BE1019643A3 - INFLUENZA PROTECTION VACCINES. - Google Patents

Abstract

Methods for producing influenza vaccines. The methods use an H1N1 strain of influenza A virus with a hemagglutinin which is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3. The vaccine can be adjuvanted with an adjuvant as an emulsion oil in water.

Description

"INFLUENZA PROTECTIVE VACCINES" TECHNICAL FIELD

This invention is in the field of manufacturing vaccines intended to protect against infection due to the influenza virus and in particular against the strain (s) of swine flu which has (have) made their (their) appearance in April 2009.

PRIOR ART

In April 2009, a human epidemic of swine flu was confirmed in many countries including Mexico and the United States, and then spread rapidly throughout the world. A pandemic was declared by the WHO in June 2009. The disease is caused by a newly identified swine flu virus A / California / 04/2009 A (H1N1). This strain of swine influenza appears to have no cross-reactive immunological reaction with current human influenza vaccine strains, including A (H1N1) antigen in current human seasonal vaccines. The virus has been referred to in various ways as "swine flu", "new swine flu H1N1", "human swine flu", "new influenza A (H1N1)" and "influenza A (H1N1) v".

There is a need to provide methods for making a vaccine to prevent subsequent human-to-human transmission of this swine influenza and variants thereof. DISCLOSURE OF THE INVENTION

The invention provides methods for making influenza vaccines. The methods use an H1N1 strain of influenza A virus with a hemagglutinin that is more closely related to SEQ ID NO: 1 than SED ID NO: 3. The vaccine can be adjuvanted with an emulsion adjuvant of oil in water. The hemagglutinin elicits an immune response of a recipient, and the adjuvant increases the heterovarant coverage of that response so that protection can be provided against the homologous strain and also against variants thereof such as derived strains. that can appear naturally. The invention has been useful for preparing the FOCETRIA ™ and CELTURA ™ vaccine products. Antigenic Components The invention utilizes hemagglutinin of influenza A virus as the vaccine antigen. The antigen is prepared from influenza virions.

Various forms of influenza vaccine are currently available (for example, see Chapters 17 & 18 of Reference 1). The antigen in the vaccines produced according to the invention takes the form of an inactivated virus. Chemical means for inactivating a virus include treatment with an effective amount of one or more of the following agents: detergents, formaldehyde, formalin, β-propiolactone, or UV rays. Additional chemical means for inactivation include treatment with methylene blue, psoralen, carboxyfullerene (C60) or any combination thereof. Other methods of viral inactivation are known in the art, such as, for example, binary ethylamine, acetylethyleneimine or gamma irradiation.

The vaccine may comprise a whole virion, a fragmented virion, or purified surface antigens (including hemagglutinin and generally including neuramidase). Fragmented virion and purified surface antigens (i.e., virion subunit vaccines) are particularly useful for the invention.

Virions can be harvested from fluids containing the virus by various methods. For example, a purification process may include zonal centrifugation using a linear sucrose gradient solution that includes detergent to dissociate the virions. The antigens can then be purified, after optional dilution, by diafiltration.

The fragmented virions are obtained by treating the virions with detergents (e.g., ethyl ether, polysorbate 80, deoxycholate, tri-N-butyl phosphate, Triton X-100, Triton N101, cetyltrimethylammonium bromide, Tergitol NP9, etc.). to produce virion subunit preparations, including the "Tween-ether" fragmentation process. Influenza virus fragmentation methods are well known in the art, for example, see references 2-7, etc. Virus fragmentation is generally accomplished by disrupting or fragmenting whole viruses, whether infectious or non-infectious, with dissociative concentration of a fragmenting agent. Dissociation results in total or partial solubilization of the virus proteins, altering the integrity of the virus. The preferred fragmenting agents are nonionic and ionic surfactants (eg cationic), for example alkyl glycosides, alkylthioglycosides, acylated sugars, sulfobetaines, betaines, polyoxyethylene alkyl ethers, Ν, Ν-dialkyl glucamides, Hecameg, alkylphenoxy- polyethoxyethanols, quaternary ammonium compounds, sarcosyl, CTAB (cetyltrimethylammonium bromides), tri-A / -butyl phosphate, Cetavlon, myristyltrimethylammonium salt, lipofectin, lipofectamine, and DOT-MA, octyl- or nonylphenoxypolyoxyethanols (e.g. Triton surfactants, such as Triton X-100 or Triton N101), polyoxyethylene sorbitan esters (Tween surfactants), polyoxyethylene ethers, polyoxyethylene esters, and the like. A useful fragmentation procedure utilizes the consequent effects of sodium deoxycholate and formaldehyde, and fragmentation can occur during the initial purification of the virion (e.g., in a linear sucrose gradient solution). Thus, a fragmentation process may include the clarification of virion-containing material (to remove material that is not virions), the concentration of harvested virions (for example by using an adsorption method such as CaHPO4 adsorption). , separation of whole virions from non-virion material, fragmentation of virions using a fragmentation agent during a density gradient centrifugation step (e.g., using a gradient sucrose which contains a fragmenting agent such as sodium deoxycholate), and then filtration (eg ultrafiltration) to remove unwanted material. The fragmented virions can be usefully resuspended in isotonic sodium chloride buffered sodium phosphate solution. BEGRIVAC ™, FLUARIX ™, FLUZONE ™ and FLUSHIELD ™ products are fragmented virion vaccines.

Purified surface antigen vaccines include hemagglutinin surface antigens and, typically, influenza virus neuramidase. The processes for preparing these proteins in purified form are well known in the art. FLUVIRIN ™, AGRIPPAL ™ and INFLUVAC ™ products are subunit vaccines.

The influenza antigens can also be presented as virosomes [8] (viral-type viral-type liposomal particles), as in the INFLEXAL V ™ and INVAVAC ™ products, but it is preferred not to use virosomes with the present invention. . Thus, in certain embodiments, the influenza antigen is not in the form of a virosome.

The haemagglutinin antigen in the vaccine may be from any suitable strain. In certain embodiments, hemagglutinin is a hemagglutinin which, when administered to a human subject in a non-adjuvanted form, may elicit the development of anti-hemagglutinin antibodies which may cross-react with hemagglutinin A / California / 04/2009 (SEQ ID NO: 1); in these embodiments, an adjuvant can enhance the immune response so that a human subject produces a broad spectrum of antibodies, which can help protect against drifts of A / California / 04/2009 strain. In other embodiments, the hemagglutinin is from A / California / 04/2009 (SEQ ID NO: 1). In other embodiments, the haemagglutinin comprises an amino acid sequence HA1 having at least 1% sequence identity with SEQ ID NO: 2, where i is 85 or greater, for example 85, 88 , 90, 92, 94, 95, 96, 97, 98, 99 or more (e.g., 100).

H1N1 strains with appropriate HA antigens include A / California / 04/2009, A / California / 7/2009, A / Texas / 5/2009, A / England / 195/2009, and A / New York / 18 / 2009.

Hemagglutinin is more closely related to SEQ ID NO: 1 (A / California / 04/2009) than to SEQ ID NO: 3 (A / Chile / 1/1983). A hemagglutinin that is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3 (i.e., which has a greater sequence identity with SEQ ID NO: 1 than with SEQ ID NO: 3 when compared using the same algorithm and the same parameters) is hereinafter referred to as hemagglutinin H1 *. SEQ ID NO: 1 and 3 are 80.4% identical.

Useful complete sequences of hemagglutinin H1 for use with the invention include SEQ ID NO: 1 and SEQ ID NO: 6, as well as those comprising an amino acid sequence having at least 1% sequence identity with SEQ ID NO: 2 as discussed above, or having at least 1% sequence identity with SEQ ID NO: 7. Ideally, the haemagglutinin does not include a hyper basic region around the HA1 / HA2 cleavage site. . Preferred haemagglutinins have a binding preference for oligosaccharides with a terminal disaccharide Sia (a2.6) Gal rather than oligosaccharides with a terminal disaccharide Sia (a2.3) Gal (see below).

SEQ ID NO: 6 (including SEQ ID NO: 7) is a useful hemagglutinin H1 *. It differs from SEQ ID NO: 1 at residues 214, 226 and 240 (i.e. 99.47% identity).

In addition to comprising a hemagglutinin H1 (such as hemagglutinin H1 *), compositions made using the invention may comprise antigen (s) from one or more (e.g., 1, 2, 3, 4 or more) additional strains of the influenza virus, including influenza A virus and / or influenza virus B. Thus, a composition may comprise an antigen from one or more characteristic strains of a normal seasonal vaccine plus at least one haemagglutinin H1 *, for example a 4-valence vaccine with two H1 strains (one which is a haemagglutinin H1 *, one which is not a haemagglutinin H1 *), one strain H3N2 and one strain influenza B, or a 5-valence vaccine with two H1 strains (one which is an H1 * hemagglutinin, one which is not an H1 * haemagglutinin), one H3N2 strain, and two strains of influenza B virus (one B / Victoria / 2/87-like strain and a B / Yamagata / 16/88-like strain). When a vaccine comprises more than one influenza strain, the different strains are typically grown separately and are mixed after the viruses have been harvested and the antigens prepared. Thus, a process of the invention may include the step of mixing antigens of more than one strain of influenza.

