KR20120027276A - Adjuvanted vaccines for protecting against influenza - Google Patents

Abstract

A vaccine containing H1 subtype Influenza A virus hemagglutinin is adjuvanted as an oil-in-water emulsion adjuvant. The vaccine is suitable for immunizing humans against a virus called swine influenza. The vaccine may be a monovalent vaccine. The vaccine may comprise two different H1 subtype influenza A virus hemagglutinin, wherein (i) the first H1 subtype influenza A virus hemagglutinin is SEQ ID NO: 1 rather than SEQ ID NO: 3 More closely associated with (ii) the second H1 subtype influenza A virus hemagglutinin is more closely associated with SEQ ID NO: 3 than SEQ ID NO: 1. The monovalent vaccine may be administered with a trivalent A / H1N1-A / H3N2-B seasonal influenza vaccine.

Description

ADJUVANTED VACCINES FOR PROTECTING AGAINST INFLUENZA}

The present invention relates to the field of vaccines adjuvanted against influenza virus infections, in particular to strains, such as the swine flu strain, which emerged in April 2009.

In April 2009, it was confirmed that many countries, including Mexico and the United States, developed swine flu in humans, and then spread rapidly around the world. In June 2009, it was declared an infectious disease by the WHO. The disease is caused by newly identified swine influenza virus A / California / 04/2009 A (H1N1). This strain of swine flu, including A (H1N1) in current human seasonal vaccines, appears to lack immunological cross-reactivity with current human influenza vaccine strains. The virus is called variously, such as swine influenza, new swine-derived H1N1 influenza, human-swine influenza, and new influenza A (H1N1) and influenza A (H1N1).

There is a need for a vaccine that can prevent the transmission of these swine flu viruses and their variants from human to human.

According to a first aspect of the invention, a vaccine containing hemagglutinin of the H1 subtype influenza A virus is immunized with an oil-in-water emulsion adjuvant. This hemagglutinin induces an immune response at the receptor, and the adjuvant increases the heterovariant coverage of the immune response. Although certain H1 antigens cannot protect against their swine flu, the adjuvant increases the immune response, so that the vaccine hemagglutinin can protect against the low immune cross-reactivity of the swine flu with hemagglutinin. . Furthermore, if the vaccine contains hemagglutinin that immune cross reacts with swine flu swine flu hemagglutinin, not only protection against homologous strains, but also their variants such as naturally occurring drift strains is possible.

Accordingly, the invention provides a method of immunizing a swine flu patient (generally human) comprising administering to a patient a vaccine comprising (i) H1 subtype influenza A virus hemagglutinin and (ii) an oil-in-water emulsion adjuvant To provide.

In some embodiments, H1 hemagglutinin is more closely associated with SEQ ID NO: 1 than SEQ ID NO: 3, and in other embodiments more closely related to SEQ ID NO: 3 than SEQ ID NO: 1.

The present invention provides an immunogenic composition comprising (i) an H1 subtype influenza A virus hemagglutinin that is more closely related to SEQ ID NO: 1 than SEQ ID NO: 3 and (ii) an oil-in-water emulsion adjuvant. The composition may be a monovalent vaccine (ie, comprising hemagglutinin antigens from a single influenza virus species), but in some embodiments, for example, comprising H3N2 influenza A virus hemagglutinin and influenza B virus hemagglutinin It may be a multivalent vaccine.

According to a second aspect of the invention, the invention provides an immunogenic composition comprising two different H1 subtype influenza A virus hemagglutinin, wherein (i) the first H1 subtype influenza A virus hemagglutinin More closely related to SEQ ID NO: 1 than SEQ ID NO: 3, and (ii) the second H1 subtype influenza A virus hemagglutinin is more closely related to SEQ ID NO: 3 than SEQ ID NO: 1 The composition contains an oil-in-water emulsion adjuvant as an immune adjuvant. This mixture of immunopotentiated H1 hemagglutinin provides a broader spectrum of protection than currently available for H1 influenza A virus species. The composition may also comprise (iii) H3N2 and / or (iv) influenza B virus antigens. In some embodiments, the composition comprises (iii) H3N2, (iv) B / Victoria / 2/87 like influenza B virus species, and (v) B / Yamagata / 16/88 like influenza B virus species.

According to a third aspect of the invention, a monovalent vaccine containing H1 subtype influenza A virus hemagglutinin is administered with a trivalent A / H1N1-A / H3N2-B seasonal influenza vaccine, wherein all of these vaccines are oil-in-water It is boosted with an emulsion. The monovalent vaccine comprises an H1 subtype influenza A virus hemagglutinin that is more closely associated with SEQ ID NO: 1 than SEQ ID NO: 3, and the trivalent vaccine is more with SEQ ID NO: 3 than SEQ ID NO: 1 Closely related H1 subtype Influenza A virus hemagglutinin. The monovalent vaccine may be administered before, after or concurrently with the trivalent vaccine. If two vaccines are administered separately, the dosing interval may be 2-26 weeks. In one useful embodiment, a patient receiving a trivalent seasonal vaccine (immunized with FLUAD ™ product) first receives a monovalent vaccine (immunized). As shown herein, pre-administration of an immunoenhanced trivalent seasonal vaccine may enhance the efficacy of the monovalent H1N1 vaccine with erythrocyte coagulation which is more closely related to SEQ ID NO: 1 than SEQ ID NO: 3 .

In a related embodiment, the monovalent vaccine containing H1 subtype influenza A virus hemagglutinin is administered with a tetravalent A / H1N1-A / H3N2-BB seasonal influenza vaccine, wherein the two B strains are B / Victoria / 2/87 like strains and B / Yamagata / 16/88 like strains, both of which are immunopotentiated with oil-in-water emulsions. The monovalent vaccine comprises an H1 subtype influenza A virus hemagglutinin that is more closely associated with SEQ ID NO: 1 than SEQ ID NO: 3, and the tetravalent vaccine is more with SEQ ID NO: 3 than SEQ ID NO: 1 Closely related H1 subtype Influenza A virus hemagglutinin. The monovalent vaccine may be administered before, after or concurrently with the trivalent vaccine. If two vaccines are administered separately, the dosing interval may be 2-26 weeks. In one useful embodiment, a patient who first received a monovalent vaccine then receives a tetravalent vaccine.

According to a fourth aspect of the invention, a monovalent vaccine comprising H1 subtype influenza A virus hemagglutinin is administered according to a two-dose regimen, wherein all administrations of the monovalent vaccine are combined with an oil-in-water emulsion Are administered together. The monovalent vaccine comprises an H1 subtype influenza A virus hemagglutinin that is more closely related to SEQ ID NO: 1 than SEQ ID NO: 3. Two doses are administered at 1-6 week intervals, for example at 1, 2, 3, 4, 5, and 6 weeks intervals. In some embodiments, the H1 hemagglutinin is the same for both monovalent vaccines, and in other embodiments, the H1 hemagglutinin in the two monovalent vaccines may differ in different amino acid sequences, eg up to 20 amino acids. (Eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions).

Antigen Ingredient

The present invention uses influenza A virus hemagglutinin as the vaccine antigen. This antigen can generally be prepared from influenza virions, or hemagglutinin can be expressed in recombinant hosts (eg, in insect cell lines using baculovirus vectors), and in tablet form (see references 1, 2 and 3). ) Or virus like particulates (VLPs; see references 4 and 5). Generally, however, the antigen will be derived from virions.

Currently, various forms of influenza virus vaccines are used (see references 17 and 18). Known viruses are generally based on live or inactivated viruses. Antigens in the vaccines of the invention take the form of inactivated viruses. Inactivated vaccines are based on whole virions, split virions, or purified surface antigens. Chemical means for inactivating the virus include treating with an effective amount of one or more detergents, formaldehyde, formalin, β-propiolactone, or UV. Other chemical means include treatment with methylene blue, psoralen, carboxyfullerene (C60) or a combination thereof. Other methods for virus inactivation are known and include, for example, diethylamine, acetylethyleneimine or gamma radiation.

The vaccine may comprise whole virions, split virions, or purified surface antigens (hemagglutinin and generally neuraminidase). Split virions and purified surface antigens (ie subvirion vaccines) are particularly useful in the present invention.

Virions can be harvested from virus containing solutions by various methods. As an example, the purification process may be by zonal centrifugation using a linear sucrose gradient solution containing a virion breaker. The antigen can then be purified by selective dilution, diafiltration.

Split virions, including the 'Tween-ether' splitting process, are ethylether, polysorbate 80, deoxycholate, tri-N-butyl phosphate, Triton X-100, Triton Nl 01, cetyltrimethylammonium bromide, Tergitol Subvirion formulations can be made by treating with substances such as NP9. Methods for splitting influenza viruses are known in the art, see, eg, references 7-12 and the like. Splitting a virus can generally be achieved by destroying or fragmenting an infectious or non-infectious whole virus with a splitting concentration of splitting agent. This disruption alters the integrity of the virus and allows for partial or complete lysis of the viral protein. Preferred splitting agents are nonionic and ionic (eg cationic) surfactants and specific examples include: alkylglycosides, alkylthiothioglycosides, acyl sugars, sulfobetaines, betaines, polys Oxyethylene alkyl ether, N, N-dialkyl-glucamide, hecameg, akylphenoxy-polyethoxyethanol, quaternary ammonium compound, sarcosyl, cetyl trimethyl ammonium bromides (CTABs), tri-N-butyl phosphate , Cetavlon, myristyltrimethylammonium salt, lipofectin, lipofectamine, and DOT-MA, octyl- or nonylphenoxy polyoxyethanol (e.g. Triton surfactants such as Triton X-100 or Triton N101), polyoxyethylene Sorbitan ester (twin surfactant), polyoxyethylene ether, polyoxyethylene ester, etc. are mentioned. One useful splitting process utilizes the continuous effects of sodium deoxycholate and formaldehyde, which can occur during the initial virion purification step, such as sucrose density gradient. Thus, the splitting process involves the purification of the virion-containing material (to remove the non-virion material), and the concentrated, non-viration of the harvested virion (eg, using an adsorption method such as CaHPO4 adsorption). Separation of whole virion from ionic material, virion splitting with splitting agent in density gradient centrifugation step (sucrose gradient containing splitting agent such as sodium deoxycholate), filtration to remove unnecessary material (Eg, ultrafiltration). Typically, split virions can be resuspended in isotonic sodium chloride solution buffered with sodium phosphate. BEGRIVAC ™, FLUARIX ™, FLUZONE ™ and FLUSHIELD ™ products are split vaccines.

Purified surface antigen vaccines also include influenza surface antigen hemagglutinin and, generally, neuraminidase. Processes for preparing these proteins in purified form are well known in the art. FLUVIRIN ™, AGRIPP AL ™ and INFLUVAC ™ products are subunit vaccines.

Influenza antigens may also be provided in the form of virosomes (nucleic acid free virus-like liposome particles), such as the INFLEXAL V ™ and INVAVAC ™ products, but preferably no virosomes are used with the present invention. Thus, in some embodiments, the influenza antigen is not in the form of virosomes.

The hemagglutinin antigen of the vaccine may be from any suitable strain. In some embodiments, hemagglutinin does not cross-react with A / California / 04/2009 hemagglutinin (SEQ ID NO: 1; GI: 227809830) when administered to a human subject in a non-immune enhanced form. Induces agglutinin antibodies; In these embodiments, the adjuvant of the vaccine increases the immune response, allowing the human subject to produce antibodies that do not cross react with A / California / 04/2009 hemagglutinin. In another embodiment, hemagglutinin induces anti-hemagglutinin antibodies that do not cross-react with A / California / 04/2009 hemagglutinin (SEQ ID NO: 1) when administered to a human subject in a non-immune enhanced form Can do it; In these embodiments, the adjuvant of the vaccine increases the immune response to produce a broader spectrum of antibodies that can help human subjects to protect against drift species of A / California / 04/2009. In another embodiment, the hemagglutinin is from A / California / 04/2009 (SEQ ID NO: 1). In another embodiment, the hemagglutinin comprises a HA1 amino acid sequence having at least i % sequence identity with SEQ ID NO: 2, wherein i is at least 85, such as 85, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more (eg 100). Available from any of the following known viral species:

A / swine / Guangxi / 17/2005, A / Swine / Ohio / 891/01, A / Swine / Indiana / 9K035 / 99, A / Swine / Indiana / P12439 / 00, A / swine / Minnesota / 1192/2001, A / SW / MN / 23124-T / 01, A / swine / Guangxi / 13/2006, A / swine / Minnesota / 00194/2, A / SW / MN / l 6419/01, A / Swine / Illinois / 100085 A / 01, A / swine / OH / 511445/2007, A / Swine / Illinois / 100084/01, A / Swine / North Carolina / 93523/01, A / Turkey / MO / 24093/99, A / swine / Korea / PZ4 / 2006, A / swine / Korea / PZ7 / 2006, A / swine / Kansas / 00246/2004, A / swine / Iowa / 24297/1991, A / swine / Korea / CY08 / 2007, A / swine / Korea / JL02 / 2005, A / swine / Maryland / 23239/1991, A / swine / Korea / S 11/2005, A / turkey / IA / 21089-3 / 1992, A / swine / Wisconsin / 1915/1988, A / Swine / Iowa / 930/01, A / swine / Korea / Hongsong2 / 2004, A / Ohio / 3559/1988, A / swine / Iowa / 17672/1988, A / turkey / NC / 19762/1988, A / swine / St-Hyacinthe / 106/1991, A / swine / Korea / JL04 / 2005, A / swine / Korea / JL01 / 2005, A / WI / 4755/1994, A / swine / California / T9001707 / 1991, A / swine / Korea / Asan04 / 2006, A / MD / 12/1991, A / Swine / Wisconsin / 235/97, A / swine / Kansas / 3228/1987, A / Swine / Indiana / 1726/1988, A / swine / On tario / 11112/04, A / Swine / Wisconsin / 163/97, A / SW / MO / 1877/01, A / swine / Shanghai / 3/2005, A / turkey / NC / 17026/1988, A / swine / Iowa / 31483/1988, A / swine / Guangdong / 2/01, A / swine / Iowa / 1/1987, A / swine / Iowa / 3/1985, A / swine / Tennessee / 31/1977. .

