Obtain 120 freezing tubes containing 1 mL of main species.

Main species quality control

Perform sterility test and hemagglutination test on the main species. For the first test, the main species of 4 cryovials were thawed, two cryovials were used to inoculate two flasks containing 100 mL of liquid thioglycolate medium NIH, and the other two cryovials were used to inoculate antibiotic-free DMEM . Both media were incubated at 35 ° C for 8 to 15 days. No growth or changes were observed in the medium after the incubation period, so it was considered sterile (free of bacteria, fungi and protozoa).

Perform a hemagglutination test to determine the potency of hemagglutinin in the main species. In this analysis, serial dilutions of 1: 2 to 1: 1024 of the main species were prepared in PBS (pH = 7.4) in a 96-well U-shaped microplate. The same volume of 0.5% (v / v) human O red blood cell suspension in PBS was added to each dilution. The microplate was incubated at 4 ° C for 2 hours, and the results showed that the hemagglutination titer was 1: 8.

Example 12: Preclinical pilot trials using the HZFLUJAL vaccine (Pre- preclinical trial) Inactivation of virus used in preclinical pilot tests

To conduct a preclinical pilot test, the main species of one cryotube was thawed and used to infect a VERO culture on an 80 cm ² flask after 7 days in a CO ₂ incubator at 33 ° C. The culture was not activated by formalin at a final concentration of 0.02% (v / v) at 35 ° C for 18 h. Formalin was cleared by dialysis against DMEM four times in 48 h. Thereafter, the hemagglutinin titer was quantified, which was once again 1: 8. The inactivated virus suspension was adjusted to 10 ⁶ number of viral particles / 25 μL and used as a vaccine in the group.

Animal model of pre-clinical pilot test

Female BALB / c mice from 7 to 8 weeks were used in preclinical pilot trials. Place each group on a rack with a cage Hepa filter cover. All animal operations should avoid unnecessary pain. At the completion of the protocol, all mice were euthanized.

Analysis of serum conversion rate in pre-clinical pilot trials

Seroconversion analysis was performed in 9 groups (5 mice per group).

Group having 1 10 ⁶ + vaccinia virus particle of transfer factor + sublingual administration

Group 2 vaccine with 10 ⁶ virus particles + transfer factor + intramuscular administration

Vaccine group 3 with ¹⁰⁶ viral particles of sublingual administration +

Group 4 vaccine with 10 ⁶ virus particles + intramuscular administration

Group 5 commercial vaccine + sublingual administration

Group 6 commercially available vaccine + intramuscular administration

Group 7 DMEM + sublingual administration of fetal bovine serum

Group 8 DMEM + intramuscular administration without fetal bovine serum

Group 9 untreated

Analysis of dose response in preclinical pilot trials

Dose response analysis was performed in 5 groups (3 mice per group).

Group A vaccine with 10 ⁷ virus particles + transfer factor + sublingual administration

Group B vaccine with 10 ⁵ virus particles + transfer factor + sublingual administration

Group C vaccine with 10 ⁴ viral particles + transfer factor + sublingual administration

Group D vaccine with 10 ³ virus particles + transfer factor + sublingual administration

Vaccine Group E with ¹⁰² viral particles of transfer factor + sublingual administration +

From the beginning of the immunization protocol, all mice were maintained in the animal experiment room with biological safety requirements for immunoassay for 4 days. After the acclimation time, clinical data such as body weight and appearance were recorded, and blood samples without anticoagulant were taken from each mouse before immunization. Thereafter, each group is inoculated by using separate administration routes. Four blood samples were obtained from each mouse at 7, 14, 21 and 28 days after immunization.

Serum was obtained by centrifuging blood samples and used to detect IgG2 antibodies against influenza A H1N1 virus. In addition, secretory samples were collected from mice for detection of secreted immunoglobulin A (1gA).

Example 13: Detection of anti-influenza p / AH1N1 antibody produced by CIATEJ / OPKO / vaccine

The example vaccines described herein as CIATEJ / OPKO / vaccines were produced in VERO cells, and the ability to generate anti-influenza AH1N1 antibodies was tested. When an antigen produced by CIATEJ / OPKO in VERO cells is used as a vaccine in mice, it can induce antibody production.

The titer of antibodies induced by CIATEJ / OPKO antigen is greater than that of commercially available vaccines. Figure 5 shows the total antibodies against influenza p / AH1N1 detected by ELISA. The CIATEJ / OPKO / vaccine line is produced in VERO cells, TF = transfer factor. Immunization was performed in 7-8 week old BALB / c female mice. The immunization is carried out sublingually or intramuscularly.

Figure 6A shows the IgM antibody against influenza p / AH1N1 detected in serum by ELISA. The CIATEJ / OPKO / vaccine line is produced in VERO cells, TF = transfer factor. Immunization was performed in 7-8 week old BALB / c female mice. The immunization is carried out intramuscularly.