The influenza virus may be resistant to antiviral therapy (eg resistant to oseltamivir [9] and / or sanamivir).

In some embodiments, the strains used with the invention will therefore have hemagglutinin with a binding preference for oligosaccharides with a terminal disaccharide.

Sia (a2.6) Gal rather than for oligosaccharides with a terminal disaccharide Sia (a2.3) Gal. To determine whether a virus has a binding preference for oligosaccharides with a terminal disaccharide

Sia (a2,3) Gal, different assays can be used, see for example references 10 to 13. Depending on the type of assay, it will be performed directly with the virus itself or it can be performed indirectly with purified haemagglutinin from of the virus.

In some embodiments, hemagglutinin H1 has a different glycosylation than that seen in egg-derived viruses. Thus, HA (and other glycoproteins) can include glycoforms that are not seen in chicken eggs. Useful HA includes canine glycoforms.

In addition to understanding the hemagglutinin antigen, vaccines made using the invention typically also include a neuramidase protein, for example the vaccine will include a viral neuramidase. The invention can protect against one or more NA subtypes of the influenza A, N1, N2, N3, N4, N5, N6, N7, N8 or N9 virus, but it will generally protect against N1 (e.g. H1N1) or N2 (for example an H1N2 virus). Whole virion, split virion or subunit vaccines all include hemagglutinin and neuramidase. When a vaccine comprises a neuramidase antigen, the neuramidase may have at least one sequence identity with SEQ ID NO: 4, where j is 75 or greater, for example 75, 80, 85, 88, 90, 92 , 94, 95, 96, 97, 98, 99 or more (e.g. 100). Many sequences of this kind are available. In some embodiments, the neuramidase is more closely related to SEQ ID NO: 4 than to SEQ ID NO: 5. SEQ ID NO: 4 and 5 are 82% identical.

The vaccines may also comprise a template protein, such as M1 and / or M2 (or a fragment thereof), and / or a nucleoprotein. A porcine model has shown that the addition of M2 to a vaccine against the inactivated H1N1 swine flu virus (adjuvanted with an oil emulsion in water) can improve the effectiveness of the vaccine [14],

The influenza virus can be a reassortant strain, and can be obtained by reverse genetics techniques. Inverse genetics techniques [e.g., 15-19] allow influenza viruses with desired genomic segments to be prepared in vitro using plasmids or plasmid-free systems. Typically, the technique comprises the expression of (a) DNA molecules that encode desired viral RNA molecules, e.g., polite promoters or bacteriophage promoter polymerases, and (b) DNA molecules that encode viral proteins, for example polll promoters, so that the expression of both types of DNA in a cell leads to the assembly of a complete intact infectious virion. DNA preferably provides all of the viral RNA and viral proteins, but it is also possible to use a helper virus to provide a portion of RNA and proteins.

An influenza A virus used with the invention may comprise one or more RNA segments of A / PR / 8/34 virus (typically 6 segments of A / PR / 8/34, the HA and N segments of a vaccine strain, ie 6: 2 reassortment), especially when the viruses are grown in eggs. A vaccine typically protects against a strain that is capable of transmitting from humans to humans, and thus the genome of the strain will generally comprise at least one RNA segment that is derived from a mammalian influenza virus (e.g. of a human being).

The viruses used as the source of the antigens can be grown on eggs or on a cell culture. The current standard method of influenza virus culture uses embryonated chicken eggs free from specific pathogens (SPF), the virus being purified from the egg contents (allantoic fluid). More recently, however, viruses have been cultured in these animal cell cultures and, for the sake of speed and allergies of patients, this culture method is preferred. If the viral culture on eggs is used, then one or more amino acids can be introduced into the allantoic fluid of the egg at the same time as the virus [7].

When cell culture is used, the viral growth substrate will typically be a mammalian cell line. Suitable cells of mammalian origin include, but are not limited to, those of the hamster, cattle, primates (including humans and monkeys) and dogs. Mammalian cell lines preferred for growing influenza viruses include: MDCK cells [20-23], derived from a dog kidney (Madin Darby canine kidney), Vero cells [24-26], derived from African green monkey kidney (Cercopithecus aethiops), or PER.C6 cells [27] derived from human embryonic retinoblasts. These cell lines are widely available, for example from the American Type Cell Culture (ATCC) collection, Coriell Cell

Repositories or the European collection of animal cell cultures (ECACC). For example, the ATCC provides various types of Vero cells under catalog numbers CCL-81, CCL-81.2, CRL-1586 and CRL-1587, and provides MDCK cells under catalog number CCL-34. PER.C6 cells can be obtained from ECACC under deposit number 96022940.

The cell lines that have the greatest preference for growing influenza viruses are the MDCK cell lines. The original MDCK cell line can be obtained from ATCC as CCL-34, but derivatives of this cell line can also be used. For example, reference 20 discloses an MDCK cell line that has been adapted to be cultured in a suspension culture ('MDCK 33016', deposited as DSM ACC 2219). Similarly, reference 28 discloses a cell line derived from the MDCK line which grew in suspension in a serum-free culture ('B-702', deposited as FERM BP-7449). Reference 29 discloses non-tumorigenic MDCK cells, including 'MDCK-S' (ATCC PTA-6500), 'MDCK-SF101' (ATCC PTA-6501), 'MDCK-SF102' (ATCC PTA-6502), and 'MDCK- SF103 '(PTA-6503). Reference 30 discloses MDCK cell lines with high susceptibility to infection, including MDCK.5FT cells (ATCC CRL-12042). Any of these MDCK cell lines can be used.

When a virus has been cultured on a mammalian cell line, then the composition may advantageously be free of egg proteins (eg ovalbumin and ovomucoid) and chicken DNA, thus reducing allergenicity.

When the virus has been grown on a cell line, then the growth culture, and also the viral inoculum used to start the culture, will preferably be free (i.e. will have been tested and will have a negative result in with regard to contamination by herpes simplex virus, respiratory syncytial virus, parainfluenza virus type 3, SARS coronavirus, adenovirus, rhinovirus, reoviruses, polyomaviruses, birnaviruses, circoviruses and / or parvovirus [31]. The absence of herpes simplex virus is particularly preferred.

For growth on a cell line, for example on MDCK cells, the virus can be grown on cells in suspension [20, 32, 33] or in an adherent culture. An MDCK cell line suitable for suspension culture is MDCK 33016 (deposited as DSM ACC 2219). As an alternative, a culture on microcarriers can be used.

Cell lines supporting replication of the influenza virus are preferably grown in culture media without serum and / or protein. Serum-free medium is referred to in the context of the present invention as a medium in which there are no serum additives of human or animal origin. It is understood that without protein means cultures in which the multiplication of cells occurs to the exclusion of proteins, growth factors, other protein additives and proteins that are not serum proteins, but which may include optionally, trypsin-like proteins or other proteases that may be necessary for viral growth. Cells growing in such cultures naturally contain proteins themselves.

Cell lines supporting replication of influenza virus are preferably grown below 37 ° C [34] during viral replication, for example at 30-36 ° C, 31-35 ° C, or 33 + _1 ° C.

The method for propagating a virus in cultured cells generally comprises the steps of inoculating the cultivated strain with the strain to be cultured, culturing the infected cells for a desired period of time for virus propagation, as determined, for example, by viral or antigen expression (eg between 24 and 168 hours after inoculation) and to collect the propagated virus. Cultured cells are inoculated with a virus (measured by PFU or TCID 50) in a cell ratio of 1: 500 to 1: 1, preferably 1: 100 to 1: 5, more preferably 1: 50 to 1:10. virus is added to a cell suspension or is applied to a monolayer of the cells, and the virus is absorbed on the cells for at least 60 minutes but generally for less than 300 minutes, preferably between 90 and 240 minutes at a temperature of 25 ° C to 40 ° C, preferably 28 ° C to 37 ° C. Culture of infected cells (eg monolayers) can be removed either by freeze-thaw or enzymatic action to increase the viral content of culture supernatants harvested. The fluids harvested are then either inactivated or stored frozen. The cultured cells may be infected with a multiplicity of infection ("MOI") of from about 0.0001 to 10, preferably from 0.002 to 5, more preferably from 0.001 to 2. More preferably, the cells are infected with a MOI. about 0.01. Infected cells can be harvested 30 to 60 hours after infection. The cells are preferably harvested 34 to 48 hours after infection. Better still, the cells are harvested 38 to 40 hours after infection. Proteases (typically trypsin) are generally added during cell culture to allow release of the virus, and proteases may be added at any convenient stage during cultivation.

Haemagglutinin (HA) is the major immunogen in inactivated influenza vaccines and vaccine doses are standardized by reference to levels of HA, typically measured by single radial immunodiffusion (SRID) assay. Current vaccines typically contain about 15 μg HA per strain, although lower doses are also used, for example for children or in emergency situations. Fractional doses such as 1/4 (ie, 7.5 μg HA per strain),% (ie, 3.75 μg per strain), and 1 / e were used [ 35, 36], as well as higher doses (eg 3x or 9x doses [37, 38]), therefore vaccines can range from 0.1 to 150 pg HA per influenza strain, preferably 0.1 and 50 μg, for example 0.1 -20 μg, 0.1 -15 μg, 0.1-10 μg, 0.1-7.5 μg, 0.5-5 μg, 3.75-15 μg etc. Particular doses include, for example, about 45, about 30, about 15, about 10, about 7.5, about 5, about 3.8, about 3.75, about 1.9, about 1.5, etc. pg by strain.