In addition, H1N1 species with suitable HA antigens are A / California / 04/2009 itself, A / California / 7/2009, A / Texas / 5/2009, A / England / 195/2009, and A / New York / 18 / 2009 is included.

Preferred embodiments are anti-hemagglutinin which does not cross react with A / California / 04/2009 hemagglutinin (SEQ ID NO: 1) when administered to human subjects in non-immune enhanced form, for example as described above. As such hemagglutinin can be derived from an hemagglutinin antibody comprising an amino acid sequence having at least i % sequence identity with SEQ ID NO: 2.

In some embodiments, hemagglutinin is more closely related to SEQ ID NO: 1 (A / California / 04/2009) than SEQ ID NO: 3 (A / Chile / 1/1983), and in other embodiments, erythrocytes Aggregates are more closely related to SEQ ID NO: 3 than SEQ ID NO: 1. More closely related to SEQ ID NO: 1 than SEQ ID NO: 3 (ie, having a higher degree of sequence identity with SEQ ID NO: 1 than SEQ ID NO: 3 when using the same algorithm and parameters) Erythrocytes are referred to as 'Hl ^* ' hemagglutination. SEQ ID NOs: 1 and 3 have 80.4% identity.

Useful full-length H1 hemagglutinin sequences for use in the present invention include amino acid sequences having at least i % sequence identity to SEQ ID NO: 2 or at least i % sequence identity to SEQ ID NO: 7 as described above. As well as SEQ ID NO: 1 and SEQ ID NO: 6.

Ideally, hemagglutinin should not contain hyper-basic regions near the HA1 / HA2 cleavage site. Preferred hemagglutinin prefers binding to oligosaccharides with Sia (α2,6) Gal terminal disaccharides over oligosaccharides with Sia (α2,3) Gal terminal disaccharides (see below).

SEQ ID NO: 6 (including SEQ ID NO: 7) is a useful H1 ^* hemagglutinin. This is different from SEQ ID NO: 1 at residues 214, 226 and 240 (ie 99.47% sequence identity).

The compositions of the present invention not only comprise H1 hemagglutinin, such as H1 ^* hemagglutinin, and oil-in-water emulsion adjuvant, but also one or more (1, 2, 3, 4 or the like) of influenza A virus and / or influenza B virus. Antigens derived from the above additional influenza virus species.

Thus, the composition may contain at least one H1 ^* hemagglutinin, for example two H1 species (one H1 ^* hemagglutinin and one H1 ^{*) in} addition to the normal seasonal vaccine and the oil-in-water emulsion adjuvant characterized by one or more species. A tetravalent vaccine with one H3N2 species and one influenza B virus species, or two H1 species (one H1 ^* hemagglutinin and one H1 ^* not hemagglutinin) and H3N2 species Pentavalent vaccines with two influenza B virus species (B / Victoria / 2/87 like species and B / Yamagata / 16/88 like species). The present invention also provides a bivalent vaccine comprising H1 ^* hemagglutinin and H5 hemagglutinin and an oil-in-water emulsion adjuvant. If the vaccine comprises one or more influenza species, the different species are generally grown separately and mixed after the virus is harvested to produce the antigen. Thus, the process of the present invention may comprise mixing one or more antigens derived from influenza species.

When the vaccine of the present invention comprises two influenza B, one may include B / Victoria / 2/87 similar species and the other B / Yamagata / 16/88 similar species. They are usually antigenically distinct, but can be described by differences in amino acid sequence to distinguish the two lines, eg, the B / Yamagata / 16/88 analogue is usually (but not always) the 'Lee40' HA sequence Numbering based on (Ref. 14) has a HA protein deleted at amino acid residue 164. In some embodiments of the invention having two or more influenza B virus species as antigens, at least two influenza B virus species may have distinct hemagglutinin but may also have associated neuraminidase. For example, they may all have B / Victoria / 2/87 like neuraminidase (reference 15), or they may all have B / Yamagata / 16/88 like neuraminidase. For example, two B / Victoria / 2 / 87-like neuraminidase may all have one or more of the following sequence specificities: (1) not serine at residue 27 but preferably leucine, (2) residues Not glutamate at 44 but preferably lysine, (3) not threonine at residue 46 but preferably isoleucine, (4) not proline at residue 51, preferably serine, (5) not arginine at residue 65 Preferably histidine, (6) not glycine at residue 70 but preferably glutamate, (7) leucine at residue 73 and preferably phenylalanine, and / or (8) proline at residue 88 but preferably glutamine. Similarly, in some embodiments, neuraminidase may have a deletion at residue 43 or have threonine; Although recent viral species have reacquired threonine at residue 43, the amino acid deletion at residue 43, resulting from deletion of three nucleotides in the neuraminidase (NA) gene, is a B / Victoria / 2/87 like species. It is reported as a feature of (Ref. 15). Conversely, the opposite features can be shared by two B / Yamagata / 16/88 like neuraminidase, such as S27, E44, T46, P51, R65, G70, L73 and / or P88. These amino acids are numbered based on the 'Lee40' neuraminidase sequence (Ref. 16).

Influenza viruses from which hemagglutinin proteins are purified may be resistant to antiviral therapies (eg, resistant to oseltamivir (Ref. 17) and / or zanamivir).

In some embodiments, the viral species used in the present invention have hemagglutinin having binding priority to oligosaccharides with Sia (α2,6) Gal terminal disaccharides as compared to oligosaccharides with Sia (α2,3) Gal terminal disaccharides. . Human influenza viruses bind to polysaccharide receptors with Sia (α2,6) Gal terminal disaccharides (sialic acid to α-2,6 linkage to galactose), but egg and Vero cells are Sia (α2, 3) has a polysaccharide receptor with Gal terminal disaccharides.

The growth of human influenza virus in cells such as MDCK provides selective pressure on the hemagglutinin to maintain native Sia (α2,6) Gal binding, unlike egg passaging. Various assays can be used to determine if a virus has a binding preference for oligosaccharides with Sia (α2,6) Gal terminal disaccharides compared to oligosaccharides with Sia (α2,3) Gal terminal disaccharides. For example, reference 18 describes a solid-phase enzyme-linked assay for influenza virus receptor-binding activity, which provides a sensitive and quantitative measure of the affinity constant. have. Reference 19 used a solid phase assay to assess whether the virus bound to two different sialic glycoproteins (ovomucoid with Sia (α2,3) Gal determinant; and swine α2- with Sia (α2,6) Gal determinant macroglobulin), as well as two receptor analogs, free sialic acid (Neu5Ac) and 3'-sialylated lactose (Neu5Acα2-3Galβ1-4Glc), describe assays that assess virus binding. Reference 20 reports an assay using glycan arrays that can clearly determine receptor affinity for α2,3 or α2,6 linkages. Reference 21 reports an assay based on aggregation of human erythrocytes that have been modified to contain Sia (α2,6) Gal or Sia (α2,3) Gal. Depending on the type of assay, the assay can be performed directly with the virus itself, or indirectly with the hemagglutinin purified from the virus.

In some embodiments, the H1 hemagglutinin has a glycosylation pattern that is different from the pattern observed in egg-derived viruses. Thus, hemagglutinin HA (and other glycoproteins) may comprise glycoforms that are not observed in chicken eggs. Useful hemagglutinin includes glycoforms in dogs.

In addition to including hemagglutinin antigens, the vaccines of the invention generally comprise neuraminidase proteins, such as viral neuraminidase. The present invention may protect against one or more influenza A virus neuraminidase subtypes N1, N2, N3, N4, N5, N6, N7, N8 or N9, but usually N1 (eg H1N1 virus) or N2 (eg H1N2 virus). Whole virions, split virions and subunit vaccines all contain hemagglutinin and neuraminidase. If the vaccine comprises a neuraminidase antigen, this neuraminidase may have at least j % sequence identity with SEQ ID NO: 4, where j is for example 75, 80, 85, 88, 90 , 75, such as 92, 94, 95, 96, 97, 98, 99 or more (eg 100). Many such sequences are used. In some embodiments, neuraminidase is more closely related to SEQ ID NO: 4 than SEQ ID NO: 5. SEQ ID NOs: 4 and 5 have 82% sequence identity to each other.

The vaccine may also include matrix proteins such as M1 and / or M2 (or fragments thereof), and / or nucleoproteins. The swine model shows that the addition of M2 to an inactivated H1N1 swine influenza virus vaccine (immunized with oil in water emulsion) may increase the efficacy of the vaccine (Ref. 22).

Influenza viruses can be reassortant species and can be obtained by reverse genetics techniques. Reverse genetics techniques (Refs. 23-27) can be used to make influenza viruses in vitro with the desired genomic fragments using plasmids or using plasmid-free systems. In general, this technique involves the process of (a) expressing a DNA molecule encoding a desired viral RNA molecule from a poll promoter, and (b) a DNA molecule encoding a viral protein from a poll promoter, wherein both types are intracellularly expressed. DNA is expressed and complete assembly of infectious virus. Preferably, this DNA provides all viral RNAs and proteins, but it is also possible to use a helper virus to provide some RNAs and proteins. Plasmid based methods using separate plasmids to prepare each viral RNA are preferred (Ref. 28-30), and these methods also all or part of viral proteins (eg, PB1, PB2, PA and NP proteins). Plasmids are used to express, and in some methods up to 12 plasmids are used. If dog cells are used, dog poll promoter can be used (Ref. 31).

In order to reduce the number of plasmids required, one approach is [32], multiple RNAs on the same plasmid (eg, sequences encoding all 8 influenza A vRNA fragments). RNA polymerase II on polymerase I transcription cassette (for viral RNA synthesis), and other plasmids (eg, sequences encoding 1, 2, 3, 4, 5, 6, 7 or all 9 influenza A mRNA transcripts) There is a combination of multiple protein-coding regions with promoters. This method comprises: (a) PB1, PB2 and PA mRNA-coding regions on a single plasmid; And (b) all 8 vRNA-encoding fragments on a single plasmid. It is also possible to include NA and HA fragments on one plasmid and six other fragments on another plasmid.

As an alternative to the use of the pol I promoter to encode viral RNA fragments, it is possible to use bacteriophage polymerase promoters [33]. For example, promoters of SP6, T3 or T7 polymerase can be conveniently used. Although cells must be transfected with plasmids encoding exogenous polymerase, the bacteriophage polymerase promoter may be more convenient for many cell types (e.g. MDCK) because of the species-specificity of the pol I promoter.

As another technique, methods using dual pol I and pol II promoters which simultaneously encode viral RNAs and mRNAs expressible from a single template are possible [34, 35].

Influenza A viruses used in the present invention are A / PR / 8/34 viruses (typically six fragments of A / PR / 8/34, including HA and N fragments of vaccine strains such as 6: 2 pathogens) In particular one or more RNA fragments of virus-growing eggs. It may also include one or more RNA fragments from A / WSN / 33 virus or any other virus strain capable of producing a pathogen virus for vaccine production. Typically, the present invention protects against viral strains that are infectious between humans, the genome of which will usually comprise at least one RNA fragment derived from a mammalian (e.g. human) influenza virus.

Viruses used as sources of antigen can be grown on eggs or cell cultures. The current basic method for influenza virus growth is the use of viruses purified from egg products (uremic fluids) in specific pathogen-free (SPF) embryonated hen eggs. Recently, however, viruses have been grown in animal cell cultures, which is desirable for speed and allergy reasons. If egg-based viral growth is used, more than one amino acid will be introduced with the virus into the egg's ureter [12].

In cell culture, the viral growth substrate will typically be a cell line of mammalian origin. Suitable source mammalian cells include, but are not limited to, hamsters, cattle, primates (including humans and monkeys), and canine cells. Various types of cells may be used, such as kidney cells, fibroblasts, retinal cells, lung cells, and the like. Examples of suitable hamster cells are cell lines with BHK21 or HKCC. Suitable monkey cells (eg African green monkey cells) are Vero cell lines, such as kidney cells. Suitable dog cells are MDCK cell lines, such as kidney cells. Thus, suitable cell lines include, but are not limited to, MDCK, CHO, 293T, BHK, Vero, MRC-5, PER.C6, WI-38, and the like. Preferred mammalian cell lines for growth of influenza viruses are MDCK cells derived from Madin Darby canine kidney [36-39], African green monkeys ( Cercopithecus). aethiops ) Vero cells derived from kidney [40-42], PER.C6 cells [43] derived from human embryonic retinal cells, and the like. These cell lines are widely available, for example from American Type Cell Culture (ATCC) collection, Coriell Cell Repositories or European Collection of Cell Cultures (ECACC). For example, ATCC provides a variety of other Vero cells under catalog numbers CCL-81, CCL-81.2, CRL-1586 and CRL-1587, and supplies MDCK cells under catalog number CCL-34. PER.C6 is available from ECACC under accession number 96022940. As an alternative, less preferred for mammalian cell lines, viruses can be grown in avian cell lines, including cell lines derived from ducks (eg duck's retinal cells) or hens [44-46]. Examples of avian cell lines include avian embryonic stem cells [44, 47] and duck retinal cells [45]. Suitable avian embryonic stem cells include EBx cell lines derived from chicken embryonic stem cells EB45, EB14, and EB14-074 [48]. Chicken embryo fibroblasts (CEF) can also be used.

The most preferred cell line for influenza virus growth is the MDCK cell line. The original MDCK cell line is available from CCL-34 of ATCC, but derivatives of this cell line may also be used. For example, Reference 36 discloses an MDCK cell line, which has been adapted for growth in suspension culture ('MDCK 33016' deposited as DSM ACC 2219). Similarly, reference 49 discloses an MDCK-derived cell line, which grows as a suspension in serum-free culture ('B-702' deposited with FERM BP-7449). Reference 50 discloses non-carcinogenic MDCK cells, which are 'MDCK-S' (ATCC PTA-6500), 'MDCK-SF101' (ATCC PTA-6501), 'MDCK-SF102' (ATCC PTA-6502) And 'MDCK-SF1003' (PTA-6503). Reference 51 discloses an MDCK cell line with high susceptibility to infection, including 'MDCK.5F1' cells (ATCC CRL-12042). Any of these MDCK cell lines can be used.