When the transfer factor was added to the vaccine CIATEJ / OPKO, it increased the IgG antibody content by 5 times, and during the first week of immunization, the transfer factor increased the IgM antibody titer by 3 times in the same manner. Figure 6B shows IgG antibodies against influenza p / AH1N1 detected in serum by ELISA. The CIATEJ / OPKO / vaccine line is produced in VERO cells, TF = transfer factor. Immunization was performed in 7-8 week old BALB / c female mice. The immunization is carried out intramuscularly.

Intramuscular administration of CIATEJ / OPKO showed that more IgG _2A antibodies were induced than sublingual administration. Figure 7A shows the IgG _2A antibody against influenza p / AH1N1 detected in serum by ELISA. The CIATEJ / OPKO / vaccine line is produced in VERO cells, TF = transfer factor. Immunization was performed in 7-8 week old BALB / c female mice. The immunization is carried out sublingually or intramuscularly.

Figure 7B shows the dose dependence of IgG _2A against influenza p / AH1N1 detected in serum by ELISA. The CIATEJ / OPKO / vaccine line is produced in VERO cells, TF = transfer factor. Immunization was performed in 7-8 week old BALB / c female mice. The immunization is administered sublingually.

The intramuscular administration of CIATEJ / OPKO showed that the level of induced IgA antibody was comparable to that of sublingual administration. Figure 8A shows the IgA antibody against influenza p / AH1N1 detected by ELISA in respiratory mucosa. The CIATEJ / OPKO / vaccine line is produced in VERO cells, TF = transfer factor. Immunization was performed in 7-8 week old BALB / c female mice. The immunization was performed sublingually or intramuscularly (on the 21st day after immunization).

Figure 8B shows the dose-dependent IgA of influenza p / AH1N1 detected in respiratory mucosa by ELISA. The CIATEJ / OPKO / vaccine line is produced in VERO cells, TF = transfer factor. Immunization was performed in 7-8 week old BALB / c female mice. The immunization was performed sublingually or intramuscularly (on the 21st day after immunization).

references

Magna Pure LC Total Nucleic Acid Isolation Kit. December 2008 edition, Kit for isolation of total viral nucleic acids from mammalian serum, plasma and whole blood, catalog number 03 038 505 001.

SuperScript III Platinum One Step Quantitative RT-PCR System. Catalog number 11738-088.

World Health Organization. CDC protocol of Real time for Influenza A (H1N1). April 29, 2009.

CENAVECE-InDRE. Lineamientos para la red Nacional de laboratorios de salud p blica e institutos nacionales de salud para vigilancia de influenza en Mexico 2009.

Ammerman N., Beier M. and Azad F. 2008 Growth and Maintenance of VERO Cell Lines. Curr Protoc Microbiol., Pages 1-10.

Erlich H., M ller M., Helen M., Tambyah P., Joukhadar M., Montomoli M., Fisher D., Berezuk D., Fritsch M., L w-Baselli A., Vartian N., Bobrovsky R., Borislava G., Pavlova, P llabauer E., Kistner O. and Barrett N. 2008 A Clinical Trial of a Whole-Virus H5N1 Vaccine Derived from Cell Culture for the Baxter H5N1 Pandemic Influenza Vaccine Clinical Study Team. N. Engl. J. Med. 358; 24., Pages 2573-2584.

Institu to de Medicina Tropical "Pedro Kouri". 2003. Manual de procedimientos para el diagnostico de laboratorio de la infecciones respiratyorias agudas de etiolog a viral. Organizaci n panam ricana de la salud. Organizaci n mundial de la salud.

Kistner O., Howard K., Spruth M., Wodal W., Br hl P., Gerencer M., Crowe B., Savidis H., Livey I., Reiter M., Mayerhofer I., Tauer C., Grillberger L., Mundt G., Falkner F. and P. Noel. 2007 Cell culture (Vero) derived whole virus (H5N1) vaccine based on wild-type virus strain induces cross-protective immune responses. Baxter AG, Biomedical Research Center, Uferstrasse, Austria. Vaccine. 2007 10; 25 (32). No. 6028-6036 page.

Reina J., Fern ndez V., Blanco I. y Munar M. 1997. Comparison of Madin-Darby Canine Kidney Cells (MDCK) with a Green Monkey Continuous Cell Line (Vero) and Human Lung Embryonated Cells (MRC-5) in the Isolation of Influenza A Virus from Nasopharyngeal Aspirates by Shell Vial Culture. American Society for Microbiology Volume 35, Number 7, page 1900-1901.

Ryan John. Ph. D. Introduction to animal cell culture. Corning Incorporated Life Sciences 900 Chelmsford St. Lowell, Bulletin Technical. MA 01851, pages 1-24.

Ryan John. Ph. D. Subculturing Monolayer cell cultures Protocol Corning Incorporated Life Sciences 900 Chelmsford St. Lowell, MA 01851, pages 1-5. Sheets R. 2000 History and Characterization of the VERO Cell Line. Vaccines and Related Biological Products Advisory Committee Meeting to be held.