The HA used with the invention may be naturally occurring HA as found in a virus or may have been modified.

When a virus has been grown on a cell line, then the common practice is to reduce the amount of residual DNA from the cell line in the final vaccine to reduce any oncogenic activity of the DNA. Thus, the composition preferably contains less than 10 ng, preferably less than 1 ng, and most preferably less than 100 μg of residual DNA of the host cell per dose, although amounts of DNA from the host cell under trace form may be present. In general, the DNA of the host cell that it is desirable to exclude is DNA that is longer than 100 bp.

Contaminant DNA may be removed during vaccine preparation using standard purification procedures, eg, chromatography, etc. Removal of the residual DNA from the host cell can be improved by nuclease treatment, for example using DNase. A convenient method for reducing DNA contamination of the host cell is disclosed in references 39 &40; it includes a two-step treatment, first the use of a DNase (eg Benzonase) that can be used during viral growth and then a cationic detergent (eg CTAB) that can be used during the dissociation of virions. Treatment with an alkylating agent, such as β-propiolactone, can also be used to remove DNA from the host cell and can also be advantageously used to inactivate virions [41] while avoiding the use of formaldehyde.

Adjuvants in the form of oil emulsion in water

Compositions made using the invention may comprise (or be used with) an adjuvant in the form of an oil emulsion in water that can function to improve the immune responses (humoral and / or cellular) obtained in a patient who receives the composition.

Various suitable emulsions are known and typically comprise at least one oil and at least one surfactant, the oil (the oils) and the surfactant (the surfactants) being biodegradable (metabolizable) and biocompatible. The oil droplets in the emulsion are generally less than 5 μm in diameter, and the emulsion advantageously comprises oil droplets of submicron diameter, these small sizes being obtained with a microfluidizer to provide stable emulsions. Droplets having a size smaller than 220 nm are preferred since they can be sterilized by filtration.

Specific emulsion-type adjuvants which are useful with the invention include, but are not limited to: • A submicronic emulsion of squalene, Tween 80 and Span 85. The composition of the bulk emulsion may be about 5% squalene, about 0.5% polysorbate 80 and 0.5% Span 85. In terms of weight, these ratios become 4.3% squalene, 0.5% polysorbate 80 and , 48% Span 85. This adjuvant is known as 'MF59' [42-44], as described in more detail in Chapter 10 of Reference 45 and Chapter 12 of Reference 46. MF59 Emulsion advantageously comprises citrate ions, for example 10 mM sodium citrate buffer.

An emulsion comprising squalene, an α-tocopherol, and polysorbate 80. These emulsions may have from 2 to 10% of squalene, from 2 to 10% of tocopherol and from 0.3 to 3% of Tween 80 and the ratio Weight of squalene: tocopherol is preferably <1 (eg 0.90) since it provides a more stable emulsion. Squalene and Tween 80 may be present in a volume ratio of about 5: 2, or a weight ratio of about 11: 5. Such an emulsion can be made by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90 ml of this solution with a mixture of (5 g of DL-α-tocopherol and 5 ml of squalene), then microfluidizing the mixture. The resulting emulsion may have submicron oil droplets, for example having a mean diameter between 100 and 250 nm, preferably about 180 nm.

An emulsion comprising squalene, an aqueous solvent, a hydrophilic nonionic surfactant based on polyoxyethylenated alkyl ether (for example a polyoxyethylenated cetostearyl ether (12)) and a hydrophobic nonionic surfactant (for example an ester sorbitan or a mannide ester, such as sorbitan monoleate or 'Span 80'). The emulsion is preferably thermoreversible and / or has at least 90% oil droplets (by volume) having a size less than 200 nm [47]. The emulsion may also comprise one or more of: alditol (e.g. mannitol), a cryoprotective agent (for example a sugar such as dodecylmaltoside and / or sucrose), and / or an alkylpolyglycoside. Such emulsions can be lyophilized. The emulsion may comprise squalene: polyoxyethylene: ketostearyl ether: sorbitan oleate: Mannitol 330: 63: 49: 61.

Antigens and adjuvants in a composition will typically be a mixed admixture at the time of administration to a patient. The emulsions can be mixed with the antigen during manufacture or extemporaneously at the time of administration. Thus, adjuvant and antigen can be stored separately in a packaged or dispensed vaccine, ready for final formulation at the time of use. The antigen will generally be in aqueous form so that the vaccine is finally prepared by mixing two liquids. The volume ratio of the two liquids for mixing can vary (for example between 5: 1 and 1: 5) but is generally about 1: 1.

When a composition comprises a tocopherol, any of the α, β, γ, δ, ε or ξ tocopherols may be used, but α-tocopherols are preferred. A preferred α-tocopherol is DL-α-tocopherol and the preferred salt of this tocopherol is succinate.

As mentioned above, oil emulsions in water comprising squalene are particularly preferred. In some embodiments, the concentration of squalene in a vaccine dose may be in the range of 5-15 mg (i.e., a concentration of 10 - 30 mg / ml, for a dose volume of 0.5 ml). It is nevertheless possible to reduce the concentration of squalene [48, 49], for example to include <5 mg per dose, or even <1.1 mg per dose. For example, a human dose may include 9.75 mg squalene per dose (as in FLUAD ™: 9.75 mg squalene, 1.175 mg polysorbate 80, 1.175 mg sorbitan trioleate in a dose volume of 0). , 5 ml), or it may comprise a fractional amount thereof, for example 3/4, 2/3, 1/2, 1/3, 1/4, 1/5, 1/6, 1/7 , 1/8, 1/9 or 1/10. For example, a composition may comprise 7.31 mg squalene per dose (and thus 0.88 mg and polysorbate 80 and sorbitan trioleate), 4.875 mg squalene / dose (and thus 0.588 mg and polysorbate 80 and sorbitan trioleate), 3.25 mg squalene / dose, 2.388 mg / dose, 1.95 mg / dose, 0.975 mg / dose, etc. Any of these fractional dilutions of FLUAD ™ adjuvanted to MF59 can be used with the invention.

As mentioned above, the antigen / emulsion mixture can be effected extemporaneously at the time of administration. Thus, the invention can be used to make kits comprising the antigen and adjuvant components ready to be mixed, kept separate until the moment of use. Pharmaceutical compositions

The compositions made with the invention are pharmaceutically acceptable. They generally comprise components in addition to the antigen, for example they typically comprise one or more carrier (s) and / or pharmaceutical excipient (s). A detailed discussion of these components is available in reference 50.

The compositions will generally be in aqueous form.

The composition may comprise preservatives such as thiomersal (for example at 10 μg / ml) or 2-phenoxyethanol. It is preferred, however, that the vaccine be substantially free of (i.e., less than 5 μg / ml) mercury material, for example, without thiomersal [51]. Vaccines without mercury without further preference. Vaccines without preservatives are particularly preferred.

To control the tone, it is preferred to include a physiological salt such as sodium salt. Sodium chloride (NaCl), which may be present at 1 to 20 mg / ml, is preferred. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dihydrate, magnesium chloride, calcium chloride, and the like.

The compositions will generally have an osmolality of between 200 mOsm / kg and 400 mOsm / kg, preferably between 240-360 mOsm / kg and more preferably will be in the range 290-310 mOsm / kg.

The compositions may comprise one or more buffers. Typical buffers include: phosphate buffer, tris buffer, borate buffer, succinate buffer, histidine buffer or citrate buffer. The buffers will typically be in the range of 5- 20 mM. The buffer may be in an aqueous emulsion phase.

The pH of a composition will generally be from 5.0 to 8.1, more typically from 6.0 to 8.0, for example. 6.5 and 7.5 or between 7.0 and 7.8. A method of the invention may therefore include a step of adjusting the pH of the bulk vaccine before packaging.

The composition is preferably sterile. The composition is preferably free of gluten.

The preferred vaccines have a low endotoxin content, for example less than 1 IU / ml and preferably <0.5 IU / ml.

The vaccine is preferably free of antibiotics (eg neomycin, kanamycin, polymyxin B).

The composition may comprise material for a single immunization or may include material for multiple immunizations (i.e. a 'multidose' composition). Multidose arrangements generally include a preservative in the vaccine.

Influenza vaccines are generally administered in a dosage volume of about 0.5 ml, although half a dose (i.e., about 0.25 ml) may be administered to children and unit doses accordingly, for example, a unit dose will be chosen to give a dose of 0.5 ml for administration to a patient.

Packaging of compositions or kit components

The processes of the invention may include a step in which vaccine is placed in a container, and in particular in a dispensing container for use by physicians.

Suitable containers for the vaccines include disposable files and syringes that must be sterile.

When a composition / component is in a vial, the vial is preferably glass or plastic. The vial is preferably sterilized before the composition is added thereto. The vial may comprise a single dose of vaccine or it may comprise more than one dose (a 'multidose' vial) for example 10 doses. Preferred vials are colorless glass.

A vial may have a stopper (eg a Luer Lock stopper) adapted in such a way that a pre-filled syringe can be inserted into the stopper, that the contents of the syringe can be expelled into the vial and that the contents of the vial can be removed with the syringe.

Vaccine Products and Kits The invention can be used to make various different vaccines.