Growth of the virus in mammalian cell lines advantageously results in the elimination of egg proteins (e.g. egg white albumin and ovomucoid) and chicken DNA in the composition, thus reducing the degree of allergy.

If the virus is grown in a cell line and culture for growth, and also viral inoculum is used at the start of the culture, preferably, herpes simplex virus, respiratory syncytial virus, parainfluenzavirus 3, SARS coronavirus, adenovirus, There will be no fear of rhinoviruses, leoviruses, polyomaviruses, vernaviruses, circoviruses, and / or parvoviruses [52]. Particularly preferred is the absence of simple perpesviruses.

During growth on cell lines such as MDCK, viruses can be grown in suspension [36, 53, 54] or in adherent cultures. One suitable MDCK cell line for suspension culture is MDCK 33016 (deposited as DSM ACC 2219). As an alternative, microcarrier cultures can be used.

Cell lines that support influenza virus replication are preferably grown on serum-free culture medium and / or protein free medium. The medium was cited as a serum-free medium in the specification of the present invention, with no additives from serum of human or animal origin. Protein-free is a means of culturing and the proliferation of cells occurs with the exception of proteins, growth factors, other protein additives and non-serum proteins, but optionally contains proteins such as trypsin or other proteolytic enzymes necessary for viral growth. can do. Cells growing in such cultures naturally contain proteins in and of themselves.

Cell lines that support influenza virus replication preferably grow at 37 ° C. during viral replication, for example 30-60 ° C., 31-35 ° C., or 33 ± 1 ° C.

Methods for propagating virus in cultured cells generally include inoculating a cultured cell with a strain to be cultured, growing the infected cell for a period necessary for virus propagation, eg, by viral titer or antigen expression (eg 24 to 168 hours after inoculation), the step of collecting the propagated virus. The cultured cells are inoculated with the virus and the cell ratio (measured by PFU or TCID ₅₀ ) is from 1: 500 to 1: 1, preferably from 1: 100 to 1: 5, more preferably from 1:50 to 1 : 10. The virus is added to the suspension of the cell or applied to the monolayer of the cell, and the virus is absorbed onto the cell for at least 60 minutes, but generally less than 300 minutes, preferably 25 ° C. to 40 ° C. preferably 28 ° C. to 37 ° C. In between 90 and 240 minutes. In order to increase the viral product of the obtained culture supernatant, the infected cell culture (eg monolayer) can be removed by lyolysis or enzymatic activity. The fluid obtained is stored inert or frozen. The cultured cells will be infected at an infection multiplicity (“moi”) of about 0.0001 to 10, preferably 0.002 to 5, more preferably 0.001 to 2. Even more preferably, the cells are infected at a moi of about 0.01. Infected cells can be obtained 30 to 60 hours after infection. Preferably, the cells can be obtained 34 to 48 hours after infection. Most preferably, cells can be obtained 34 to 48 hours after infection. Proteases (typically trypsin) can generally be added during cell culture for viral release and proteases can be added at any suitable stage during the culture.

Hemagglutinin (HA) is the major immunogen for inactivated influenza vaccines, and vaccine doses are standardized with reference to HA levels, typically measured by SRID assay.

Current vaccines generally contain about 15 μg of HA per strain and may also be used at lower dosages, such as when administered to a child or in an emergency situation. Used in divided doses such as 1/2 (ie 7,5 μg HA, FOCETRIA ™ per strain), 1/4 (ie 3.75 μg per strain, PREPANDRIX ™) and 1/8 [56, 57], high doses Also used (eg 3x or 9x doses [58, 59]). Thus the vaccine will comprise 0.1 to 150 μg of HA per influenza strain, preferably 0.1 to 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 And so on. Specific dosages include 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 μg and the like per strain. The same HA mass per strain is common. Low doses (ie <15 μg / dose) are most useful when there is an adjuvant in the vaccine as in the present invention. In some studies (e.g. Ref. 60), doses of up to 90 μg have been used, but compositions of the present invention will generally comprise no more than 15 μg / dose / strain.

HA used in the present invention will be a native HA such as found in viruses or will be modified.

Compositions of the present invention are particularly useful for split or surface antigen vaccines, including polyoxyethylene sorbitan ester surfactants (known as 'Tweens', for example polysorbate 80), octosinol (octosinol-0 (triton) X-100) or 10, or t-octylphenoxypolyethoxyethanol), cetyl trimethyl ammonium bromide ('CTAB'), or sodium dioxycholate. Detergents may only be present in very small amounts. Thus, the vaccine may comprise less than 1 mg / ml of octosinol-10, α-tocopheryl hydrogen succinate and polysorbate80, respectively. Trace amounts of other residual components may be antibiotics such as neomycin, kanamycin, polymycin B, and the like.

Host cell DNA

In viruses growing on cell lines, it is common practice to minimize the amount of residual cell line DNA in the final vaccine to minimize any tumor activity of the DNA. Thus, the composition preferably comprises 10 ng of residual host cell DNA (preferably less than 1 ng, and more preferably less than 100 pg) per dose, even if very small amounts of host cell DNA are present. Generally, host cell DNA is preferably excluded from the compositions of the present invention and the DNA is longer than 100 bp. The determination of residual host cell DNA is now a general regulatory requirement in biology and is at a general level of competence for those skilled in the art. The assay used for DNA measurement will generally be a validated assay [61, 62]. The performance characteristics of proven assays can be described in mathematical and quantitative terms and can identify the sources of their possible errors. Such an analysis can typically test characteristics such as accuracy, precision, and specificity.

After the assay has been adjusted (e.g. for standard quantification of known host cell DNA) and tested, quantitative DNA measurements can generally be operated. Three main techniques can be used for DNA quantification: hybridization methods such as Southern blots or slot blots [63]; Immunoassays such as the Threshold ™ system [64]; And quantitative PCR [65]. Those skilled in the art will readily know these methods, even if there is no specific description of the characteristics of each method depending on the host cell such as selection of probes for hybridization, selection of primers and / or probes for amplification, and the like. From molecular devices, the Threshold ™ system is for quantitative analysis of picogram levels of total DNA and is used as a monitoring level for biopharmaceutically contaminated DNA [64]. General assays include non-sequence-specific formation of the reaction complex between biotinylated ssDNA binding protein, urease-binding anti-ss antibody, and DNA. All assay components are included in a complete complete DNA analysis kit available from the manufacturer. Various commercial manufacturers provide quantitative PCR analysis to detect residual host cell DNA, including AppTec ™ Laboratory Services, BioReliance ™, Althea Technologies, and others. A comparison of the chemiluminescent hybridization assay and the entire DNA Threshold ™ system to determine host cell DNA contamination of human viral vaccines is described in Ref. 66.

DNA contamination can be removed through basic purification procedures such as chromatography in the vaccine manufacturing process. Residual host cell DNAdml removal can be enhanced by nuclease therapy such as using DNAase. A convenient method for reducing host cell DNA contamination is disclosed in references 67 and 68, including two-step treatment, first using DNase (eg benzonase), which can be used for viral growth, Cationic detergents (eg CTAB) that can be used in virion decay. Treatment with alkylating agents, such as β-propiolactone, can also be used to remove host cell DNA, and may advantageously be used to inactivate virions without the use of formaldehyde [69].

Vaccines comprising host cell DNA <10 ng (e.g. <lng, <100 pg) per 15 μg of hemagglutinin are preferred, as are vaccines containing <10 ng (e.g. <lng, <100 pg) of host cell DNA per 0.25 ml volume. More preferred are vaccines containing <10 ng (eg <lng, <100 pg) of host cell DNA per 50 μg of hemagglutinin, as are vaccines containing <10 ng (eg <lng, <100 pg) of host cell DNA per 0.5 ml volume. .

Oil in water Emulsion Adjuvant

The composition of the present invention comprises an oil-in-water emulsion adjuvant, which functions to enhance the immune response (humidity and / or cellular) that is induced in a patient administering the composition. Novartis Vaccines' FLU AD ™ products include oil-in-water emulsions.

 Various suitable emulsions are known and they generally comprise at least one oil and at least one surfactant, these oils and surfactants being biodegradable (metabolic) and biocompatible. Oil droplets in emulsions are generally less than 5 μm in diameter, and advantageously emulsions contain oil droplets of ultrafine particles and these small sizes can be achieved using a microfluidizer to provide a stable emulsion. Droplets having a size of less than 200 nm are preferred, and they can be subjected to filter sterilization. The present invention can be used with oils obtained from animal (such as fish) or plant sources. Sources of plant oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available examples are nut oils. You can also use jojoba oil, such as that obtained from jojoba beans. Seed oils include such things as safflower oil, cotton seed oil, sunflower seed oil, sesame seed oil and the like. In the cereal group, corn oil is most readily available, although other cereal grains may also be used, such as wheat, oats, rye, rice, tef, lymil and the like. 6-10 carbon fatty acid esters of glycerol and 1,2-propanediol, which do not occur naturally in seed oils, can be prepared by hydrolysis, separation and esterification of suitable materials starting from nuts and seed oils. Fats and oils in mammalian milk can be metabolized and used in the present invention. Processes for separation, purification, saponification, and other methods for obtaining pure oils from animal sources are known in the art. Most fish-containing metabolizable oils are easily recovered. For example, various fish oils can be used here, such as cod liver oil, shark liver oil, and whale oil such as sperm. Many branched chain oils are synthesized in 5-carbon isoprene units and are generally called terpenoids. Shark liver oils include branched, unsaturated terpenoids, including 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, known as squalene do. Another preferred oil is tocopherol (see below). Oil-in-water emulsions containing squalene are particularly preferred. Mixtures of oils may also be used.

Surfactants can be classified by their 'HLB' (hydrophile / lipophile balance). Preferred surfactants of the invention have an HLB of at least 10, preferably at least 15, and more preferably at least 16. The present invention may utilize, but is not limited to, the following surfactants: polyoxyethylene sorbitan ester surfactants (commonly called Tweens), in particular polysorbate 20 and polysorbate 80; Copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO) sold under the trade name DOWF AX ™, such as linear EO / PO block copolymers; Octocinol, which contains a diverse number of repeating ethoxy (oxy-1,2-ethanediyl) groups and the octocitol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) of particular interest Having; (Octylphenoxy) polyethoxyethanol (IGEPAL CA-630 / NP-40); Phospholipids such as phosphatidylcholine (lecithin); Polyoxyethylene patty ethers derived from such as lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), triethyleneglycol monolauryl ether (Brij 30); And sorbitan trioleate (sorbitan esters such as Span 850 and sorbitan monolaurate (commonly known as SPANs). Preferred surfactants for inclusion in the emulsion include Tween 80 (polyoxyethylene sorbitan monooleate), Span 80 (sorbitan trioleate), lecithin and Triton X-100 As mentioned above, detergents such as Tween 80 contribute to thermal stability as shown in the examples below.

Surfactants can be used in mixtures, for example in Tween 80 / Span 85 mixtures. Also suitable are combinations of polyoxyethylene sorbitan esters such as polyoxyethylene sorbitan monooleate (Tween 80) and octocitol such as t-octylphenoxypolyethoxyethanol (Triton X-100). Other useful combinations include laureth 9 plus polyoxyethylene sorbitan esters and / or octosinols.

Preferred amounts of surfactant by% are: 0.01 to 1%, in particular about 0.1% polyoxyethylene sorbitan esters (such as Tween 80); Octyl- or nonylphenoxy polyoxyethanol (Triton X-100 or other Triton series of detergents) 0.001-0.1%. Especially 0.005 to 0.02%; Polyoxyethylene ether (such as laureth 9) 0.1 to 20%, in particular 0.1 to 10% and especially 0.1 to 1% or about 0.5%.

Specific oil-in-water emulsion adjuvants useful in the present invention include, but are not limited to:

Ultrafine emulsion of squalene, Tween 80 and Span 85. The composition of the emulsion may be about 5% squalene, about 0.5% polysorbate 80 and about 0.5% Span 85 by volume. By weight, these proportions are 4.3% squalene, 0.5% polysorbate 80 and 0.48% Span 85. These adjuvants are known as 'MF59' [70-72] and are described in more detail in Chapter 10 of Reference 73 and Chapter 12 of Reference 74. The MF59 emulsion advantageously contains citrate ions, such as 10 mM sodium citrate buffer.

Emulsions comprising squalene, α-tocopherol, and polysorbate 80. This emulsion may comprise 2-10% squalene, 2-10% tocopherol and 0.3-3% Tween 80 and the weight ratio of squalene to tocopherol is preferably <1 (e.g. 0.90) to provide a stable emulsion. Squalene and Tween 80 may be present in a volume ratio of about 5: 2 or in a weight ratio of about 11: 5. One such emulsion can be made by dissolving Tween 80 in PBS to obtain a 2% solution, then mixing 90 ml of this solution with a mixture of (5 g DL-α-tocopherol and 5 ml squalene) and then microfluidizing the mixture. have. The resulting emulsion may have oil droplets of submicron, for example average diameter of 100-250 nm, preferably average diameter of 180 nm.

Emulsions including squalene, tocopherol, and triton detergents (eg Triton X-100). This emulsion may also comprise 3d-MPL (see below). This emulsion may contain a phosphate buffer.

Emulsions comprising polysorbates (eg polysorbate 80), triton detergents (eg Triton X-100) and tocopherols (eg α-tocopherol succinate). This emulsion contains these three components in a mass ratio of about 75:11:10 {e.g. 750 μg / ml polysorbate 80, HO μg / ml Triton X-100 and 100 μg / ml α-tocopherol succinate), and these concentrations should have any contribution of these components from the antigen. In addition, the emulsion may comprise squalene. This emulsion may also comprise 3d-MPL (see below). The aqueous phase may contain a phosphate buffer.

