WO2010125461A1 - Adjuvanted vaccines for protecting against influenza - Google Patents

Abstract

A vaccine containing a H1 subtype influenza A virus hemagglutinin is adjuvanted with an oil-in- water emulsion adjuvant. The vaccine is suitable for immunizing a patient against the virus referred to as 'swine flu'. The vaccine may be monovalent. The vaccine may include two different H1 subtype influenza A virus hemagglutinins, wherein (i) the first H1 subtype influenza A virus hemagglutinin is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3 and (ii) the second H1 subtype influenza A virus hemagglutinin is more closely related to SEQ ID NO: 3 than to SEQ ID NO: 1. A monovalent vaccine may be administered in conjunction with a trivalent A/H1N1-A/H3N2- B seasonal influenza vaccine.

Description

ADJUVANTED VACCINES FOR PROTECTING AGAINST INFLUENZA TECHNICAL FIELD

This invention is in the field of adjuvanted vaccines for protecting against influenza virus infection, and in particular against strains such as the swine flu strain(s) which emerged in April 2009. BACKGROUND ART

In April 2009 a human outbreak of swine flu was confirmed in many countries including Mexico and USA, and then spread rapidly across the globe. A pandemic was declared by the WHO in June 2009. The disease was caused by a newly identified swine influenza virus A/California/04/2009 A(HlNl). This swine flu strain seems to have no immunological cross-reactivity with current human influenza vaccines strains, including the A(HlNl) antigens in current human seasonal vaccines. The virus has been referred to variously as 'swine influenza', 'novel swine-origin HlNl influenza', 'human-swine influenza', 'novel influenza A(HlNl)' and 'influenza A(HlNl)v'.

There is a need for a vaccine to prevent further human-to-human transmission of this swine flu and variants of it. DISCLOSURE OF THE INVENTION

According to a first aspect of the invention, a vaccine containing a Hl subtype influenza A virus hemagglutinin is adjuvanted with an oil-in-water emulsion adjuvant. The hemagglutinin elicits an immune response in a recipient, and the adjuvant enhances the heterovariant coverage of this response. Although a particular Hl antigen might not protect against swine flu on its own, the adjuvant can enhance the immune response so that protection is achieved even if the vaccine hemagglutinin shows only low immunological cross-reactivity with the swine flu hemagglutinin. Furthermore, if the vaccine includes a hemagglutinin which is immunologically cross-reactive with the swine flu hemagglutinin then protection can be provided against the homologous strain and also against variants thereof, such as drift strains which can arise naturally. Thus the invention provides a method for immunizing a patient (typically a human) against swine flu, comprising a step of administering to the patient a vaccine comprising (i) a Hl subtype influenza A virus hemagglutinin and (ii) an oil-in-water emulsion adjuvant. In some embodiments the Hl hemagglutinin is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3; in other embodiments it is more closely related to SEQ ID NO: 3 than to SEQ ID NO: 1. The invention provides an immunogenic composition comprising (i) a Hl subtype influenza A virus hemagglutinin which is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3 and (ii) an oil- in-water emulsion adjuvant. This composition may be a monovalent vaccine (i.e. it includes hemagglutinin antigen from a single influenza virus strain) but in some embodiments it may be a multivalent vaccine e.g. a trivalent vaccine also including a H3N2 influenza A virus hemagglutinin and an influenza B virus hemagglutinin.

According to a second aspect of the invention, the invention provides an immunogenic composition comprising two different Hl subtype influenza A virus hemagglutinins, wherein (i) the first Hl subtype influenza A virus hemagglutinin is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3 and (ii) the second Hl subtype influenza A virus hemagglutinin is more closely related to SEQ ID NO: 3 than to SEQ ID NO: 1, and wherein the composition includes as an immunological adjuvant an oil-in-water emulsion adjuvant. This mixture of adjuvanted Hl hemagglutinins offers a broader spectrum of protection against Hl influenza A virus strains than currently available. This composition may also include (iii) a H3N2 and/or (iv) an influenza B virus antigen. In some embodiments, the composition includes (iii) a H3N2, (iv) a B/Victoria/2/87-like influenza B virus strain; and (v) a B/Yamagata/16/88-like influenza B virus strain.

According to a third aspect of the invention, a monovalent vaccine containing a Hl subtype influenza A virus hemagglutinin is administered in conjunction with a trivalent A/H1N1-A/H3N2-B seasonal influenza vaccine, wherein both of the vaccine(s) are adjuvanted with an oil-in-water emulsion. The monovalent vaccine includes a Hl subtype influenza A virus hemagglutinin which is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3; the trivalent vaccine includes a Hl subtype influenza A virus hemagglutinin which is more closely related to SEQ ID NO: 3 than to SEQ ID NO: 1. The monovalent vaccine may be administered before the trivalent vaccine, after the trivalent vaccine, or at the same time. Where the two vaccines are administered separately, there may be from

2-26 weeks between the administrations. In one useful embodiment, a patient first receives the trivalent seasonal vaccine (adjuvanted, such as the FLUAD™ product), and later receives the monovalent vaccine (adjuvanted). As shown herein, pre-administration of an adjuvanted trivalent seasonal vaccine can improve the efficacy of a monovalent HlNl vaccine with a hemagglutinin more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3.