A vaccine made using the invention comprises (i) a hemagglutinin H1 of subtype A influenza virus that is more closely related to SEQ ID NO: 1 than SEQ ID NO: 3 and (ii) an adjuvant in the form of an oil emulsion in water. This composition is a monovalent inactivated surface antigen vaccine. The inactivated viruses could be grown on eggs or in cell culture (for example in these MDCK cells). The vaccine can be presented in a syringe containing a unit dose of 0.5 ml, each unit dose comprising about 7.5 μg of hemagglutinin H1. The syringe may be glass with a plunger made of bromobutyl rubber. The adjuvant comprises squalene, polysorbate 80 and sorbitan trioleate, for example about 9.75 mg of squalene, about 1.18 mg of polysorbate 80 and about 1.18 mg of sorbitan trioleate per 7.5. HA. The composition may comprise a citrate buffer. The composition is ideally free of mercury although a small dose of thimerosal may sometimes be included. In some embodiments, an adjuvanted vaccine has 3.75 μg HA, especially when a 0.25 ml assay volume is used.

Another vaccine made using the invention comprises (i) an H1 hemagglutinin of subtype A influenza virus that is more closely related to SEQ ID NO: 1 than SEQ ID NO: 3 and (ii) ) an adjuvant in the form of an oil emulsion in water. This composition is a monovalent inactivated surface antigen vaccine. The inactivated viruses could be grown on eggs. The vaccine may be presented in a vial containing multiple unit doses of 0.5 ml, for example a 10 dose vial comprising thimerosal, each unit dose comprising about 7.5 μg or 15 μg or 30 μg of the hemagglutinin H1. . The adjuvant comprises squalene, polysorbate 80 and sorbitan trioleate, for example about 9.75 mg squalene, about 1.18 mg polysorbate 80 and about 1.18 mg sorbitan trioleate per 7.5 μg of HA. The composition may comprise a citrate buffer.

Another vaccine made using the invention comprises (i) an H1 hemagglutinin of subtype A influenza virus that is more closely related to SEQ ID NO: 1 than SEQ ID NO: 3 and (ii) ) an adjuvant in the form of an oil emulsion in water. This composition is a monovalent inactivated surface antigen vaccine. Inactivated viruses were grown in MDCK cells. The vaccine is presented with a unit dose containing 3.75 μg of hemagglutinin H1. The adjuvant comprises squalene, polysorbate 80 and sorbitan trioleate, for example about 4.875 mg of squalene, about 0.59 mg of polysorbate 80 and about 0.59 mg of sorbitan trioleate. The composition may comprise a citrate buffer. A unit dose may have a volume of 0.25 ml. The vaccine can be distributed as a pack of 10 x 0.25 ml dose.

Another vaccine made using the invention comprises (1) an H1 hemagglutinin of subtype A influenza virus that is more closely related to SEQ ID NO: 1 than SEQ ID NO: 3 and (ii) ) an adjuvant comprising and aluminum phosphate and aluminum hydroxide. In one useful embodiment, this composition is an inactivated whole virion monovalent vaccine. The inactivated viruses may have grown on eggs. The vaccine can be presented in a syringe containing a unit dose of 0.5 ml, each unit dose comprising about 15 μg of hemagglutinin H1. The adjuvant may comprise, in a volume of 0.5 ml, about 0.5 mg of Al +++, for example 0.45 mg of aluminum phosphate and 0.05 mg of hydrated aluminum hydroxide. The composition may comprise 50 μg of thiomersal.

Another vaccine made using the invention comprises (1) an H1 hemagglutinin of subtype A influenza virus that is more closely related to SEQ ID NO: 1 than SEQ ID NO: 3 and (ii) ) an adjuvant comprising aluminum phosphate. In a useful embodiment, this composition is an inactivated whole virion monovalent vaccine (e.g. obtained by formaldehyde inactivation). The inactivated viruses could be grown on eggs. The vaccine can be presented in a vial containing a unit dose of 0.5 ml, each unit dose containing about 6 μg of hemagglutinin H1 (eg of a reassortant strain such as X-179A). The hemagglutinin can be adsorbed on the aluminum phosphate adjuvant which can be in gel form. The composition may contain thiomersal.

Another vaccine made using the invention is a non-adjuvanted vaccine comprising a subtype A influenza virus H1 hemagglutinin that is more closely related to SEQ ID NO: 1 than SEQ ID NO: 3 ( for example, strain A / California / 7/2009). This composition is an inactivated whole virus inactivated vaccine for which inactivated viruses have been grown on Vero cells. The vaccine can be presented in a vial containing multiple doses, for example 10 x unit dose of 0.5 ml, each unit dose containing about 7.5 μg of hemagglutinin H1 (i.e., 75 μg per multidose vial). The composition may comprise trometamol, sodium chloride and polysorbate 80.

Another vaccine made using the invention is a non-adjuvanted vaccine comprising a subtype A influenza virus H1 haemagglutinin which is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3 ( for example, strain A / California / 7/2009). This composition is an inactivated inactivated fragmented virion vaccine for which inactivated viruses have been grown on eggs. The vaccine may be presented in a vial containing multiple doses (e.g., 10 x unit dose of 0.5 ml with a thimerosal preservative which may be present at 45 μg per 0.5 ml unit dose) or in syringes (one dose per syringe). Each dose of vaccine may have 7.5, 15 or 30 μg of haemagglutinin H1. The vaccine may comprise a phosphate buffer.

The invention may also be used for the manufacture of a kit comprising (i) a first kit component comprising an unadjuvanted subtype A influenza virus H1 hemagglutinin that is more closely related to SEQ ID NO: 1 to SEQ ID NO: 3, manufactured using the invention, and (ii) a second kit component comprising an adjuvant in the form of an oil emulsion in water. Both kit components can be mixed at the time of use to give a monovalent vaccine. The first kit component is a monovalent inactivated vaccine with fragmented virions. The inactivated viruses could be grown on eggs. The kit may be presented as a two-vial composition, each vial comprising an equal volume of liquid to be mixed in a volume ratio of 1: 1, for example to mix 2.5 ml of antigen with 2.5 ml of adjuvant. A unit dose of 0.5 ml of the adjuvanted monovalent vaccine may comprise 7.5 μg, 3.75 μg or 1.9 μg of the hemagglutinin H1. The adjuvant comprises squalene, DL-α-tocopherol and polysorbate 80, for example in a unit dose of 0.5 ml: about 10.7 mg of squalene, about 11.9 mg of tocopherol and about 4.9 mg polysorbate 80 (or a fractional amount thereof, for example 3/4, 2/3, 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1 / 8, 1/9, or 1/10 of these amounts of squalene, tocopherol and polysorbate 80). Thus, the components of the adjuvant may be present in a mass ratio (squalene: tocopherol: polysorbate 80) of 2.20: 2.44: 1. The components of the adjuvant may be present at 2.85 μg. of squalene, 3.16 μg of tocopherol and 1.30 μg of polysorbate 80 per μg of haemagglutinin H1. The vaccine may contain thiomersa.l preservative e.g. at about 10 μg / ml, i.e., about 5 μg in a 0.5 ml dose. Both the antigen and adjuvant components may comprise a phosphate buffer. The antigen component may comprise polysorbate 80, octoxynol 10, potassium chloride and / or magnesium chloride. One kit may comprise 50 vials of antigen (2.5 ml of suspension in each) and 50 vials of adjuvant (2.5 ml of emulsion in each). The antigen vials may be in a single package; the adjuvant flasks may be in two packages.

Another kit comprises (i) a first kit component comprising an unadjuvanted subtype A influenza virus H1 hemagglutinin which is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3, manufactured using the invention, and (ii) a second kit component comprising an adjuvant in the form of an oil emulsion in water. The two kit components can be mixed at the time of use to give a monovalent vaccine. In a useful embodiment, the first kit component is a split-virion inactivated monovalent vaccine. The inactivated viruses may have grown on eggs. The kit may be presented as a two-vial composition (eg borosilicate glass, optionally with chlorobutyl stoppers), the first vial containing a unit volume of antigen and the second vial containing 3x this unit volume of emulsion, for example to mix to give 4x the unit volume of final vaccine. Thus, 1.5 ml of antigen can be combined with 4.5 ml of emulsion to give 6 ml of vaccine. A unit dose of 0.5 ml of the unadjuvanted monovalent vaccine may comprise about 3.8 μg of the hemagglutinin H1. The adjuvant comprises squalene, sorbitan oleate, polyoxyethylene cetostearyl ether and mannitol, for example in a unit dose of 0.5 ml: about 12.4 mg squalene, about 1.9 mg of sorbitan oleate, about 2.4 mg of polyoxyethylene cetostearyl ether and about 2.3 mg of mannitol (or a fraction thereof, for example 3/4, 2/3, 1/2, 1 / 3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9 or 1/10). Thus, the components of the adjuvant can be present in a mass ratio (squalene: sorbitan oleate: polyoxyethylene cetostearyl ether: mannitol) of 124: 19: 24: 23. The vaccine may contain the thiomersal preservative e.g. at about 11.3 μg per 0.5 ml or at about 3 μg of thiomersal per μg of hemagglutinin. Both the antigen and adjuvant components may comprise a phosphate buffer.

The vaccines mentioned in this section may usefully contain a hemagglutinin comprising SEQ ID NO: 7.