Emulsions including squalene, polysorbate 80 and poloxamer 401 (“Pluronic ™ L121”). This emulsion may be formulated with a salt buffered with phosphoric acid, pH 7.4. This emulsion is a useful delivery vehicle for muramyl dipeptides and has been used with threonyl-MDP in the "SAF-I" adjuvant (0.05-1% Thr-MDP, 5% squalene, 2.5% Pluronic L121 and 0.2% polysorbate 80) (Ref. 75). This emulsion may also be used without threonyl-MDP as in the "AF" adjuvant (5% squalene, 1.25% Pluronic L121 and 0.2% polysorbate 80) (Ref. 76). Microfluidisation is preferred.

Include squalene, aqueous solvents and polyoxyethylene alkylether hydrophilic nonionic surfactants (eg polyoxyethylene (12) cetostearyl ether) and hydrophobic nonionic surfactants (eg orbitan monoleate or sorbitan esters or mannide esters such as 'Span 80') Emulsion. Preferably, this emulsion has at least 90% by volume of oil droplets that are thermoreversible and / or have a size of less than 200 nm (Ref. 77). In addition, the emulsion may comprise one or more alditol (e.g. mannitol); Cryoprotective agents (e.g. sugars such as dodecylmaltoside and / or sucrose); And / or alkylpolyglycosides. Such emulsions may be lyophilized. This emulsion may contain squalene: polyoxyethylene cetostearyl ether: sorbitan oleate: mannitol in a mass ratio of 330: 63: 49: 61.

An emulsion comprising 0.5-50% oil, 0.1-10% phospholipids, and 0.05-5% nonionic surfactant. As described in Ref. 78, preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin. Submicron sized droplets are preferred.

Oil-in-water emulsion of submicron comprising non-metabolite oils such as light mineral oil and at least one surfactant such as lecithin, Tween 80 or Span 80. QuilA saponin, cholesterol, a saponin-lipophile conjugate (e.g. GPI-0100 described in Ref. 79, produced by the addition of an aliphatic amine to desacylsaponin via a carboxyl group of glucuronic acid), dimethidioctadecylammonium bromide and And / or additives such as N, N-dioctadecyl-N, N-bis (2-hydroxyethyl) propanediamine.

Emulsions comprising mineral oils, lipophilic ethoxylated fatty alcohols of nonionics, and hydrophilic surfactants of nonionics (eg ethoxylated fatty alcohols and / or polyoxyethylene-polyoxypropylene block copolymers) 80).

Emulsions comprising mineral oils, hydrophilic ethoxylated fatty alcohols of nonionics, and lipophilic surfactants of nonionics (eg ethoxylated fatty alcohols and / or polyoxyethylene-polyoxypropylene block copolymers) 80).

• Emulsions in which saponins (eg QuilA or QS21) and sterols (eg cholesterol) are bound in helical micelles [81].

The antigen and adjuvant in the composition will typically be mixed at the time of delivery to the patient. The emulsion may be mixed with the antigen during the preparation or may be mixed immediately upon delivery. Thus, the adjuvant and antigen can be preserved separately in packaged or disseminated vaccines, ready for final formulation in use. Since the antigen will generally be in aqueous form, the vaccine is finally prepared by mixing the two liquids. The volume ratio of the two liquids to be mixed may vary (eg 5: 1 to 1: 5), but is generally about 1: 1.

After the antigen and adjuvant have been mixed, generally hemagglutinin antigen will be present in the aqueous solution, which can be distributed around the oil / water interface. In general, it is unlikely that erythrocytes enter the oil phase of the emulsion.

If the composition comprises tocopherol, any of α, β, γ, δ, ε or ξ tocopherol may be used, with α-tocopherol being preferred. Tocopherols can take several forms, for example in the form of different salts and / or isomers. Salts include organic salts such as succinate, acetate, nicotinate and the like. Both D-α-tocopherol and DL-α-tocopherol can be used. Tocopherols are advantageously included in vaccines for use in older patients (eg, 60 years of age or older) because vitamin E has been reported to have a positive effect on immune responses in this group of patients [82]. In addition, they have antioxidant properties that can help stabilize emulsions [83]. Preferred α-tocopherol is DL-α-tocopherol and the preferred salt of this tocopherol is succinate. Succinate salts have been found to work in concert with TNF-related ligands in vivo. In addition, α-tocopherol succinate is known to be compatible with influenza vaccines and to be a useful preservative in place of mercury compounds.

As mentioned above, oil-in-water emulsions comprising squalene are particularly preferred. In some embodiments, the squalene concentration in the vaccine dose may range from 5-15 mg (ie, a concentration of 10-30 mg / mL if the dose volume is 0.5 mL). However, it is also possible to reduce the concentration of squalene [84,85], for example it may comprise <5 mg per dose, or even <1.1 mg per dose. For example, a human dose may comprise 9.75 mg squalene per dose (as with FLUAD ™ products: 9.75 mg squalene, 1.175 mg polysorbate 80, 1.175 mg sorbitan trioleate in a 0.5 mL dose volume), Or a fraction of this amount, for example 3/4, 2/3, 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9, Or an amount of 1/10. For example, the composition may contain 7.31 mg squalene / dose (and thus 0.88 mg of polysorbate 80 and sorbitan trioleate, respectively) and 4.875 mg squalene / dose (and thus polysorbate 80 and sorbitan trioleate, respectively) 0.588 mg), 3.25 mg squalene / dose, 2.438 mg squalene / dose, 1.95 mg squalene / dose, 0.975 mg squalene / dose and the like. Any of these fractional dilutions of FLUAD ™ -strength MF59 can be used in the present invention.

As mentioned above, antigen / emulsion mixing can be performed on delivery at the time of delivery. Accordingly, the present invention provides a kit comprising an antigen and an adjuvant component prepared for mixing. In the kit, the adjuvant and antigen can be preserved separately until the point of use. These components are physically separated from each other in the kit, and this separation can be accomplished in a variety of ways. For example, the two components may be contained in two separate containers, such as vials. The contents of the two vials can then be mixed, for example, by taking the contents of one vial and adding the contents to another vial, or by taking the contents of both vials and mixing them in a third vessel. . In a preferred configuration, one of the kit components is in a syringe and the other component is in a container such as a vial. A syringe can be used (eg with a needle) to insert its contents into a second container for mixing, and then the mixture can be aspirated back into the syringe. The mixed contents of the syringe can then be administered to the patient, typically through a sterile new needle. Filling a syringe with one component eliminates the need to use a separate syringe for patient administration. In another preferred configuration, two kit components are held together in the same syringe in a separate state, where a double-chamber syringe is used, such as, for example, those disclosed in references 86-93 and the like. When the syringe is activated (eg during administration to a patient) the contents of the two chambers are mixed. This configuration eliminates the need for a separate mixing step in use.

Pharmaceutical composition

Compositions of the present invention may be pharmaceutically acceptable. These generally include components other than antigens and adjuvants, for example they typically comprise one or more pharmaceutical carrier (s) and / or excipient (s). A full discussion of these components can be obtained from Ref. 94.

The composition will generally be in aqueous form.

The composition may comprise a preservative such as thiomersal (eg 10 μg / mL) or 2-phenoxyethanol. However, the vaccine should preferably be substantially free of mercury material (ie less than 5 μg / mL) and preferably contain no thiomersal, for example [95]. More preferred are vaccines that do not contain mercury. Vaccines that do not contain a preservative are particularly preferred.

It is desirable to include physiological salts such as sodium salts to control toxicity. Sodium chloride (NaCl) is preferred, which may be present at 1-20 mg / mL. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate anhydrous, magnesium chloride, calcium chloride and the like.

The composition will generally have an osmolality of 200 mOsm / kg to 400 mOsm / kg, preferably 240-360 mOsm / kg, with more preferred osmolarities in the range of 290-310 mOsm / kg. Although osmotic concentrations have already been reported to have no effect on the pain caused by vaccination [96], it is still desirable to maintain osmotic concentrations within this range.

The composition may comprise one or more buffers. Typical buffers include phosphate buffers; Tris buffer; Borate buffer; Succinate buffer; Histidine buffer; Or citrate buffer. The buffer will typically be included in the range of 5-2OmM. The buffer may be present in the aqueous phase of the emulsion.

The pH of the composition will generally be 5.0-8.1, more typically 6.0-8.0, for example 6.5-7.5, or 7.0-7.8. Thus, the process of the present invention may include adjusting the pH of the bulk vaccine prior to packaging.

The composition is preferably sterilized. The composition preferably contains no gluten.

Preferred vaccines have a low endotoxin content, for example less than 1 IU / mL, preferably less than 0.5 IU / mL. International units of endotoxin measurement are well known, for example 2 nd available from NIBSC. International Can be calculated for a sample by comparison with international standards [97,98] such as Standard (Code 94 / 580-IS). Endotoxin levels of existing vaccines prepared from viruses grown in eggs range from 0.5-5 IU / mL.

The vaccine preferably does not contain antibiotics (eg neomycin, kanamycin, polymyxin B).

The composition may comprise a substance for one immunization or may comprise a substance for several immunizations (ie, a “multidose” composition). Multidose configurations generally include a preservative in the vaccine. To avoid the need for a preservative, the vaccine can be placed in a container equipped with a sterile adapter for withdrawing the material.

Influenza vaccines are typically administered in a dosage volume of about 0.5 mL, but children may be given half a dose (ie, about 0.25 mL), and the unit dose will be selected correspondingly, for example 0.5 suitable for patient administration. A unit dose that will provide a mL dose will be selected.

Composition or Kit  Packaging of ingredients

The process of the invention may comprise placing the vaccine in a container, in particular in a dispensing container for use by a physician. Suitable containers for the vaccine include vials, nasal sprays and disposable syringes, which should be sterile.

When the composition / component is placed in a vial, the vial is preferably made of glass or plastic material. It is preferred that the vial is sterilized before the composition is put into the vial. In order to avoid problems in latex-sensitive patients, the vial is preferably sealed with a latex-free stopper and it is desirable that all packaging materials be free of latex. The vial may comprise a single dose of the vaccine or may comprise two or more doses (“multidose” vials), eg 10 doses. Preferred vials are made of colorless glass.

The vial may have a suitable cap (eg, Luer lock) into which a prefilled syringe may be inserted into the cap, the contents of the syringe may be released into the vial, and the contents of the vial may enter the syringe again. . After removing the syringe from the vial, a needle can be attached so that the composition can be administered to the patient. The cap is preferably located inside the seal or cover and must be accessible only after the seal or cover is removed. The vial may be provided with a cap, in particular capable of aseptic removal of the contents in a multi-dose vial.

When packaging a composition / component in a syringe, a needle may be attached to the syringe. If no needle is attached, a separate needle may be provided with the syringe for assembly and use. A cover may be used for such a needle. Safety needles are preferred. 1-inch 23-gauge, 1-inch 25-gauge and 5 / 8-inch 25-gauge needles are typical. The syringe is provided with a peel-off label, which may be printed with a lot number, influenza season, and expiration date of the contents, which may facilitate record keeping. The plunger of the syringe preferably has a stopper that can prevent the plunger from being inadvertently removed during suction. The syringe may have a latex rubber cap and / or plunger.

Disposable syringes contain a single dose of the vaccine. The syringe will generally have a tip cap that seals the tip before attaching the needle, which tip cap is preferably made of butyl rubber. In the case where the syringe and the needle are packaged separately, it is preferred that the butyl rubber cover is fitted to the needle. Preferred syringes are those sold under the trade name "Tip-Lok" ™.

The container may be labeled with a half-dose volume, for example to facilitate delivery to the child. For example, a syringe containing a 0.5 mL dose may have an indication indicating 0.25 mL volume.

When using glass containers (eg syringes or vials), preference is given to using containers made of borosilicate glass rather than soda lime glass.

The composition may be provided with a leaflet (eg in the same box) that contains the details of the vaccine, eg, instructions for administration, details of the antigens in the vaccine, and the like. In addition, the instructions may contain warnings, for example, warnings to keep the adrenaline solution readily available in case of anaphylactic reactions following vaccination.

Treatment Methods, and Vaccine Administration

The composition of the present invention is suitable for administration to a human patient, and the present invention provides a method of causing an immune response in a patient comprising administering the composition of the present invention to a patient.

The present invention also provides a kit or composition of the present invention for use as a medicament.

Immune responses arising from the methods and uses of the invention will generally include antibody responses, preferably protective antibody responses. Methods for assessing antibody response, neutralizing dose and protective action following influenza virus vaccination are well known in the art. Human studies have shown that the antibody titer of human influenza virus to hemagglutinin correlates with its protective action (a serum sample hemagglutination-inhibition titer of about 30-40 protects about 50% of infection by homologous virus). [99]. Antibody responses are typically measured by hemagglutination inhibition, microneutralization, single radial immunovariance (SRID), and / or single radial hemolysis (SRH). Such analytical techniques are well known in the art.

The compositions of the present invention can be administered in a variety of ways. The most preferred immunization route is by intramuscular injection (for example in the arm or leg) and other available routes are subcutaneous injection, nasal [100-102], intradermal [103,104], oral [105], transdermal, Transdermal route 106, and the like. Intradermal and nasal passages are good. Intradermal administration may involve, for example, a microinjection device with a needle about 1.5 mm long.

Vaccines prepared according to the invention can be used in the treatment of both children and adults. Influenza vaccines are currently recommended for immunization of children and adults from 6 months of age. Thus, a patient may be less than 1 year old (eg, less than 6 months old), 1-5 years old, 5-15 years old, 15-55 years old, or 55 years old or older. Preferred patients vaccinated are elderly (eg, 50 years, 60 years, and preferably 65 years), young children (eg, 5 years or less, or 6 months to 24 years, or 6 months). To 4 years old, or 5-18 years old), middle aged (25-64 years), inpatient, healthcare worker, military and military, pregnant woman, chronic disease, immunodeficiency patient, 7 days before vaccination Patients who have taken antiviral compounds (eg, oseltamivir or zanamivir compounds; see below), I am allergic and travel abroad. However, the vaccine is not only suitable for this group, but may be used more commonly for the whole country.

Some older adults (approximately one-third of older people over 60) and a few young people may already have serum antibodies against the A / CA / 04/09 pandemic strain, but children are essentially free of antibodies. Immunization of young people does not elicit antibodies against this strain [107]. A useful group of recipients of the immunogenic compositions of the present invention comprising an oil-in-water adjuvant are those who do not have existing serum antibodies against the A / CA / 04/09 pandemic strain, for example, after 1960, Patients born after 1970, after 1980, after 1990, or after 2000.