All publications and patent applications cited in this specification are incorporated herein by reference, as if each individual publication or patent application clearly and individually indicated to be incorporated by reference.

Although the present invention has been explained in more detail with the help of illustrations and examples for the purpose of understanding clearly, those skilled in the art can easily understand the teaching of the present invention and can make certain changes and modifications to the present invention, Without departing from the spirit or scope of the accompanying patent application.

Figure 1 shows an example of quantification of viral load by real-time PCR.

Figure 2 shows the sample configuration for AH1N1 real-time PCR detection.

Figures 3A and 3B show the graphical window of the Light Cycler 2.0 real-time temperature cycler interface, which shows the fluorescence curve. Figure 3A) Clinical samples. Figure 3B) RNAseP (RP) detection.

Figure 4A shows the adaptation of influenza virus HZFLUJAL in the VERO cell line. The virus can effectively replicate in VERO cells until 4 subcultures. Figure 4B shows the adaptation stage of influenza virus HZFLUJAL in the MDCK cell line. After three subcultures, the influenza virus HZFLUJA can effectively replicate in MDCK cells.

Figure 5 shows the total antibodies against influenza p / AH1N1 detected by ELISA.

Figure 6A shows the IgM antibody against influenza p / AH1N1 detected in serum by ELISA.

Figure 6B shows IgG antibodies against influenza p / AH1N1 detected in serum by ELISA.

Figure 7A shows the IgG _2A antibody against influenza p / AH1N1 detected in serum by ELISA. Figure 7B shows the dose dependence of IgG _2A against influenza p / AH1N1 detected in serum by ELISA.

Figure 8A shows the IgA antibody against influenza p / AH1N1 detected by ELISA in respiratory mucosa. Figure 8B shows the dose-dependent IgA of influenza p / AH1N1 detected in respiratory mucosa by ELISA.

<110>

<120> New vaccine against pandemic influenza virus A / H1N1

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<151> 2010-11-02

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Claims (20)

An influenza virus vaccine comprising an influenza virus, the virus comprising: a) a mutant neuraminidase (NA) gene segment, which has a point mutation at amino acid residue 275, wherein the point mutation is Tyr275, and b) mutant hemagglutinin (HA) gene segment, which contains the D222E mutation. The vaccine according to claim 1, wherein the HA gene segment comprises a branched 7-specific HA sequence of influenza A H1 / N1 influenza virus. The vaccine of claim 1, wherein the HA gene segment contains the S203T mutation relative to the wild-type influenza A H1 / N1 influenza virus. The vaccine according to claim 1, wherein the HA gene segment further comprises P83S mutation, I321V mutation; P83S mutation and I321V mutation. The vaccine of claim 1, wherein the HA gene segment comprises the amino acid sequence encoded by the nucleotide sequence of segment 4 of virus 1 (SEQ ID NO: 4). The vaccine of claim 1, wherein the NA gene segment comprises the amino acid sequence encoded by the nucleotide sequence of segment 6 in virus 1 (SEQ ID NO: 6). The vaccine of claim 1, further comprising: c) a matrix (MA) gene segment, which encodes an M1 peptide, the M1 peptide comprising an A198P mutation. The vaccine of claim 7, wherein the M1 peptide comprises an amino acid sequence SEQ ID NO: 50 derived from a virus 1 isolate of influenza A H1 / N1 influenza virus. The vaccine of claim 7, wherein the MA gene segment comprises the amino acid sequence encoded by the nucleotide sequence of segment 7 in virus 1 (SEQ ID NO: 7). The vaccine according to claim 7, wherein the matrix (MA) gene segment further encodes an M2 peptide, the M2 peptide comprising the amino acid sequence SEQ ID NO: ID NO: 51. The vaccine of claim 1, wherein the mutant hemagglutinin (HA) gene segment encodes a protein that includes the amino acid sequence shown in SEQ ID NO: 34. The vaccine of claim 1, wherein the mutant neuraminidase (NA) gene segment encodes a protein that includes the amino acid sequence shown in SEQ ID NO: 39. The vaccine according to any one of claims 1 to 12, wherein the vaccine is a redistribution vaccine. The vaccine of claim 13, wherein the redistribution influenza virus is obtained by traditional redistribution method. The vaccine according to any one of claims 1 to 12, wherein the vaccine is a live attenuated vaccine or an inactivated vaccine. The vaccine of claim 15, wherein the vaccine is formulated for oral or intranasal administration. A use of an influenza virus vaccine according to any one of claims 1 to 12 for the manufacture of a medicament for immunizing patients against influenza virus. A use of the influenza virus vaccine according to any one of claims 1 to 12, for the manufacture of a medicament for immunizing a patient against a hyperviral influenza virus. Use of an influenza virus vaccine according to any one of claims 1 to 12 for the manufacture of an agent that immunizes a patient against oseltamivir resistant influenza virus. A method for preparing a vaccine that immunizes an individual against a hypervirulent influenza virus strain, which includes mixing influenza virus as defined in any one of claims 1 to 12 with a carrier and an adjuvant optionally selected step.