Preferred vaccines meet 1, 2 or 3 of the CPMP efficacy criteria. For adults (18-60 years), these criteria are: (1)> 70% seroprotection, (2)> 40% seroconversion, and / or an increase in GMT> 2.5 times. In the elderly (> 60 years), these criteria are: (1)> 60% seroprotection, (2)> 30% seroconversion and / or (3) an increase in MGT> 2 times. based on open trials with at least 50 patients The criteria apply for each strain in a vaccine.

Virus reassortants

As mentioned above, the invention may utilize a strain of reassortant influenza virus. In such reassortant virus, (a) the viral hemagglutinin gene encodes a hemagglutinin which is more closely related to SEQ ID NO: 1 than SEQ ID NO: 3 and (b) at least one other viral gene comes from the strain of influenza virus (A / Puerto Rico / 8/34). Thus, the virus may comprise at least one of the NP, M, NS, PA, PB1 and / or PB2 segments of A / PR / 8/34.

In another reassortant virus, (a) the viral hemagglutinin gene encodes a hemagglutinin protein that has at least k% sequence identity with SEQ ID NO: 1, where k is 85 or greater, e.g. , 88, 90, 92, 94, 95, 96, 97, 98, 99 or more (e.g. 100), and (b) at least one other viral gene is from the strain of influenza virus (A / Puerto Rico / 8/34). Thus, the virus may comprise at least one of the NP, M, NS, PA, PB1 and / or PB2 segments of A / PR / 8/34.

In these viruses, the viral neuramidase gene can encode a neuramidase protein that has at least one sequence identity with SEQ ID NO: 4, where 75 is or greater, for example 75, 80, 85, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more (e.g., 100). In some embodiments, neuramidase is more closely related to SEQ ID NO: 4 than SEQ ID NO: 5.

The eight influenza A genome segments encode (i) the PA subunit of the viral polymerase, (ii) the PB1 subunit of the viral polymerase, and (iii) the viral polymerase subunit PB2. (iv) viral nucleoprotein, (v) viral matrix proteins, (vi) NS1 and NS2 viral proteins, (vii) hemagglutinin and (viii) neuramidase. The preferred reassortant viruses are 6: 2 reassortants, i.e. they comprise 6 segments of a strain (eg A / PR / 8/34) but the HA and NA segments of a strain different {for example as defined above with reference to SEQ ID NOs 1 and 4). In other embodiments, there is a 7: 1 reassortant with HA as defined above. In other embodiments, the virus comprises genes of three different origins but with at least one segment (eg 1, 2, 3, 4, 5, 6) from A / PR / 8/34.

Viral segments of A / PR / 8/34, and their sequences, are widely available.

These viruses are particularly useful for preparing vaccines and can be conveniently prepared by reverse genetics. Vaccines comprising one or more of the viral genes of A / PR / 8/34 may be used to prepare inactivated influenza vaccines.

The reassortant viruses can be cultured in MDCK cells and the invention uses a method for preparing a vaccine comprising the steps of: (i) infecting a cell culture with a virus as defined above, (ii) culturing the cell culture of step (i) to produce other viruses, (iii) purifying the viruses obtained in step (ii), and (iv) treating purified viruses in step (iii) to prepare a vaccine. The vaccine can be a vaccine in bulk. It can be used to produce a monovalent final vaccine product or it can be used as a component to make a final multivalent vaccine product. The cell culture in step (i) is preferably a culture of MDCK cells but other cells (ideally mammalian cells such as PER.C6 cells) can be used as an alternative.

The invention also provides a method for preparing a vaccine comprising the steps of (i) preparing a reassortant influenza virus using reverse genetics, for example with a viral hemagglutinin gene encoding a hemagglutinin which is more closely related to the SEQ ID NO: as SEQ ID NO: 3; (ii) use the reassortant strain to make a vaccine. Step (ii) may include: culturing a virus (e.g., in eggs or in cell culture) and preparing the vaccine from the cultured virus. The cultured virus can be inactivated, and the inactivated virus can then be used to prepare the vaccine (for example after fragmentation or after purification of the antigens).

The invention also provides a method for preparing a vaccine comprising a step of using a reassortant influenza virus as defined above, which has been prepared using reverse genetics.

Overview

The term "comprising" includes "containing" as well as "consisting", for example a composition comprising X may consist exclusively of X or may contain something more, for example X + Y.

The word "essentially" does not exclude "completely", eg a composition that is "essentially free" from Y may be completely free of Y. When necessary, the word "essentially" may be omitted from the definition of the invention.

The term "about" relating to a numerical value x is optional and means, for example x ± 10%.

Subject to specific indication, a process comprising a step of mixing two or more components does not require a specific mix order. So the components can be mixed in any order. When there are three components, two components can then be combined with each other and the combination can then be combined with the third component, etc.

Where animal (and in particular bovine) material is used in cell culture, it must be obtained from sources which are free of transmissible spongiform encephalopathies (TSE) and in particular free from bovine spongiform encephalopathy (BSE). Overall, culture cells are preferred in the complete absence of animal-derived material.

When a cell substrate is used for reassortment or reverse genetics procedures, it is preferably a substrate that has been approved for use in the production of human vaccines, for example as in the general chapter 5.2.3 of the Ph Eur .

An identity between polypeptide sequences is preferably determined by the Smith-Waterman homology search algorithm as implemented in the Oxford Molecular (MPSRCH) program using an affine gap search with the parameters gap open penalty = 12 and gap extension penalty = 1.

MODES FOR CARRYING OUT THE INVENTION Focetria ™ and Celtura ™ Products

Either SPF eggs (for Focetria ™) or a suspension culture of MDCK cells (for Celtura ™) were infected with a H1N1 reassortant strain of influenza A virus with a hemagglutinin that is more closely related to SEQ ID NO: 1 as in SEQ ID NO: 3. Viruses were grown using known techniques, then collected and inactivated, and monovalent surface antigen vaccines were prepared from the purified viruses. The purified antigens were diluted and then combined with an oil-in-water emulsion comprising submicron squalene droplets (MF59 ™) to provide the bulk vaccine for the Focetria ™ product (having 7.5 μg haemagglutinin). per unit dose of 0.5 ml) and Celtura ™ product (having 3.75 μg haemagglutinin per unit dose of 0.5 ml). The products are introduced into syringes. Therefore, the products are distributed in pre-filled syringes for injection, for example in the deltoid or anterolateral part of the thigh. Both products are safe and effective and have been licensed for human use in various territories.

Others products

As indicated in reference 52, a non-adjuvanted fragmented viral monovalent vaccine was prepared from A / California / 7/2009 H1N1sw strain in embryonated chicken eggs using the same standard techniques as those used for production of seasonal trivalent inactivated vaccine CSL [53], Two different vaccines were manufactured: one with a dose of 15 μg of HA (volume of 0.25 ml) and another with a dose of 30 μg of HA (volume of 0 , 5 ml).

Reference 54 provides details of manufacture of an inactivated whole viral monovalent vaccine with aluminum phosphate adjuvant having 6 μg HA per dose.

It is understood that the invention has been described by way of example only and that modifications may be made while remaining within the scope and spirit of the invention.

REFERENCES

[1] Vaccines, (eds., Plotkin & Orenstein). 4th edition, 2004, ISBN: 0-7216-9688-0.

[2] WO02 / 28422.

[3] WO02 / 067983.

[4] WO02 / 074336.

[5] WO01 / 21151.

[6] W002 / 097072.

[7] WO2005 / 113756.

[8] Huckriede stallion (2003) Methods Enzymol373: 74-91.

[9] Herlocher et al. (2004) J Infect Dis 190 (9): 1627-30.

[10] Gambaryan & Matrosovich (1992) J. Virol Methods 39 (1-2): 111-23.

[11] Mastrosovich et al. (1999) J Virol 73: 1146-55.

[12] Stevens et al. (2006) J. Mol Biol 355: 1143-55.

[13] Couceiro & Baum (1994) Inst Inst Oswaldo Cruz 89 (4): 587-91.

[14] Kitikoon et al. (2009) Vaccine doi: 10.1016 / j.vaccine.2009.09.130.

[15] Hoffmann et al. (2002) Vaccine 20: 3165-3170.

[16] Subbarao et al. (2003) Virology 305: 192-200.

[17] Liu et al. (2003) Virology 314: 580-590.

[18] Ozaki et al. (2004) J. Virol. 78: 1851-1857.

[19] Webby stall. (2004) Lancet 363: 1099-1103.

[201] WO97 / 37000.

[21] Brands et al. (1999) Dev Biol Stand 98: 93-100.

[22] Halperin stallion (2002) Vaccine 20: 1240-7.

[23] Tree et al. (2001) Vaccine 19: 3444-50.

[24] Kistner et al. (1998) Vaccine 16: 960-8.

[25] Kistner et al. (1999) Dev Biol Stand 98: 101-110.

[26] Brühl et al. (2000) Vaccine 19: 1149-58.

[27] Pau stall. (2001) Vaccine 19: 2716-21.

[28] EP-A-1260581 (WO01 / 64846).

[29] WO2006 / 071563.

[30] WO2005 / 113758.

[31] WO2006 / 027698.

[32] W003 / 023021 [33] W003 / 023025 [34] W097 / 37001.