Preferred compositions of the invention satisfy 1, 2 or 3 in CPMP criteria for efficacy. In adults (18-60 years old) these criteria are: (1) at least 70% serum protection; (2) at least 40% seroconversion; And / or (3) a GMT increase of 2.5 times or more. In the elderly (over 60 years) these criteria are: (1) serum protection of at least 60%; (2) at least 30% seroconversion; And / or (3) a two-fold increase in GMT. These criteria are based on open label studies in at least 50 patients. Criteria apply for each strain contained in the vaccine.

Treatment can be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in the primary immunization schedule and / or in the secondary immunization schedule. In a multiple dose schedule, various doses may be provided by the same or different routes, such as prime administration parenterally and booster administration by mucosal route, prime administration by mucosal route and parenteral booster administration. Administration of two or more doses (typically two doses) is intended for use in patients who have never had an immunological dose, for example in a person who has never received any influenza vaccination, or for a new HA subtype. This is especially useful if you want to. Multiple doses will typically be administered at least one week apart (eg, about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 12 weeks, about 16 weeks, etc.).

The vaccine produced by the present invention can be administered to a patient substantially simultaneously with other vaccines (eg, during the same medical consultation, or when visiting a healthcare professional or immunization agency), for example measles. Vaccine, mumps vaccine, rubella vaccine, MMR vaccine, varicella vaccine, MMRV vaccine, diphtheria vaccine, tetanus vaccine, pertussis vaccine, DTP vaccine, conjugated H. influenzae type b vaccine, inactivated poliovirus vaccine, hepatitis B Viral vaccines, meningococcal conjugate vaccines (eg, trivalent AC-W135-Y vaccines), respiratory syncytial virus vaccines, pneumococcal conjugate vaccines and the like. Administration at the same time as the pneumococcal vaccine and / or meningococcal vaccine is particularly useful in elderly patients.

Similarly, the vaccines of the present invention are substantially concurrent with the antiviral compounds, in particular antiviral compounds (eg, oseltamivir and / or zanamivir) that are active against influenza viruses (eg, the same medical consultation process). Or when visiting a healthcare professional). Such antiviral agents include (3R, 4R, 5S) -4-acetylamino-5- including neuraminidase inhibitors such as esters (eg ethylesters) and salts (eg phosphate salts) Amino-3 (1-ethylpropoxy) -1-cyclohexene-1-carboxylic acid or 5- (acetylamino) -4-[(aminoiminomethyl) amino] -2,6-anhydro-3,4 , 5-trideoxy-D-glycero-D-galactonone-2-enoic acid. Preferred antiviral agents are (3R, 4R, 5S) -4-acetylamino-5-amino-3 (1-ethylpropoxy) -1-cyclohexene-1-carboxylic acid, ethyl ester, phosphate (1: 1) It is also referred to as oseltamivir phosphate (TAMIFLU ™). Another antiviral agent that can be administered is thymosin alpha 1 (eg, thymalpasin, a synthetic peptide of 28 amino acids, available as ZADAXIN ™) [108]. In one specific embodiment, the patient is receiving a neuraminidase inhibitor such as oseltamivir phosphate simultaneously with receiving the inactivated whole virion vaccine (eg monovalent, H1 *).

Vaccine products and Kit

As mentioned above, the present invention provides a vaccine comprising (i) an H1 subtype influenza A virus hemagglutinin that is more closely related to SEQ ID NO: 1 than SEQ ID NO: 3 and (ii) an oil-in-water emulsion adjuvant. . In a useful embodiment, the composition is a monovalent inactivated surface antigen vaccine. Inactivated viruses can be grown in eggs or in cell culture (eg, in MDCK cells [36,118]). The vaccine can be released in a syringe containing 0.5 mL unit dose (eg borosilicate glass), each unit dose containing 7.5 μg of H1 hemagglutinin (eg, reassortant). A / California / 7 / 2009-like strains such as those derived from strain X-179A or X-181). The syringe may have a bromo-butyl rubber plunger-stopper. Adjuvants include 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 HA. Include. The composition may comprise a citrate buffer. The composition is ideally free of mercury, but can sometimes contain small amounts of thimerosal. In some embodiments, the adjuvant added vaccine has a 3.75 μg HA, especially when a 0.25 mL dosage volume is used. The adjuvant-added vaccine can be administered by intramuscular route, for example deltoid muscle or anterior lateral thigh. Adjuvant added vaccines may be inoculated in a single dose or inoculated in two doses (eg, at two to six month intervals, eg at three week intervals). The syringes can each be packed in a blister pack and packed in a box, for example ten boxes.

The present invention also provides a vaccine comprising (i) an H1 subtype influenza A virus hemagglutinin that is more closely related to SEQ ID NO: 1 than SEQ ID NO: 3 and (ii) an oil-in-water emulsion adjuvant. In a useful embodiment, the composition is a monovalent inactivated surface antigen vaccine. Inactivated viruses may be grown in eggs. The vaccine can be released as a vial containing multiple 0.5 mL unit doses, for example as a 10 dose vial containing thimerosal, each unit dose containing about 7.5 μg or 15 μg or 30 μg of H1 hemagglutinin. (Eg A / California / 7 / 2009-like strains such as those derived from recross strain X-179A). Adjuvants include 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 HA. Include. The composition may comprise a citrate buffer. The adjuvant-added vaccine can be administered by intramuscular route, for example deltoid muscle or anterior lateral thigh. Adjuvant added vaccines may be inoculated in a single dose or inoculated in two doses (eg, at two to six month intervals, eg at three week intervals).

In addition, the present invention provides a kit comprising: (i) a first kit component comprising a beer adjuvant H1 subtype influenza A virus hemagglutinin that is more closely related to SEQ ID NO: 1 than SEQ ID NO: 3 and (ii) an oil-in-water emulsion adjuvant It provides a kit comprising a second kit component comprising a. The two kit components can be mixed at the point of use, thereby providing a monovalent vaccine of the present invention. In a useful embodiment, the first kit component is a monovalent split virion inactivating vaccine. Inactivated viruses may be grown in eggs. The kit may be released as a two-vial composition (eg, borosilicate glass, optionally with a butyl rubber stopper), each vial containing an equal volume of liquid for mixing in a 1: 1 volume ratio, for example For example, 2.5 mL antigen and 2.5 mL adjuvant may be mixed. The 0.5 mL unit dose of the adjuvant-added monovalent vaccine may comprise about 7.5 μg, 3.75 μg or 1.9 μg of H1 hemagglutinin (eg, A / California, such as from recrossing strain X-179A). / 7 / 2009-like strain). Adjuvants include, for example, squalene, DL-α-tocopherol and polysorbate 80 in a 0.5 mL unit dose: about 10.7 mg squalene, about 11.9 mg tocopherol and about 4.9 mg polysorbate 80 (or an amount thereof) 3/4, 2/3, 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1 of the amounts of fractions, for example squalene, tocopherol and polysorbate 80 / 8, 1/9 or 1/10). Thus, the adjuvant component may be present in a mass ratio of 2.20: 2.44: 1 (squalene: tocopherol: polysorbate 80). The adjuvant component may be present as 2.85 μg squalene, 3.16 μg tocopherol and 1.30 μg polysorbate 80 per μg H1 hemagglutinin. The vaccine may comprise a thimersal preservative, for example at about 10 μg / ml, ie at about 5 μg in a 0.5 mL dose. Vaccines with adjuvant may be inoculated in a single dose, or inoculated in two doses (eg, at 1, 2 or 3 week intervals, or at intervals greater than 3 weeks, eg For example, at intervals of 3-26 weeks). Adults 18-60 years of age may find it useful to receive a single dose, and older people over 60 may receive two doses. Children aged 3-9 years may receive a half dose, for example, 0.25 mL volume containing 1.875 μg of HA. Both the antigen and the adjuvant component may comprise a phosphate buffer. The antigenic component may comprise polysorbate 80, octoxynol 10, potassium chloride and / or magnesium chloride. Kits of the invention may comprise 50 antigen vials (each containing 2.5 mL suspension) and 50 adjuvant vials (each containing 2.5 mL emulsion). The antigen vial may be in one pack and the adjuvant vial may be in two packs. The adjuvant-added vaccine can be administered by intramuscular route, for example deltoid muscle or anterior lateral thigh.

The present invention also provides a vaccine comprising (i) an H1 subtype influenza A virus hemagglutinin that is more closely related to SEQ ID NO: 1 than SEQ ID NO: 3 and (ii) an oil-in-water emulsion adjuvant. In a useful embodiment, the composition is a monovalent inactivated surface antigen vaccine. Inactivated virus was grown in MDCK cells [36,118]. The vaccine is released in unit doses containing 3.75 μg of H1 hemagglutinin (eg A / California / 7 / 2009-like strains such as those from recross strain X-179A). The adjuvant includes squalene, polysorbate 80 and sorbitan trioleate, and includes about 4.875 mg squalene, about 0.59 mg polysorbate 80 and about 0.59 mg sorbitan trioleate. The composition may comprise a citrate buffer. The adjuvant-added vaccine can be administered by intramuscular route, for example deltoid muscle or anterior lateral thigh. Adjuvant added vaccines may be inoculated in one dose or inoculated in two doses (eg, two to six month intervals, eg three week intervals). The unit dose may have a volume of 0.25 mL, in which the patient immunizes one unit dose (eg, patients aged 3-17 or 18-40 years old) or two unit doses (patients older than 40 years) once You can get it at poetry. The vaccine can be supplied as a pack of 10 0.25 mL doses.

The present invention also provides a kit comprising a (i) a first kit component comprising a beerjuven H1 subtype influenza A virus hemagglutinin that is more closely related to SEQ ID NO: 1 than SEQ ID NO: 3 and (ii) an oil-in-water emulsion adjuvant. A second kit component is included. The second kit component can be mixed in use to provide the monovalent vaccine of the invention. In a useful embodiment, the first kit component is a monovalent inactivated split virion. Inactivated virus could be grown in eggs. The kit may comprise a two-vial composition (eg, a first vial containing a unit volume of antigen and a second vial containing a 3x unit volume of emulsion, e. Optionally borosilicate glass with chlorobutyl stopper). Thus 1.5 ml of antigen can be combined with 4.5 ml of emulsion to give 6 ml of vaccine. The 0.5 ml unit dose monovalent adjuvant vaccine may comprise about 3.8 μg of H1 hemagglutinin (eg, A / California / 7 / 2009-like strains such as recross strain X-179A). The adjuvant is, for example, 0.5 ml unit dose: about 12.4 mg squalene, about 1.9 mg sorbitan oleate, about 2.4 mg polyoxyethylene cetostearyl ether, and about 2.3 mg mannitol (or a partial amount thereof, eg For example, 3/4, 2/3, 1/2, 1/3, 1/4, 1/5, 1/6 of these amounts of squalene, sorbitan oleate, polyoxyethylene cetostearyl ether and mannitol, 1/7, 1/8, 1/9, or 1/10); squalene, sorbitaoleate, polyoxyethylene cetostearyl ether, and mannitol. Thus, the adjuvant component may be present in a mass ratio of 124: 19: 24: 23 (squalene: sorbitan oleate: polyoxyethylene cetostearyl ether: mannitol). The vaccine may comprise a thimerosal preservative, for example about 11.3 μg / 0.5 ml, or about 3 μg of thimerosal / μg of hemagglutinin. Both antigen and adjuvant components may comprise phosphate buffer. Subjects may receive a single dose of adjuvant vaccine or, more generally, two doses (eg, separated by at least 3 weeks, eg 3-26 weeks). Subjects aged 3-60 years may receive a single dose usefully, while older people over 60 may receive two doses. Children over 6 months of age and under 3 years of age can receive a 0.25 ml volume with half the dose, eg about 1.9 μg HA. Adjuvant vaccines can be administered intramuscularly, for example in deltoid muscles or an anterolateral thigh.

The invention also provides a method of making an influenza vaccine comprising mixing a first kit component as defined in the preceding phrase and a second kit component as defined in the preceding phrase.

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

Mixed-Source Vaccine

Some embodiments of the invention mentioned above are multivalent, ie they comprise HA antigens from one or more influenza virus strains. Viruses used to make multivalent vaccines can all be grown using the same substrate (eg, all grown in eggs, or all grown in MDCK culture, etc.) or they can be grown on different substrates ( For example, grow one strain in an egg, grow another strain in a cell culture; or grow one strain in an MDCK culture or another strain in a Vero culture). For example, growth substrates can be selected according to the growth preference of a particular strain, for example, if the H1N1 strain grows better in cell culture than eggs, but influenza B viruses exhibit opposite preferences, they May be grown on different substrates and then mixed.

In one embodiment, the H1 * strain (eg, H1N1) is grown in a cell culture (eg, in an MDCK culture such as suspension culture [36,118]), and in another strain (eg, an H3N2 strain). , Influenza B strains, etc.) can be grown in eggs. Antigens made from strains can then be mixed to provide a multivalent influenza vaccine. This procedure is particularly suitable for preparing 4-valent vaccines with two H1 strains (one H1 * hemagglutinin, one not H1 * hemagglutinin), H3N2 strain, and one influenza B strain.

Accordingly, the present invention provides a vaccine comprising hemagglutinin obtained from at least two different strains of influenza virus, wherein the first hemagglutinin is made from influenza virus grown in eggs and the second hemagglutinin is in cell culture. It is made from grown influenza virus. Thus, there are two different strains of influenza virus, one in cell culture and one in egg. The virus is purified from two sources and then mixed to provide a vaccine.

The first and second hemagglutinin may both come from influenza A virus, both from influenza B virus, or one from influenza A virus and the other from influenza B virus. Preferably at least one of the first and second hemagglutinin is from influenza A virus. If both the first and second hemagglutinin are influenza A viruses, they will typically be H1 hemagglutinin and H3 hemagglutinin, eg, H1N1 strain and H3N2 strain.