[35] WO01 / 22992.

[36] Hehme ef al. (2004) Virus Res. 103 (1-2): 163-71.

[37] Treanor et al. (1996) J Infect Dis 173: 1467-70.

[38] Keitel et al. (1996) Clin Diagn Lab Immunol3: 507-10.

[39] EP-B-0870508.

[40] US 5948410.

[41] WO2007 / 052163.

[42] WO90 / 14837.

[43] Podda & Del Giudice (2003). Expert Rev Vaccines 2: 197-203.

[44] Podda (2001) Vaccine 19: 2673-2680.

[45] Vaccine Design: The Subunit and Adjuvant Approach (eds., Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X).

[46] Adjuvant Vaccine: Preparation Methods and Research Protocols (Volume 42 of the Molecular Medicine Methods). ISBN: 1-59259-083-7. Ed O'Hagan.

[47] US-2007/014805.

[48] WO2007 / 052155.

[49] WO 2008/128939.

[50] Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472.

[51] Banzhoff (2000) Immunology Letters 71: 91-96.

[52] Greenberg stallion (2009) NEJM 361 (10.1056 / NEJMoa0907413).

[53] Talbot et al. (2008) Vaccine 26: 4057-61.

[54] Vajo et al. (2010) Lancet 375: 49-55.

Seqlist DE-BE-FR.tXt SEQUENCE LISTING

<110> NOVARTIS AG

<120> VACCINES FOR PROTECTING AGAINST INFLUENZA

<130> FILEREF

<140> APPNUM <141> 2010-04-27 <150> US 61 / 214,787 <151> 2009-04-27 <150> US 61 / 216,198 <151> 2009-05-13 <150> US 61 / 238,628 < 151> 2009-08-31 <150> US 61 / 279,665 <151> 2009-10-22 <160> 7 <170> Patentin version 3.5 <210> 1 <211> 566

<212> PRT

<213> Influenza A virus <400> 1

Met Lys Al to Ile Leu val val Leu Leu Tyr Tyr Phe Ala Thr Ala Asn 15 10 15

Ala Asp Thr Leu Cys Is Gl Gln Tyr His Ala Asn Asp Ser Thr Asp Thr 20 25 30

Val Asp Thr Val Leu Glu Lys Asn Thr Thr Val Thr His Ser Val Asn 35 40 45

Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gl y Val 50 55 60

Ala Pro Leu Leu Leu Gl y Lys Cys Asn Ile Ala Gl y Trp Ile Leu Gl y 65 70 75 80

Asn Pro Glu Cys Glu Ser Ser Ser Thr Ser Ser Ser Ser Ser Ser 85 85 95

Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe 100 105 110

Isle Asp Tyr Glu Glu Leu Arg Glu Gin Leu Ser Ser Val Ser Ser Phe 115 120 125

Glu Arg Phe Glu Isle Phe Pro Lys Thr Ser Ser Pro Trp Asn His Asp 130 135 140

Seqlist DE-BE-FR.txt

Ser Asn Lys Gly Val Thr Al has Ala Cys Pro His Al has Gly Al has Lys Ser 145 150 155 160

Phe Tyr Lys Asn Leu Ile Trp Leuval Lys Lys Gly Asn Ser Tyr Pro 165 170 175

Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val 180 185 190

Leu Trp Gly Ile His His Ser Ser Pro Ser Ala Asp Gin Gin Ser Leu 195 200 205

Tyr Gin Asn Ala Asp Thr Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser 210 215 220

Lily Lys Phe Lys Pro Glu Ile Ala Island Arg Pro Lys Val Arg Asp Gin 225 230 235 240

Glu Gly Arg Met Asn Tire Tyr Trp Thr Leuk Glu Pro Gly Asp Lys 245 250 255

Thr Phe Island Glu Ala Thr Gly Asn Leu val val Pro Arg Tyr Ala Phe 260 265 270

Ala Met Glu Arg Asn Ala Gly Ser Gly Island Isle Ser Ser Asp Thr Pro 275 280 285 val His Asp Cys Asn Thr Thrs Cys Gin Thr Pro Lys Gly Ala Asn Island 290 295 300

Thr Ser Leu Pro Phe Gin Asn Island His Pro Island Thr Island Gly Lys Cys 305 310 315 320

Pro Lys Tyr val Lys Ser Thr Lys Leu Arg Ala Leu Thr Gly Leu Arg 325 330 335

Asn Island Ser Pro Gin Ser Arg Gly Leu Phe Gly Ala Island Ala Gly 340 345 350

Phe Glu Island Gly Gly Trp Thr Gly Val Asp Gly Trp Tyr Gly Tyr 355 360 365

His His Gin Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser 370 375 380

Thr Gin Asn Ala Island Asp Glu Island Thr Asn Lys Val Asn Ser Val Island 385 390 395 400

Glu Lys Met Asn Thr Gin Phe Thr Ala Val Gly Lys Glu Asa Phe His 405 410 415 seqlist de-be-fr.txt

Leu Glu Lys Arg Glu Asn Leu Asn Lys Val Asp Asp Asp Gl y Phe 420 425 430

Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Leu Leu Leu Glu Asn 435 440 445

Glu Arg Thr Asp Asp Asp His Asp Asn Asn Lys Asn Leu Tyr Glu 450 455 460

Lys Val Arg Ser Gin Leu Lys Asn Asn Ala Lys Glu Island Gly Asn Gly 465 470 475 480

Cys Phe Glu Phe Tyr His Lys Asp Cys Asn Thr Cys Met Glu Ser Val 485 490 495

Lys Asn Gly Thr Asp Tyr Asp Tyr Lys Tyr Ser Glu Glu Ala Lys Leu 500 505 510

Asn Arg Glu Glu Asp Asp Gly val Lys Leu Glu Ser Thr Arg Tyr Island 515 520 525

Gin Ile Leu Ala Island Tyr Ser Thr val Ala Ser Ser Leu Val Leu val 530 535 540 val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu 545 550 555 560

Gin Cys Arg Ile Cys Island 565 <210> 2 <211> 326

<212> PRT

<213> Influenza A virus <400> 2

Asp Thr Leucys Island Gly Tyr His Ala Asn Asn Ser Thr Asp Asp val 15 10 15

Asp Thr val Leu Glu Lys Asn val Thr val Thr His Ser ser val Asn Leu 20 25 30

Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly Val Ala 35 40 45

Pro Leu Leu Leu Gly Lys Cys Asn Island Ala Gly Trp Ile Leu Gly Asn 50 55 60

Pro Glu Cys Glu Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Isle Val 65 70 75 80

Seqlist DE-BE-FR.txt

Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe Ile 85 90 95

Asp Tyr Glu Glu Leu Arg Glu Gin Leu Ser Ser Val Ser Ser Phe Glu 100 105 110

Arg Phe Glu Isle Phe Pro Lys Thr Ser Ser Pro Trp Asn His Asp Ser 115 120 125

Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser Phe 130 135 140

Tyr Lys Asn Leu Trp Leu Val Lys Lys Gly Asn Ser Tire Pro Lys 145 150 155 160

Leu Ser Lys Ser Tire Ile Asn Asp Lys Gly Lys Glu Val Leu Val Leu 165 170 175

Trp Gly Isle His His Ser Ser Pro Ser Ala Asp Gin Gin Ser Leu Tyr 180 185 190

Gin Asn Ala Asp Thr Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser Lys 195 200 205

Lys Phe Lys Pro Glu Island Ala Island Arg Pro Lys Val Arg Asp Gin Glu 210 215 220

Gly Arg Met Asn Tire Tyr Trp Thr Leuval Glu Pro Gly Asp Lys Ile 225 230 235 240

Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe Ala 245 250 255

Met Glu Arg Asn Ala Gly Ser Gly Island Isle Ser Ser Asp Thr Pro val 260 265 270

His Asp Cys Asn Thr Thr Cys Gin Thr Pro Lys Gly Ala Asn Thr 275 280 285

Ser Leu Pro Phe Gin Asn Island His Pro Island Thr Island Gly Lys Cys Pro 290 295 300

Lys Tyr val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg Asn 305 310 315 320

Pro Island Ser Gin Ser Island 325 <210> 3 <211> 566

<212> PRT

Seqlist DE-BE-FR.txt <213> Influenza A virus <400> 3

Met Lys Al Leuk Leu Leu Val Leu Leu Leuk Leu Al Ser Asp 15 15

Ala Asp Thr Island Cys Island Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30

Val Asp Thr val Leu Glu Lys Asn Val Thr val Thr His Ser val Asn 35 40 45

Leu Leu Glu Asp Asn His Asn Gly Lys Leu Cys Lys Leu Lys Gly Ile 50 55 60

Ala Pro Leu Gin Leu Gly Lys Cys Ser Ile Ala Gly Trp Ile Leu Gly 65 70 75 80

Asn Pro Glu Cys Glu Ser Leu Phe Ser Lily Lys Ser Trp Ser Tyr Ile 85 90 95

Ala Glu Thr Pro Asn Ser Glu Asn Gly Thr Cys Tyr Pro Gly Tyr Phe 100 105 110

Ala Asp Tyr Glu Glu Leu Arg Glu Gin Leu Ser Ser Val Ser Ser Phe 115 120 125

Glu Arg Phe Glu Isle Phe Pro Lys Glu Ser Ser Trp Pro Lys His Asn 130 135 140 val Thr Lys Gly val Thr Ala Ala Cys Ser His Lys Gly Lys Ser Ser 145 150 155 160