If the first and second hemagglutinin comprise influenza A virus hemagglutinin, one of these may be H1 * hemagglutinin. It is preferred that the two influenza A hemagglutinins are both not H1 * hemagglutinin, and more preferably both influenza A hemagglutinin are not H1 hemagglutinin. If the vaccine comprises H1 * hemagglutinin, it is preferably a second hemagglutinin, ie the H1 * strain is grown in cell culture and the H1 * vaccine antigen is then a non-H1 * vaccine prepared from eggs. Combined with the antigen. In another embodiment, the H1 * hemagglutinin is the first hemagglutinin, ie the H1 * strain is grown in eggs and the H1 * vaccine antigen is then combined with a non-H1 * vaccine antigen prepared from cell culture.

Suitable cell culture hosts are described above and include MDCK cells, for example MDCK 33016, which is useful for preparing viruses that can be grown in suspension and have H1 * hemagglutinin.

This mixed source approach is particularly useful for vaccine production, including H1 * strains, non-H1 * H1 strains, H3 strains, and influenza B strains. The H1 * strain can be grown in cell culture, and the other three strains (ie, usually trivalent mixtures for vaccines of recent seasons) can be grown in eggs by conventional methods.

Normal

The term "comprising" includes "comprising" as well as "comprising", for example, a composition "comprising" comprising X may consist exclusively of X or some additional one, for example X + Y. It may also include.

The word "substantially" does not exclude "completely", for example a composition that is "substantially free of Y" may be completely free of Y. If necessary, the word "substantially" may be omitted from the definition of the present invention.

The term "about" with respect to the numerical value x is optional and means for example x ± 10%.

"GI" numbering was used above. The GI number, or "Genlnfo Identifier", is a series of numbers assigned consecutively in each sequence record processed by the NCBI when a sequence is added to its database. The GI number does not have similarity with the registration number of the sequence record. If the sequence is updated (eg, corrected or added to a comment or information), it receives a new GI number. Thus, the sequence associated with a given GI number never changes.

Unless specifically stated otherwise, a process comprising mixing two or more components does not require any particular order of mixing. Thus the components can be mixed in any order. If there are three components, the two components can be mixed with each other and the combination can then be combined with the third component or the like.

If animal (and especially bovine) materials are used in cell culture, they must be obtained from a source free from infectious spongiform encephalopathy (TSE), in particular without mad cow disease (BSE). Overall, culture cells that are entirely free of animal-derived material are preferred.

If the compound is administered to the body as part of a composition, the compound may alternatively be replaced with a suitable prodrug.

If the cell culture is to be used for recrossing or reverse genetics process, it is preferably approved for use in human vaccine production, for example as Ph Eur general chapter 5.2.3.

Identity between polypeptide sequences is preferably by the Smith-Waterman homology search algorithm as supplemented in the MPSRCH program (Oxford Molecular), using an affine gap search with a gap open penalty = 12 and a gap expansion penalty = 1. Is determined.

1 shows HI titers obtained after immunization with an adjuvant H1N1 sw antigen with beer adjuvant (0.5 or 1 μg HA dose) or MF59 (0.5 μg). PBS control was also used. Black bars represent titers after one immunization; Gray bars represent titers after 2 immunizations.
2 shows lung viruses loaded into ferrets immunized with various prime / boost regimens. Animal groups A to H are described below. The y-axis represents Log ₁₀ TCID ₅₀ / gr. 3 shows nasal viral load at the same ferret and y-axis is log ₁₀ Represents a CDU. 4 shows HI titers at the same ferret.
Figure 5 shows IgG serum antibody titers after two H1N1sw boosting administrations in mice primed with seasonal H1N1 (Brisbane) (ELISA). Priming and boosting strains and adjuvant additions are shown.

Ferret  Research

Reference 109 reports a ferret model for investigating influenza vaccines. Ferrets were primed with adjuvant (squalene-containing oil-in-water emulsion, MF59 ™) or beer-juvenant seasonal vaccine, or PBS. After three weeks (21 days) these ferrets received a seasonal or monovalent epidemic ('H1N1sw') vaccine of adjuvant or beerjuvenant trivalent, or a booster dose of PBS. A total of eight animal groups A to H were used:

On day 49, ferrets were immunoassayed with H1N1sw strain (106 TCID50) and lung pathology was evaluated in each group. Unlike the seasonal H1N1, which only infects the nose and organs, the H1N1sw virus also infects the lungs. H1N1sw virus is not lethal for ferrets.

The average percentage of affected lung milk tissues is:

It was.

Thus, compared to the PBS group, all ferrets pre-primed with an adjuvant or beerjuven seasonal vaccine and then boosted with homologous seasonal or H1N1sw have a significant reduction in lung lesions. The best effect was observed when the boosting dose was H1N1sw, and both the priming and boosting vaccines had an adjuvant added (group C). Even in the absence of priming, a single administration of adjuvant H1N1sw provides similar protection.

Pulmonary viral load was evaluated and the results are shown in FIG. 2. In comparison to PBS, administration of one of the adjuvant H1N1sw vaccines reduced pulmonary viral load by 2-3 logs (compare group G & H). If the H1N1sw vaccination preceded the administration of the Beerjuven seasonal influenza vaccine, the viral load in the lungs was reduced to almost undetectable levels (Group F), and if the seasonal vaccine was before the adjuvant was added (Group C) The load was undetectable.

Viral loads can also be assessed from nasal specimens (FIG. 3), compared to PBS, one administration of the adjuvant H1N1sw vaccine, but not the nonjuvenant vaccine, reduced the viral load in the nasal sample by 1 log. If the H1N1sw vaccination was preceded by vaccination with the Beerjuven season season vaccine, the nasal viral load was further reduced (group F). If the H1N1sw vaccination was preceded by vaccination with an adjuvant seasonal vaccine, the nasal viral load is not detectable (group C). Similar results were found in the neck specimens.

HI antibody response was also measured on day 49 (FIG. 4). One dose of the adjuvant H1N1sw vaccine was more immune than the beer adjuvant H1N1sw vaccine. HI titers for H1N1sw virus were increased by at least 1 log in ferrets previously immunized with the adjuvant seasonal vaccine.

HI antibody responses against seasonal sections of H1N1 and H3N2 seasonal strains were also evaluated. Cross-reacting antibodies between seasonal H1N1 and H1N1sw were not detected by HI.

Thus, a single administration of the adjuvant H1N1sw vaccine was more immunogenic and more efficient than the beerjuvenant vaccine as measured by viral load in the lungs, nose, and throat. Immunogenicity and efficiency were both enhanced by pre-immunization with seasonal influenza vaccines, and this effect was better if the seasonal vaccine had an adjuvant. This improvement in immunogenicity and efficiency is not due to the antibody cross-reaction (by HI) between the seasonal H1N1 and H1N1sw.

This result may explain why older people are better protected against H1N1sw virus despite the small cross-reactivity of antibodies. Because this effect can be attributed to prior immunological experience with seasonal viruses, despite little or no cross-reactivity of the antibodies, they also preliminary clinical trials that show good response after a single dose in healthy adults. Explain the negative results. The results also suggest that better protection is achieved in the presence of an adjuvant and if the patient has been previously immunized with an adjuvant seasonal vaccine. The data show that for best and sustained protection, one or more adjuvant H1N1sw vaccines may be used for immunologically naïve individuals (eg, children) and immunologically weak individuals, even though one dose may still be clinically useful. It is proposed that the administration of. A more detailed description of this Ferret study is found in Ref. 110.

Mouse Study I

The benefit of using an oil-in-water emulsion adjuvant with the H1N1sw vaccine is evident from immunogenicity studies conducted in mice. In unprimed mice, one dose of emulsion-adjuvant H1N1sw vaccine, without any prior exposure to influenza antigen, provides HI titers related to protection in humans. However, two doses were required to reach this titer without an adjuvant. Thus, human protection can be achieved with a single adjuvant dose even in children. In contrast, the swine flu vaccine in 1976 required two doses for children and young adults. In addition, since the adjuvant enables single dose immunization even in unprimed subjects, subjects already exposed to influenza (eg, the general adult population) may also have only one week of vaccine to achieve a strong response. Bunt administration is required.

The vaccine was prepared from H1N1sw A / California / 07/2009 H1N1-like virus grown in eggs. The vaccine was with or without the adjuvant of the oil-in-water emulsion comprising squalene (MF59 ™). Vaccines were normalized by SRID at either HA administration of 0.5 μg or 1 μg. 6-7 week old Balb / c mice were immunized intramuscularly on Day 1 with phosphate buffered saline, 0.5 or 1.0 μg (HA content) of antigen alone, or 50 μl of adjuvant with 0.5 μg of antigen. The dose volume was 100 μl. Serum was obtained on day 13. Mice were boosted to the second dose and matched for the first time on day 14. Serum was collected again on day 21. Serum was assessed by hemagglutination inhibition (HI) using whole virus and turkey erythrocytes inactivated against antigen.

Single immunization with 0.5 μg adjuvant antigen resulted in a 1:63 mean agonistic antibody (HI) titer in serum obtained two weeks after immunization. HI titers of 1:40 are associated with human protection from seasonal influenza [111]. After 2 weeks, the second immunization with an adjuvant vaccine increased the average HI titer to 1: 1280 in serum obtained 1 week after the boost. Single immunization with the antigen without adjuvant did not result in significant HI titers, but after 2 weeks the second immunization later resulted in a HI titer of 1: 160. There was no significant difference in titers induced by immunization with 0.5 or 1.0 μg of Beerjubant antigen.

These data are consistent with the results of human immunization with vaccines for other potential infectious influenza strains. Without the adjuvant, vaccines against H5 avian influenza strains result in low antibody titers; MF59 increases the rapidity, titer and broadness of the induced antibodies [112,113]. During the much less human outbreak of swine-borne influenza in 1976, the adjuvant vaccine was not available. Single doses of the vaccine in 1976 resulted in lower antibody titers in younger people, but resulted in significantly higher titers in older subjects, perhaps because older subjects experienced more major influenza infections or immunizations [114]. Mouse immunization data support the inclusion of an adjuvant such as MF59 in the H1N1 sw infectious immunization campaign, especially for children and young adults with little or no prior exposure to influenza infection or immunization. These individuals are particularly vulnerable to morbidity and mortality in current infectious diseases [115]. A single dose given to an immunologically naïve mouse with an MF59-adjuvant infectious antigen produces an antibody response associated with protection from seasonal influenza in humans; Without adjuvant, two doses are required. In this study, 0.5-1 μg of the beerjuvenant antigen did not have an administration response observed. This finding in mice increases the likelihood that a dosing-sparing regimen that can increase the number of doses available is effective in human clinical trials.

Mouse research II

Mice primed with seasonal H1N1 (A / Brisbane / 59/2007; administered with 0.2 μg HA) monovalent vaccine (with or without MF59 adjuvant) were treated with the same vaccine or with the same monovalent vaccine (also with MF59 adjuvant) ) Were prepared from infectious H1N1 sw strains by boosting twice (Days 36 and 66) (A / California / 04/2009 hemagglutinin).

ELISA analysis of the immune response (FIG. 5) suggests that mice are effectively primed for higher titer responses in the H1N1sw vaccine, and this priming is particularly important if the H1N1sw vaccine lacks an adjuvant. In unprimed mice or beer primed mice primed with seasonal H1N1, the H1N1sw vaccine showed high titer response only with an adjuvant.

Therefore, adjuvant addition of the H1N1sw vaccine appears to be important for a strong immune response. In addition, adjuvant additions seem to be important for seasonal vaccines to allow priming for strong antibody responses to infectious vaccines.

In summary, immunization with two doses of beerjube infectious vaccine resulted in little functional antibodies in mice that were not primed or primed with beerjuven seasonal vaccines. However, in mice primed with an adjuvant seasonal vaccine, two doses of the beer adjuvant infectious vaccine provided a good response. Mice reacted strongly with two doses of the adjuvant infectious vaccine, whether or not they were primed. Although adjuvant seasonal vaccines may not efficiently induce antibodies against infectious strains, they may therefore be primed for higher titers to infectious vaccines. These data support the use of oil-in-water adjuvant for infectious immunization and also prepare a population for infectious immunization in seasonal campaigns.

Mouse research III

Three groups of 40 six-week-old female BALB / c mice received one intramuscular injection of the trivalent seasonal vaccine in either the 2005/06 season or the 2009/10 season (both northern hemisphere). Influenza-naive control mice received PBS. Vaccines were administered at 1 / 1Oth human dose (1.5 μg HA / strain) on day 0. On day 40 mice were divided into four subgroups of 10 animals each and re-inoculated with the monovalent inactivated H1N1sw vaccine. The four groups were combined with sorbitan oleate, polyoxyethylene cetostearyl ether and mannitol to produce high or low doses (3 μg HA or 0.3 μg HA) with or without submicron oil-in-water emulsion adjuvant containing squalene. received. All animals then received a second H1N1sw dose on day 61. The presence of HI antibodies against the second and infectious H1N1 strains was assessed at days 40, 61, 75, and 102. A complete detailed description of this mouse is given in Ref. 116.

The results confirmed that a single injection of the H1N1 vaccine was sufficient to elicit an HI antibody response to protection levels with or without an adjuvant. HI antibody titers (GMT) for the H1N1sw strain were> 40 in all groups except the group of naïve mice immunized with 0.3 μg HA of the Beerjuven vaccine.

Antibodies caused by previous seasonal influenza vaccination did not cross-react with the H1N1sw strain, but priming with seasonal influenza vaccine resulted in a higher antibody response to the Beerjubant H1N1sw vaccine. In contrast, previous seasonal immunizations did not appear to affect the immunogenicity of the adjuvant H1N1sw vaccine in mice, as was due to the strong primary response induced by the adjuvant vaccine in these groups.

In conclusion, Mouse Study III supports the use in humans of the split-virion inactivated H1N1sw vaccine formulated with an aqueous squalene emulsion.