Phe Tyr Arg Asn Leu Leu Trp Leu Thr Glu Lily Asn Gly Ser Tyr Pro 165 170 175

Asn Leu Ser Lys Ser Tyr val Asn Asn Lys Glu Lys Glu val Leu val 180 185 190

Leu Trp Gly val His His Pro Ser Asn Island Glu Asp Gin Lys Thr Island 195 200 205

Tyr Arg Lys Glu Asn Ala Tyr val Ser val val Ser Ser His Tyr Asn 210 215 220

Arg Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val Arg Asn Gin 225 230 235 240

Glu Gly Arg Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr 245 250 255

Seqlist DE-BE-FR.txt

Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe 260 265 270

Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Thr Island Ser Asn Ala Ser 275 280 285

Met Asp Glu Asp Cys Ala Lys Cys Gin Pro Thr Gin Gly Ala Island Asn 290 295 300

Ser ser Leu Pro Phe Gin Asn val His Pro val Thr Island Gly Glu Cys 305 310 315 320

Pro Lys Tyr Val Arq Ser Thr Lys Leu Arg Met Val Thr Gly Leu Arg 325 330 335

Asn Island Pro Ser Gin Island ser Arg Gly Leu Phe Gly Ala Island Ala Gly 340 345 350

Phe Island Glu Gly Gly Trp Thr Gly Met Island Asp Gly Trp Tyr Gly Tyr 355 360 365

His His Gin Asn Glu Gin Gly Ser Gly Tire Ala Ala Asp Gin Lys Ser 370 375 380

Thr Gin Asn Ala Island Asn Gly Island Thr Asn Lys Val Asn Ser Island Island 385 390 395 400

Glu Lys Met Asn Thr Gin Phe Thr Ala Val Gly Lys Glu Phe Asn Lys 405 410 415

Leu Glu Lys Arg Glu Asn Leu Asn Lys Asp Asp Asp Gly Phe 420 425 430

Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Leu Leu Leu Glu Asn 435 440 445

Glu Arg Thr Leu Asp Phe His Asp Ser Asn val Lys Asn Leu Tyr Glu 450 455 460

Lys Val Lys Ser Gin Leu Lys Asn Asn Ala Lys Glu Island Gly Asn Gly 465 470 475 480

Cys Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met Glu Ser Val 485 490 495

Lys Asn Gly Thr Asp Tyr Asp Tyr Lys Tyr ser Glu Glu Ser Lys Leu 500 505 510

Asn Arg Glu Lily Asp Asp Gly Val Lys Leu Glu Ser Met Gly Val Tyr 515 520 525

Seqlist DE-BE-FR.txt

Gin Ile Leu Ala Tyr Island Ser Thr val Ala Ser Leu Leu val Leu Leu 530 535 540

Val Ser Leu Gly Ala Ser Phe Trp Island Met Cys Ser Asn Gly Ser Leu 545 550 555 560

Gin Cys Arg Ile Cys Island 565 <210> 4 <211> 469

<212> PRT

<213> Influenza A virus <400> 4

Met Asn Pro Asn Gin Lys Island Isle Thr Island Gly Ser val Cys Met Thr 15 10 15

Ile Gly Met Ala Asn Leu Ile Leu Gin Ile Gly Asn Ile Ile Ser Ile 20 25 30

Trp Ser Her Ser Island Gin Island Leu Gly Asn Gin Asn Gin Island Glu Thr 35 40 45

Cys Asn Gin Ser val Thr Island Tyr Asn Gln Asn Thr Trp val Asn Gin 50 55 60

Thr Tyr val Asn Ser Ser Asn Thr Asn Phe Ala Ala Gly Gin Ser val 65 70 75 80 val Ser val Lys Leu Ala Gly Asn Ser Ser Leu Cys Pro val Ser Gly 85 90 95

Trp Ala Island Tyr Ser Lys Asp Asn Ser val Arg Island Gly Ser Lys Gly 100 105 HO

Asp val Phe val Ile Arg Glu Pro Phe Ile Ser Cys Ser Pro Leu Glu 115 120 125 CVS Arq Thr Phe Phe Leu Thr Gin Gly Ala Leu Leu Asn Asp Lys His 130 135 140

Ser Asn Gly Thr Ile Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser 145 150 155 160

Cvs Pro Gly Glu Val Pro Pro Pro Ser Asn Ser Ser Arg Phe Glu Ser 165 170 175

Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Ile Asn Trp Leu Thr 180 185 190

Gly Island Ser Gly Pro Asp Asn Gly Ala Valley Ala Val Leu Lys Tyr Seqlist DE-BE-FR.txt 195 200 205

Asn Gly Island Island Thr Asp Thr Island Lys Ser Trp Arg Asn Asn Island Leu 210 215 220

Arg Thr Gin Glu Ser Glu Cys Al Cys Val Asn Gly Ser Cys Phe Thr 225 230 235 240

Val Met Thr Asp Gly Pro Asn Ser Gly Gin Ala Ser Tyr Lys Phe Island 245 250 255

Arq Island Glu Lys Gly Lys Island Val Lys Ser Glu Val Met Asn Ala Pro 260 265 270

Asn Tire His Tyr Glu Glue Cys Ser Cys Tire Pro Asp ser ser Glu Island 275 280 285

Thr Cys val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp val 290 295 300

Ser Phe Asn Gin Asn Leu Glu Tyr Gin Island Gly Tyr Isle Cys Ser Gly 305 310 315 320

Ile Phe Gly Asp Asn As Pro Pro Asn Asp Lys Thr Gly Ser Cys Gly 325 330 335

Pro val Ser ser Asn Gly Ala Asn Gly val Lys Gly Phe Ser Phe Lys 340 345 350

Tyr Gly Asn Gly val Trp Gly Arg Island Thr Lys Ser Ser Ser Ser Arg 355 360 365

Asn Gly Phe Glu Met Trp Island Asp Asn Pro Gly Trp Thr Gly Thr Asp 370 375 380

Asn Asn Phe Ser Island Lys Gin Asp Island Val Gly Island Asn Glu Trp Ser 385 390 395 400

Gly Tyr Ser Gly Ser Phe val Gin His Glu Pro Leu Thr Gly Leu Asp 405 410 415

Cys Island Arg Pro Cys Phe Trp Val Glu Leu Arg Arg Island Arg Lys Pro 420 425 430

Glu Asn Thr Island Trp Thr Ser Ser Gly Ser Ser Ser Phe Cys Gly Val 435 440 445

Asn Ser Asp Thr val Gly Trp Pro Ser Trp Asp Gly Pro Ala Glu Leu 450 455 460

Phe Thr Island Asp Lys

Seqlist DE-BE-FR.txt 465 <210> 5 <211> 470

<212> PRT

<213> Influenza A virus <400> 5

Met Asn Pro Asn Gin Lys Island Thr Island Gly Isle Ser Island Cys Met Thr 15 10 15

Ile Gly Ile Ile Ser Leu Ile Leu Gin Ile Gly Asn Ile Ile Ser Ile 20 25 30

Trp Val Ser Ser Ser Gin Island Gly Gly Ser Asn His Thr Gly Island 35 40 45

Cys Asn Gin Arg Island Thr Island Tyr Glu Asn Thr Ser Trp Val Asn Gin 50 55 60

Thr Tyr val Asn Island Asn Asn Thr Asn Val Val Ala Gly Lys Asp Thr 65 70 75 80

Thr Ser Val Leu Leu Ala Gly Asn Ser Ser Leu Cys Pro Arg Gly Island 85 90 95

Trp Ala Island Tyr Ser Lys Asp Asn Ser Island Arg Island Gly Ser Lys Gly 100 105 HO

Asp val Phe Val Ile Arg Glu Pro Phe Ser Cys Ser Ser His Leu Glu 115 120 125

Cys Arg Thr Phe Phe Leu Thr Gin Gly Ala Leu Leu Asn Asp Lys His 130 135 140

Ser Asn Gly Thr val Lys Asp Arg Ser Pro Tyr Arg Ala Leu Met Ser 145 150 155 160

Cys Pro Gly Glu Ala Pro Ser Pro Tyr Asn ser Arg Phe Glu Ser 165 170 175 Ala Trp Ser Ser Ala Ser Ala Cys His Gly Met Gly Trp Leu Thr 180 185 190

Gly Island Ser Gly Pro Asp Asp Gly Ala Valley Ala Val Leu Lys Tyr 195 200 205

Asn Gly Island Thr Island Glu Thr Isle Lys Ser Trp Arg Lys Arg Ile Leu 210 215 220

Arg Thr Gin Glu Ser Glu Val Cys Val Asn Gly Ser Cys Phe Thr 225 230 235 240

Seqlist DE-BE-FR.txt Met Thr Asp Gly Pro Ser Asn Gly Pro Ala Ser Tyr Arg Ile Phe 245 250 255

Lys Island Glu Lys Gly Lys Island Thr Lys Ser Island Glu Leu Asp Ala Pro 260 265 270

Asn Ser His Tyr Glu Glu Cys Ser Asp Tyr Cys Thr Thr Gly Thr val 275 280 285

Met Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val 290 295 300

Ser Phe Asn Gin Asn Leu Asp Tyr Gin Island Gly Tyr Isle Cys Ser Gly 305 310 315 320

Val Phe Gly Asp Asn Pro Arg Pro Lys Asp Gly Lys Gly Ser Cys Asp 325 330 335

Pro Val Thr Asp Val Asp Gly Ala Asp Gly Val Lys Gly Phe Tyr Ser Arg 340 345 350

Tyr Gly Asn Gly val Trp Gly Arg Island Thr Lys Ser Asn Ser Ser Arg 355 360 365

Lys Gly Phe Glu Met Asp Trp Island Asp Asn Gly Trp Asp Asp Thr Asp 370 375 380

Ser Asn Phe Leu val Lys Gin Asp val Val Ala Met Thr Asp Trp Ser 385 390 395 400

Gly Tyr Ser Gly Ser Phe val Gin His Glu Pro Leu Thr Gly Leu Asp 405 410 415

Cys Met Arg Pro Cys Phe Trp Val Glu Leu Val Arg Gly Arg Pro Arg 420 425 430

Glu Gly Thr Thr val Trp Ser Ser Gly Ser Ser Ser Ser Phe Cys Gly 435 440 445

Val Asn Ser Asp Thr Ala Asn Serp Pro Trp Asp Gly Ala Glu Leu 450 455 460

Pro Phe Thr Asp Lys Island 465 470 <210> 6 <211> 566

<212> PRT

<213> Influenza A virus <400> 6

Seqlist DE-BE-FR.txt

Met Lys Ala Island Leu val val Leu Leu Tyr Tyr Phe Ala Thr Ala Asn 15 10 15

Ala Asp Thr Leucys Island Gly Tyr His Ala Asn Asn Ser Thr Thr Asp 20 25 30

Val Asp Thr Val Leu Glu Lys Asn val Thr val Thr His Ser val Asn 35 40 45

Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly val 50 55 60

Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly 65 70 75 80

Asn Pro Glu Cys Glu Ser Ser Ser Thr Ser Ser Ser Ser Ser Ser 85 85 95

Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe 100 105 HO

Tire Tyr Asp Glu Glu Leu Arg Glu Gin Leu Ser Ser Ser Ser Ser Phe 115 120 125

Glu Arg Phe Glu Isle Phe Pro Lys Thr Ser Ser Pro Trp Asn His Asp 130 135 140

Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser 145 150 155 160

Phe Tyr Lys Asn Leu Ile Trp Leuval Lys Lys Gly Asn Ser Tyr Pro 165 170 175

Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val 180 185 190

Leu Trp Gly Ile His His Ser Ser Pro Ser Ala Asp Gin Gin Ser Leu 195 200 205

Tyr Gin Asn Ala Asp Ala Tyr val Phe Val Gly Ser Ser Arg Tyr Ser 210 215 220

Lys Thr Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Arg 225 230 235 240

Glu Gly Arg Met Asn Tire Tyr Trp Thr Val Glu Pro Gly Asp Lys 245 250 255

Thr Phe Island Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe 260 265 270

Seqlist DE-BE-FR.txt

Ala Met Glu Arg Asn Ala Gly Ser Gly island island island Asp Asp Pro 275 280 285 val His Asp Cys Asn Thr Thr Cys Gin Thr Pro Lys Gly Ala island Asn 290 295 300

Thr Ser Leu Pro Phe Gin Asn Island His Pro Island Thr Island Gly Lys Cys 305 310 315 320

Pro Lys Tyr val Lys Ser Thr Lys Leu Arg Ala Leu Thr Gly Leu Arg 325 330 335

Asn Island Ser Pro Gin Ser Arg Gly Leu Phe Gly Ala Island Ala Gly 340 345 350

Phe Glu Island Gly Gly Trp Thr Gly Val Asp Gly Trp Tyr Gly Tyr 355 360 365

His His Gin Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser 370 375 380

Thr Gin Asn Ala Island Asp Glu Island Thr Asn Lys Val Asn Ser val Island 385 390 395 400

Glu Lys Met Asn Thr Gin Thr Phe Thr Ala Gly Lys Glu Phe Asn His 405 410 415

Leu Glu Lys Arg Glu Asn Leu Asn Lys Asp Asp Asp Gly Phe 420 425 430

Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Leu Leu Leu Glu Asn 435 440 445

Glu Arq Thr Asp Asp Asp His Asp Asn Asn Val Lys Asn Leu Tyr Glu 450 455 460

Lys Val Arq Ser Gin Leu Lys Asn Asn Ala Lys Glu Island Gly Asn Gly 465 470 475 480

Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val 485 490 495

Lys Asn Gly Thr Asp Tyr Asp Tyr Lys Tyr Ser Glu Glu Ala Lys Leu 500 505 510

Asn Arg Glu Glu Asp Asp Gly val Lys Leu Glu Ser Thr Arg Tyr Island 515 520 525

Gin Ile Leu Ala Tyr Island Ser Thr val Ser Ala Ser Ser Leu Val Val 530 535 540

Seqlist DE-BE-FR.tXt

Val Ser Leu Gly Ala Ser Phe Trp Island Met Cys Ser Asn Gly Ser Leu 545 550 555 560

Gin Cys Arg Ile Cys Island 565 <210> 7 <211> 526

<212> PRT

<213> influenza A virus <400> 7

Asp Thr Leu Cys Island Gly Tyr His Ala Asn Asn Ser Thr Thr Asp Val 15 10 15

Asp Thr val Leu Glu Lys Asn val Thr val Thr His Ser ser val Asn Leu 20 25 30

Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly Val Ala 35 40 45

Pro Leu Leu Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly Asn 50 55 60 Pro Glu Cys Glu Ser Ser Ser Thr Ser Ala Ser Ser Trp Ser Tyr Island Val 65 70 75 80

Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe Island 85 90 95

Asp Tyr Glu Glu Leu Arg Glu Gin Leu ser ser val Ser Ser Phe Glu 100 105 110

Arq Phe Glu Island Phe Pro Lys Thr ser Ser Serp Pro Asn His Asp Ser 115 120 125

Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser Phe 130 135 140

Tyr Lys Asn Leu Ile Trp Leu val Lys Lys Gly Asn Ser Tyr Pro Lys 145 150 155 160

Leu Ser Lys Ser Tyr Asn Asp Asp Lys Gly Lys Glu Leu Val Leu Leu 165 170 175

Trp Gly Island His His Ser Ser Pro Ser Ala Asp Gin Gin Ser Leu Tyr 180 185 190

Gin Asn Ala Asp Ala Tyr val Phe Val Gly ser ser Arg Tyr Ser Lys 195 200 205

Seqlist DE-BE-FR.tXt

Thr Phe Lys Pro Glu Isle Ala Island Arg Pro Lys Val Arg Asp Arg Glu 210 215 220

Gly Arq Met Asn Tire Tyr Trp Thr Leuval Glu Pro Gly Asp Lys Ile 225 230 235 240

Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe Ala 245 250 255

Met Glu Arg Asn Ala Gly Ser Gly Island Isle Ser Ser Asp Thr Pro val 260 265 270

His Asp Cys Asn Thr Thr Cys Gin Thr Pro Lys Gly Ala Asn Thr 275 280 285

Ser Leu Pro Phe Gin Asn Island His Pro Island Thr Island Gly Lys Cys Pro 290 295 300

Lys Tyr val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg Asn 305 310 315 320

Ser Island Pro Gin Ser Island 325

Claims (11)

A method for making an influenza vaccine, wherein the vaccine is adjuvanted with an adjuvant in the form of an oil emulsion in water, comprising a step of preparing vaccine antigens from a influenza having a hemagglutinin comprising an amino acid sequence HA1 having at least 95% sequence identity with SEQ ID NO: 2. A method for making a vaccine, wherein the vaccine is adjuvanted with an adjuvant in the form of an oil emulsion in water, comprising the steps of: (i) preparing a reassortant influenza virus using reverse genetics, wherein the virus has a hemagglutinin comprising an amino acid sequence HA1 having at least 95% sequence identity with SEQ ID NO: 2; and (ii) using the reassortant strain to make a vaccine by growing a virus in eggs or in cell culture, and preparing the vaccine from the cultured virus. A method for making a vaccine, comprising a step of using a reassortant influenza virus which is prepared using reverse genetics, wherein the reassortant virus has a hemagglutinin comprising an amino acid sequence HA1 having at least 95 % sequence identity with SEQ ID NO: 2, and wherein the vaccine is adjuvanted with an adjuvant as an oil emulsion in water. The method of any one of the preceding claims, wherein the vaccine is prepared from inactivated influenza virions. The method of claim 4, wherein the vaccine comprises whole virions, fragmented virions or purified surface antigens. The method of any one of the preceding claims, wherein the hemagglutinin comprises SEQ ID NO: 7. The method of any one of the preceding claims, wherein the virus has neuramidase that is more closely related to SEQ ID NO: 4 than SEQ ID NO: 5. The method of any one of the preceding claims, wherein the virus comprises one or more RNA segments of A / PR / 8/34 virus. The method of any one of the preceding claims, wherein the virus is grown on eggs. The method of any one of claims 1 to 9, wherein the virus is grown in cell culture. The method of claim 10, wherein the virus is grown in MDCK cell culture or Vero cell culture.