Focetria ™ and Celtura ™ products

Either SPF eggs (for Focateria ™) or suspension cultures (for Celtura ™) of MDCK cells were infected with a recrossing H1N1 influenza A virus strain with hemagglutinin associated more closely with SEQ ID NO: 1 than SEQ ID NO: 3 I was. Viruses were grown using known techniques, then collected and inactivated, and monovalent surface antigen vaccines were prepared from purified viruses. Purified antigens were diluted and then combined with an oil-in-water emulsion containing submicron squalene droplets (MF59 ™) and Focetria ™ product (with 7.5 μg hemagglutinin / 0.5 ml unit dose) and Celtura ™ product (3.75 μg Bulk vaccine) was provided for hemagglutinin / 0.25 ml unit dose. Fill the syringe with the product. The product is therefore divided from prefilled syringes for injection. These two monovalent adjuvant products are authorized for human use in various regions.

Person Study I ( Leicester , England)

Monovalent surface antigen vaccines were prepared from the A / California / 7/2009 H1N1sw strain as reported in Ref. 117. The vaccine strain has HA, NA and PB1 gene segments from A / California / 7/2001 H1N1sw and the remaining five segments are from A / PR8 / 8/34. The virus is grown in MDCK cells. Viruses and antigens were prepared using the procedure used to prepare the trivalent OPTAFLU ™ product [118]. Two vaccines were prepared: an adjuvant vaccine with MF59 oil-in-water emulsion containing 7.5 μg HA and submicron squalane drops; And viajuven vaccine with 15 μg HA in buffer. All vaccines had a volume of 0.5 ml. Half the dose of adjuvant vaccine was used for some subjects (ie in 0.25 ml volumes). The HA content of the final vaccine was determined by reverse phase HPLC since no SRID reagent was available.

175 patients were divided into seven groups. Patients received a single dose (day 0) or two identical doses (day 0; day 7, 14 or 21). The seven groups A to G were as follows (dose; adj = adjuvant):

Immunogenicity was assessed on days 0, 14 and 21. Interim evaluation measured immunogenicity immediately prior to administration of the 21st dose. Thus groups A to C completed their regimen, while group D received only one 7.5 μg adjuvant dose. E to G groups were not evaluated at this intermediate stage. Antibody response was assessed by haemagglutination (HI) assay as geometric mean titers (GMT), geometric mean ratios, serum conversion (%) and serum protection (%). Antibody response was also assessed as micro-neutralization (MN) as GMTs, the proportion of patients with titers ≧> 40 (%) or serum conversion. In the interim evaluation the antibody response by HI was as follows:

In the interim evaluation the antibody response by MN was as follows:

Pre-immunized antibodies were detected in 14% and 39% of patients, respectively, by HI assay (titer> 1: 8) and MN assay (titer> 1:10), with the frequency of receiving age or previous seasonal vaccines. It was independent of experience. On day 14, geometric mean titers (GMTs) measured using HI and MN assays were compared to patients who received only a single dose compared to patients who received two 7.5 μg adjuvant doses (for group D). Compared to groups A to C) and there was no significant difference in titers between groups. On day 21, there was no significant difference in titer among patients receiving a single dose or two doses. All patients had MN antibodies at titers greater than 1:40 on day 21.

Thus, at an intermediate stage, the immune response was consistent with serum protection against 2009 H1N1sw virus within two weeks after administration of a single dose of adjuvant vaccine. Single or two doses of adjuvant vaccine containing 7.5 μg of HA administered on various schedules resulted in robust antibody titers. Double doses (15 μg HA) provided higher antibody levels than single doses, but serum protective titers were achieved in at least 80% of patients in all groups.

Person study II

Monovalent inactivated antigen vaccines were prepared from A / California / 7/2009 H1N1sw virus grown in eggs. Antigen was diluted to an HA concentration of 30 μg / ml and mixed with an MF59 oil-in-water emulsion containing submicron squalene drop in citric acid buffer to give an adjuvant bulk with an HA concentration of 15 μg / ml. The adjuvant vaccine was packaged in syringes in individual 0.5 ml doses to provide a vaccine with 7.5 μg HA per 0.5 ml dose. Adjuvant vaccines (eg from FOCETRIA ™) are administered intramuscularly, for example in the deltoid muscle or in the anterior lateral thigh.

Person study III

Monovalent inactivated isolated vaccines from the H1N1sw state were provided to human adult volunteers (ages 18-60). Patients received either an adjuvant vaccine or a beer adjuvant vaccine. The adjuvant vaccine had 5.25 μg HA with submicron oil-in-water emulsions containing squalene (AS03); Beerjuven vaccine had 21 μg HA. The vaccine was administered on days 0 and 21. HI titers for A / California / 7/2009, as well as serum conversion and serum protection were evaluated on these days. Full details of this human study are given in Ref. 119. The vaccine was well tolerated and the immunogenicity results were as follows:

Thus both the adjuvant vaccine and the beer adjuvant vaccine were immunogenic in adults, and a single dose of either 5.25 μg HA (adjuvant) or 21 μg HA (non adjuvant) was sufficient to meet the eligibility criteria. . Adjuvant vaccines with four fold fewer antigens elicited a comparable immune response to the beer adjuvant vaccine.

Person study IV

Adjuvant monovalent H1N1sw vaccine (7.5 μg HA; FOCETRJA ™) was administered to human patients (adults and the elderly) concurrently with, or 3 months later, the trivalent (3 × 15 μg HA) 2009/10 seasonal vaccine (adjuvant or beer adjuvant). Provided. All vaccines were inactivated surface antigen vaccines and the adjuvant was squalene-containing oil-in-water emulsion (MF59 ™). Immunogenicity of all vaccines was assessed by hemagglutination inhibition on Days 1 and 22 and patient diaries were used to monitor safety and response. Full details of this human study are given in Ref. 120.

A single dose of the adjuvant H1N1sw vaccine met eligibility criteria for adult and elderly patients after 3 months of seasonal vaccination in adults or concurrently with seasonal vaccines without affecting the acceptability or immunogenicity of either vaccine.

Person Study V: Combination Therapy

Patients with end-stage renal disease with chronic dialysis were given 7.5 μg per dose of the FOCETRJA ™ vaccine containing H1N1 * hemagglutinin in the form of a monovalent inactivated surface antigen vaccine. The vaccine includes an MF59 ™ submicron oil-in-water emulsion adjuvant containing squalene. Some patients also received thymalfasin (thymosin alpha 1; commercially available as ZADAXIN ™ product), a peptide known for the treatment of hepatitis virus.

Two different doses of thimalfasin were tested to provide a random 3-arm open level study.

Timalpasin was given twice, 1 injection 7 days before vaccination and 2 injections on vaccination day. All patients who did not achieve an antibody titer of at least 1:40 on day 21 received the second vaccination that day.

In comparison to patients receiving only the FOCETRJA ™ vaccine, the addition of thymolfasin at two doses resulted in a statistically significant increase in the percentage of patients seroconverted both 21 and 42 days after vaccination. 93% of patients receiving low doses of thymalpasin (3.2 mg) and 94% of patients receiving high doses (6.4 mg) achieved seroconversion to H1N1 * virus after 42 days and received only vaccine Compared to 77%.

Person study VI

Two multicentral randomized dose range studies evaluated adjuvants (with MF59) and beerjuven egg induction and cell culture induction monovalent H1N1sw vaccines in healthy children from 6 months to 12 years of age. The goal was to identify the desired vaccine formulation (with or without adjuvant), dosage and schedule (single or two doses) in healthy children and adults.

At enrollment, patients stratified into (i) four age cohorts: 9-17 years, 3-8 years, 12-35 months and 6-11 months; (ii) Randomly, three vaccine groups were given 3.75 μg HA + Vi dose MF59, 7.5 μg HA + total dose MF59 or 15 μg HA beer adjuvant. Children aged 9-17 years and infants 6-11 months received only the adjuvant vaccine. Patients received two vaccinations 21 days apart. Vaccines were prepared in eggs or in MDCK cell culture (suspension culture).

Immunogenicity was measured 21 days after each vaccination by hemagglutination inhibition (HI). Geometric mean HI titers (GMT) and geometric mean ratios (GMR) of HI vaccination / pre-vaccination were calculated. Seroconversion, i.e., percent of patients with HI ≥1: 40 post vaccination and patients negative (HI <1:10) at baseline, or at least 4 for patients positive (HI≥1: 10) at baseline Fold increase was also evaluated. Serum protection ratio (SP), i.e., patient's shock with HI titer> 1:40, was also evaluated.

Intermediate presentations were obtained from patients aged 3-8 years and 9-17 years (388 patients who received a cell-guided vaccine and 403 patients who received an egg-induced vaccine). GMT and GMR values in patients receiving cell-induced vaccines were as follows:

GMT and GMR values in patients receiving egg-induced vaccines were as follows:

In both studies the adjuvant vaccine had a SP ratio ≧ 70% after 3 weeks of first and second vaccination in the 9-17 and 3-8 year old cohorts. In both studies, the viajuvant vaccine achieved an SP ratio ≧ 70% in the 3-8 year old cohort three weeks after the second vaccine dose. All vaccines (ages 3-17) in the two age cohorts had a SC ratio ≧ 40% three weeks after the first and second vaccinations in both studies. GMT increased strongly after 3 weeks of each dose and all vaccines had GMRs ≧ 2.5 in both cohorts.

Adjuvant egg induction (FOCETRIA ™) and cell culture induction (CELTURA ™) vaccines elicited a rapid and strong immune response at lower HA doses than the beer adjuvant vaccine. The immunogenicity of all adjuvant vaccines meets the European Regular Pandemic Influenza Vaccine Assessment Criteria (patients with HI titers ≧ 1: 40; seroconversion> 40% and GMR> 2.5) in a single dose.

Person study VII  ( Costa rica )

This study aimed to determine safety and antibody response after administration of an adjuvant (with MF59) or beer adjuvant H1N1sw vaccine in a pediatric population. Vaccines were prepared from viruses grown in eggs. The patients were divided into two age groups (child ages 3-8 years and adult ages 9-17 years) and randomly (a) a single 7.5 μg dose of the adjuvant vaccine, (b) a single 15 μg beer adjuvant dose, or ( c) A 30 μg beer adjuvant dose (2 × l 5 μg dose) was used. Three weeks later, patients received an MF59-adjuvant vaccine (surface antigen vaccine, egg-induced) with 7.5 μg of H5N1 platelet aggregates. Blood samples for serum testing were collected on days 1 (immunization), days 22, 29 and 43. Antibody titers against H1N1 vaccine antigens were assessed for hemagglutination inhibition (HI). Percentages of patients with geometric mean titers (GMTs), serum turnover (SC) ratios and HI titers ≧ 1: 40 of anti-agglutinating inhibitory antibodies were calculated. SC ratios and HI titers ≧ 1: 40 were compared with the Center for Biologies Evaluation and Research (CBER) normal criteria available. Lower binding of 95% CI to SC ratio should be ≥40%. The lower binding of 95% CI to the percentage with HI titer ≧ 1: 40 should be ≧ 70%.

194 children and 196 adults were enrolled. After the first dose (Day 22), 93% of children who received the 7.5 μg adjuvant vaccine achieved HI titers ≧ 1: 40, compared to 72-74% of children who received the Beerjuven vaccine . The SC ratio (day 22) (91%) for adjuvant vaccine at children ages 3-8 years was higher than for beerjuvenant vaccine (71-72%). On day 29, all patients who gave 7.5 μg of adjuvant vaccine achieved HI titers> 1:40; All vaccines met the CBER criteria. SC ratios after the second vaccine dose ranged from 83-95% across all study groups. GMTs were elevated after each vaccination, especially in patients who received 7.5 μg adjuvant vaccine in children. All three H1N1 vaccines developed a high HI antibody response in the pediatric population within two doses of the vaccine, and after one dose only the adjuvant vaccine achieved an HI antibody response that met the CBER immunogenicity criteria. These criteria were also met with a lower total dose of antigen (7.5 μg) in the adjuvant vaccine compared to the beer adjuvant vaccine.

Person study VIII  (United States of America)

Dose range studies were performed to assess the optimal dose of monovalent H1N1sw vaccine with or without oil-in-water adjuvant (MF59) in the pediatric population. A total of 1357 healthy children, ages 3 to <9 years, were enrolled. Children were randomized equally into eight groups and given intramuscular vaccination on days 1 and 22. The vaccine was formulated as 3.75, 7.5, 15 or 30 μg HA with or without MF59 or at half dose. Immunogenicity (HI assay) [HI titers ≥1: 40 (95% CI subbinding ≥ 70%) and serum conversion rate (95% CI subbinding> 40%) according to CBER criteria were determined on day 22 and It was evaluated on day 43. Serum conversion was defined as HI titer <1:10 before vaccination and titer ≧ 1: 40, or ≧ 4-fold increase in HI titer before vaccination ≧ 1: 10 and titer after vaccination. HI antibody responses were expressed as geometric mean titers (GMTs) and geometric mean ratios (GMRs) before and after vaccination titers. Comparisons were made in pairs of GMT ratios between each group and 95% CI was evaluated for a non-degradable margin of 0.5, and then 0.67. Differences between vaccine groups were assumed to be statistically significant if the two-sided 95% CI around the GMT ratio did not contain 1, indicating statistically significant superiority or deterioration.

GMT and GMR results were as follows:

Baseline seropositive rates (HI titers ≧ 10) in each group were comparable (18% -27%). All the adjuvant groups met the HI titer ≧ 1: 40 criteria after a single dose, while the beer adjuvant group met the serum protection criteria only after two doses. Patients in all vaccine groups (except the 7.5 μg group of non-juvenants) met the seroconversion criteria after dose 1 and all groups met this criteria after 2 doses. One group comparison of pairs of GMTs on day 22 using two-sided 95% CI shows that all adjuvant vaccines are superior to beer adjuvant vaccines. The adjuvant group met the eligibility criteria after a single dose and the vaccine dose with half the dose of 7.5 μg antigen and MF59 adjuvant clearly showed a good response.

It is to be understood that the invention has been described by way of example only and that modifications may be made without departing from the scope and concept of the invention.

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                         SEQUENCE LISTING <110> NOVARTIS AG   <120> ADJUVANTED VACCINES FOR PROTECTING AGAINST INFLUENZA <130> P054585WO <140> PCT / IB2010 / ______ <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 Ala Ile Leu Val Val Leu Leu Tyr Thr Phe Ala Thr Ala Asn 1 5 10 15 Ala Asp Thr Leu Cys Ile 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 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 Leu Ser Thr Ala Ser Ser Trp Ser Tyr Ile                 85 90 95 Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe             100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe         115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 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 Leu Val 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 Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu         195 200 205 Tyr Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser     210 215 220 Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln 225 230 235 240 Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys                 245 250 255 Ile Thr Phe 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 Ile Ile Ile Ser Asp Thr Pro         275 280 285 Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn     290 295 300 Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys 305 310 315 320 Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg                 325 330 335 Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly             340 345 350 Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr         355 360 365 His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser     370 375 380 Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile 385 390 395 400 Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His                 405 410 415 Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe             420 425 430 Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn         435 440 445 Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu     450 455 460 Lys Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile 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 Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu             500 505 510 Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr         515 520 525 Gln Ile Leu Ala Ile 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 Gln Cys Arg Ile Cys Ile                 565 <210> 2 <211> 326 <212> PRT <213> Influenza A virus <400> 2 Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr Val 1 5 10 15 Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 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 His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly Asn     50 55 60 Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp Ser Tyr Ile Val 65 70 75 80 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 Gln Leu Ser Ser Val Ser Ser Phe Glu             100 105 110 Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp 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 Ile Asn Asp Lys Gly Lys Glu Val Leu Val Leu                 165 170 175 Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu Tyr             180 185 190 Gln Asn Ala Asp Thr Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser Lys         195 200 205 Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln Glu     210 215 220 Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val 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 Ile Ile Ile Ser Asp Thr Pro Val             260 265 270 His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn Thr         275 280 285 Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile 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 Ile Pro Ser Ile Gln Ser                 325 <210> 3 <211> 566 <212> PRT <213> Influenza A virus <400> 3 Met Lys Ala Lys Leu Leu Val Leu Leu Cys Ala Leu Ser Ala Thr Asp 1 5 10 15 Ala Asp Thr Ile Cys Ile 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 Gln 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 Lys 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 Gln Leu Ser Ser Val Ser Ser Phe         115 120 125 Glu Arg Phe Glu Ile 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 Lys 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 Ile Glu Asp Gln Lys Thr Ile         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 Gln 225 230 235 240 Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr                 245 250 255 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 Ile Thr Ser Asn Ala Ser         275 280 285 Met Asp Glu Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly Ala Ile Asn     290 295 300 Ser Ser Leu Pro Phe Gln Asn Val His Pro Val Thr Ile Gly Glu Cys 305 310 315 320 Pro Lys Tyr Val Arg Ser Thr Lys Leu Arg Met Val Thr Gly Leu Arg                 325 330 335 Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly             340 345 350 Phe Ile Glu Gly Gly Trp Thr Gly Met Ile Asp Gly Trp Tyr Gly Tyr         355 360 365 His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser     370 375 380 Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Ile Ile 385 390 395 400 Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys                 405 410 415 Leu Glu Lys Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe             420 425 430 Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val 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 Gln Leu Lys Asn Asn Ala Lys Glu Ile 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 Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu             500 505 510 Asn Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser Met Gly Val Tyr         515 520 525 Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu     530 535 540 Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu 545 550 555 560 Gln Cys Arg Ile Cys Ile                 565 <210> 4 <211> 469 <212> PRT <213> Influenza A virus <400> 4 Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Cys Met Thr 1 5 10 15 Ile Gly Met Ala Asn Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile             20 25 30 Trp Ile Ser His Ser Ile Gln Leu Gly Asn Gln Asn Gln Ile Glu Thr         35 40 45 Cys Asn Gln Ser Val Ile Thr Tyr Glu Asn Asn Thr Trp Val Asn Gln     50 55 60 Thr Tyr Val Asn Ile Ser Asn Thr Asn Phe Ala Ala Gly Gln 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 Ile Tyr Ser Lys Asp Asn Ser Val Arg Ile Gly Ser Lys Gly             100 105 110 Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser Pro Leu Glu         115 120 125 Cys Arg Thr Phe Phe Leu Thr Gln 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 Cys Pro Ile Gly Glu Val Pro Ser Pro Tyr Asn 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 Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr         195 200 205 Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu     210 215 220 Arg Thr Gln Glu Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr 225 230 235 240 Val Met Thr Asp Gly Pro Ser Asn Gly Gln Ala Ser Tyr Lys Ile Phe                 245 250 255 Arg Ile Glu Lys Gly Lys Ile Val Lys Ser Val Glu Met Asn Ala Pro             260 265 270 Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Ser Ser Glu Ile         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 Gln Asn Leu Glu Tyr Gln Ile Gly Tyr Ile Cys Ser Gly 305 310 315 320 Ile Phe Gly Asp Asn Pro Arg 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 Ile Gly Arg Thr Lys Ser Ile Ser Ser Arg         355 360 365 Asn Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Gly Thr Asp     370 375 380 Asn Asn Phe Ser Ile Lys Gln Asp Ile Val Gly Ile Asn Glu Trp Ser 385 390 395 400 Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp                 405 410 415 Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys             420 425 430 Glu Asn Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val         435 440 445 Asn Ser Asp Thr Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro     450 455 460 Phe Thr Ile Asp Lys 465 <210> 5 <211> 470 <212> PRT <213> Influenza A virus <400> 5 Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Ile Cys Met Thr 1 5 10 15 Ile Gly Ile Ile Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile             20 25 30 Trp Val Ser His Ser Ile Gln Thr Gly Ser Gln Asn His Thr Gly Ile         35 40 45 Cys Asn Gln Arg Ile Ile Thr Tyr Glu Asn Ser Thr Trp Val Asn Gln     50 55 60 Thr Tyr Val Asn Ile Asn Asn Thr Asn Val Val Ala Gly Lys Asp Thr 65 70 75 80 Thr Ser Val Thr Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Arg Gly                 85 90 95 Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly             100 105 110 Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu         115 120 125 Cys Arg Thr Phe Phe Leu Thr Gln 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 Ile Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser                 165 170 175 Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr             180 185 190 Ile Gly Ile Ser Gly Pro Asp Asp Gly Ala Val Ala Val Leu Lys Tyr         195 200 205 Asn Gly Ile Ile Thr Glu Thr Ile Lys Ser Trp Arg Lys Arg Ile Leu     210 215 220 Arg Thr Gln Glu Ser Glu Cys Val Cys Val Asn Gly Ser Cys Phe Thr 225 230 235 240 Ile Met Thr Asp Gly Pro Ser Asn Gly Pro Ala Ser Tyr Arg Ile Phe                 245 250 255 Lys Ile Glu Lys Gly Lys Ile Thr Lys Ser Ile Glu Leu Asp Ala Pro             260 265 270 Asn Ser His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp 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 Gln Asn Leu Asp Tyr Gln Ile Gly Tyr Ile 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 Val Asp Gly Ala Asp Gly Val Lys Gly Phe Ser Tyr Arg             340 345 350 Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Asn Ser Ser Arg         355 360 365 Lys Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Asp Thr Asp     370 375 380 Ser Asn Phe Leu Val Lys Gln Asp Val Val Ala Met Thr Asp Trp Ser 385 390 395 400 Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu 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 Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly         435 440 445 Val Asn Ser Asp Thr Ala Asn Trp Ser Trp Pro Asp Gly Ala Glu Leu     450 455 460 Pro Phe Thr Ile Asp Lys 465 470 <210> 6 <211> 566 <212> PRT <213> Influenza A virus <400> 6 Met Lys Ala Ile Leu Val Val Leu Leu Tyr Thr Phe Ala Thr Ala Asn 1 5 10 15 Ala Asp Thr Leu Cys Ile 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 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 Leu Ser Thr Ala Ser Ser Trp Ser Tyr Ile                 85 90 95 Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe             100 105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe         115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro 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 Leu Val 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 Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu         195 200 205 Tyr Gln 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 Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys                 245 250 255 Ile Thr Phe 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 Ile Ile Ile Ser Asp Thr Pro         275 280 285 Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn     290 295 300 Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys 305 310 315 320 Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg                 325 330 335 Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly             340 345 350 Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr         355 360 365 His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser     370 375 380 Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile 385 390 395 400 Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His                 405 410 415 Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe             420 425 430 Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn         435 440 445 Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu     450 455 460 Lys Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile 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 Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu             500 505 510 Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr         515 520 525 Gln Ile Leu Ala Ile 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 Gln Cys Arg Ile Cys Ile                 565 <210> 7 <211> 326 <212> PRT <213> Influenza A virus <400> 7 Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr Val 1 5 10 15 Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His 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 His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly Asn     50 55 60 Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp Ser Tyr Ile Val 65 70 75 80 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 Gln Leu Ser Ser Val Ser Ser Phe Glu             100 105 110 Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp 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 Ile Asn Asp Lys Gly Lys Glu Val Leu Val Leu                 165 170 175 Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu Tyr             180 185 190 Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser Lys         195 200 205 Thr Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Arg Glu     210 215 220 Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val 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 Ile Ile Ile Ser Asp Thr Pro Val             260 265 270 His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn Thr         275 280 285 Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile 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 Ile Pro Ser Ile Gln Ser                 325

Claims (23)

administering to the patient a vaccine comprising (i) a H1 subtype influenza A virus hemagglutinin and (ii) an oil-in-water emulsion adjuvant that is more closely related to SEQ ID NO: 1 than SEQ ID NO: 3 How to immunize a person. An immunogenic composition comprising (i) H1 subtype influenza A virus hemagglutinin and (ii) an oil-in-water emulsion adjuvant that is more closely related to SEQ ID NO: 1 than SEQ ID NO: 3. A composition according to claim 2, which is a monovalent vaccine. 4. The composition of claim 2 or 3, wherein the hemagglutinin comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2. 5. The composition of claim 2, wherein the oil-in-water emulsion adjuvant comprises squalene and has droplets up to 250 nm in diameter. The composition of claim 2, wherein the composition has a hemagglutinin concentration of about 7.5 μg / ml or about 15 μg / ml. 3. The composition of claim 2, which is a trivalent vaccine comprising also H3N2 influenza A virus hemagglutinin and influenza B virus hemagglutinin. An immunogenic composition comprising two different H1 subtype influenza A virus hemagglutinin and an oil-in-water emulsion adjuvant, wherein (i) the first H1 subtype influenza A virus hemagglutinin is less than SEQ ID NO: 3 More closely related to SEQ ID NO: 1, and (ii) the second H1 subtype influenza A virus hemagglutinin is more closely related to SEQ ID NO: 3 than SEQ ID NO: 1 Composition. 9. The composition of claim 8, further comprising (iii) H3N2 influenza A virus hemagglutinin and (iv) influenza B virus hemagglutinin. The method of claim 8, further comprising: (iii) H3N2 influenza A virus hemagglutinin; (iv) B / Victoria / 2 / 87-like influenza B virus hemagglutinin; And (v) B / Yamagata / 16/88 like influenza B virus hemagglutinin. (i) administering to the patient a monovalent vaccine comprising H1 subtype influenza A virus hemagglutinin and (ii) administering a trivalent A / H1N1-A / H3N2-B seasonal influenza vaccine to the patient. A method of immunizing a human against an influenza virus comprising: (a) the monovalent vaccine comprises H1 subtype influenza A virus hemagglutinin, which is more closely related to SEQ ID NO: 1 than SEQ ID NO: 3, b) the trivalent vaccine comprises an H1 subtype influenza A virus hemagglutinin that is more closely related to SEQ ID NO: 3 than SEQ ID NO: 1, and (c) the monovalent vaccine comprises an oil-in-water emulsion adjuvant (d) The trivalent vaccine comprises an oil-in-water emulsion adjuvant. The method of claim 11, wherein the monovalent vaccine is administered at least 4 weeks before the trivalent vaccine. 12. The method of claim 11, wherein the monovalent vaccine is administered at least 4 weeks after the trivalent vaccine. With the use of the H1 subtype influenza A virus hemagglutinin in the preparation of monovalent vaccines for immunizing humans, the vaccine comprises an oil-in-water emulsion adjuvant and the monovalent vaccine comprises SEQ ID NO: Use characterized in that it comprises the H1 subtype influenza A virus hemagglutinin more closely related to 1. 15. The H1 subtype of claim 14, wherein the person has previously received a trivalent A / H1N1-A / H3N2-B seasonal influenza vaccine, and the trivalent vaccine is more closely related to SEQ ID NO: 3 than SEQ ID NO: 1 Use characterized in that it comprises the type influenza A virus hemagglutinin. The method, composition or use according to any one of the preceding claims, wherein the emulsion comprises oil droplets of submicron diameter and the emulsion comprises squalene. A vaccine comprising hemagglutinin obtained from at least two different strains of influenza virus, wherein the first hemagglutinin is prepared from an influenza virus grown in eggs and the second hemagglutinin is in cell culture. A vaccine prepared from a grown influenza virus, wherein the second hemagglutinin is more closely related to SEQ ID NO: 1 than SEQ ID NO: 3. 18. The vaccine of claim 17 wherein the cell culture is an MDCK cell culture. (i) purified H1 subtype influenza A virus hemagglutinin expressed in a recombinant host, H1 hemagglutinin more closely related to SEQ ID NO: 1 than SEQ ID NO: 3, and (ii) oil-in-water emulsion Immunogenic composition comprising an adjuvant. 20. The composition of claim 19, wherein the hemagglutinin is expressed in insect cell lines using baculovirus vectors. 21. The composition of claim 19 or 20, wherein the composition comprises recombinant influenza virus neuraminidase. A method of immunizing a subject comprising administering to the subject two separate doses of the influenza vaccine, wherein (a) the two doses are administered at 1-6 week intervals, and (b) each vaccine is SEQ ID NO: Contains the H1 subtype influenza A virus hemagglutinin, which is more closely related to SEQ ID NO: 1 than 3, and (c) the subject is at least subjected to a neuraminidase inhibitor such as oseltamivir phosphate while receiving two vaccine doses How to take three times. 23. The method of claim 22, wherein one or both of the vaccines administered comprise an oil-in-water emulsion adjuvant.

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