In a related embodiment, a monovalent vaccine containing a Hl subtype influenza A virus hemagglutinin is administered in conjunction with a 4-valent A/H1N1-A/H3N2-B-B seasonal influenza vaccine, wherein the two B strains are a B/Victoria/2/87-like strain and a B/Yamagata/16/88-like strain, and wherein both of the vaccines are adjuvanted with an oil-in-water emulsion. The monovalent vaccine includes a Hl subtype influenza A virus hemagglutinin which is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3; the 4-valent vaccine includes a Hl subtype influenza A virus hemagglutinin which is more closely related to SEQ ID NO: 3 than to SEQ ID NO: 1. The monovalent vaccine may be administered before the trivalent vaccine, after the trivalent vaccine, or at the same time. Where the two vaccines are administered separately, there may be from 2-26 weeks between the administrations. In one useful embodiment, a patient first receives the monovalent vaccine and later receives the 4-valent vaccine.

According to a fourth aspect of the invention, a monovalent vaccine containing a Hl subtype influenza A virus hemagglutinin is administered by a two-dose regimen, where both doses of monovalent vaccines are adjuvanted with an oil-in-water emulsion. The monovalent vaccine includes a Hl subtype influenza A virus hemagglutinin which is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3. The two doses are administered 1-6 weeks apart e.g. 1 week apart, 2 weeks apart, 3 weeks apart, 4 weeks apart, 5 weeks apart, 6 weeks apart. In some embodiments the Hl hemagglutinin is identical in both monovalent vaccines; in other embodiments the Hl hemagglutinins in the two monovalent vaccines have different amino acid sequences e.g. they may differ by up to 20 amino acids from each other (e.g. 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 components The invention uses influenza A virus hemagglutinin as a vaccine antigen. The antigen will typically be prepared from influenza virions but, as an alternative, haemagglutinin can be expressed in a recombinant host (e.g. in an insect cell line using a baculovirus vector) and used in purified form [1,2,3] or in the form of virus-like particles (VLPs; e.g. see references 4 and 5). In general, however, antigens will be from virions. Various forms of influenza virus vaccine are currently available (e.g. see chapters 17 & 18 of reference 6). Known vaccines are generally based either on live virus or on inactivated virus. The antigen in vaccines of the invention take the form of an inactivated virus. Inactivated vaccines may be based on whole virions, 'split' virions, or on purified surface antigens. Chemical means for inactivating a virus include treatment with an effective amount of one or more of the following agents: detergents, formaldehyde, formalin, β-propiolactone, or UV light. Additional chemical means for inactivation include treatment with methylene blue, psoralen, carboxyfullerene (C60) or a combination of any thereof. Other methods of viral inactivation are known in the art, such as for example binary ethylamine, acetyl ethyleneimine, or gamma irradiation.

The vaccine may comprise whole virion, split virion, or purified surface antigens (including hemagglutinin and, usually, also including neuraminidase). Split virion and purified surface antigens (i.e. subvirion vaccines) are particularly useful with the invention.

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

Split virions are obtained by treating virions with detergents (e.g. ethyl ether, polysorbate 80, deoxycholate, tri-N-butyl phosphate, Triton X-100, Triton Nl 01, cetyltrimethylammonium bromide, Tergitol NP9, etc.) to produce subvirion preparations, including the 'Tween-ether' splitting process. Methods of splitting influenza viruses are well known in the art e.g. see refs. 7-12, etc. Splitting of the virus is typically carried out by disrupting or fragmenting whole virus, whether infectious or non-infectious with a disrupting concentration of a splitting agent. The disruption results in a full or partial solubilisation of the virus proteins, altering the integrity of the virus. Preferred splitting agents are non-ionic and ionic (e.g. cationic) surfactants e.g. alkylglycosides, alkylthioglycosides, acyl sugars, sulphobetaines, betains, polyoxyethylenealkylethers, N,N-dialkyl-Glucamides, Hecameg, alkylphenoxy-polyethoxyethanols, quaternary ammonium compounds, sarcosyl, CTABs (cetyl trimethyl ammonium bromides), tri-N-butyl phosphate, Cetavlon, myristyltrimethylammonium salts, lipofectin, lipofectamine, and DOT-MA, the octyl- or nonylphenoxy polyoxyethanols (e.g. the Triton surfactants, such as Triton X-100 or Triton Nl 01), polyoxyethylene sorbitan esters (the Tween surfactants), polyoxyethylene ethers, polyoxyethlene esters, etc. One useful splitting procedure uses the consecutive effects of sodium deoxycholate and formaldehyde, and splitting can take place during initial virion purification {e.g. in a sucrose density gradient solution). Thus a splitting process can involve clarification of the virion-containing material (to remove non-virion material), concentration of the harvested virions (e.g. using an adsorption method, such as CaHPO₄ adsorption), separation of whole virions from non-virion material, splitting of virions using a splitting agent in a density gradient centrifugation step {e.g. using a sucrose gradient that contains a splitting agent such as sodium deoxycholate), and then filtration {e.g. ultrafiltration) to remove undesired materials. Split virions can usefully be resuspended in sodium phosphate-buffered isotonic sodium chloride solution. The BEGRIVAC™, FLUARIX™, FLUZONE™ and FLUSHIELD™ products are split vaccines.

Purified surface antigen vaccines comprise the influenza surface antigens haemagglutinin and, typically, also neuraminidase. Processes for preparing these proteins in purified form are well known in the art. The FLUVIRIN™, AGRIPP AL™ and INFLUVAC™ products are subunit vaccines.

Influenza antigens can also be presented in the form of virosomes [13] (nucleic acid free viral-like liposomal particles), as in the INFLEXAL V™ and INVA VAC™ products, but it is preferred not to use virosomes with the present invention. Thus, in some embodiments, the influenza antigen is not in the form of a virosome.

The hemagglutinin antigen in the vaccine may be from any suitable strain. In some embodiments the hemagglutinin is one which, when administered to a human subject in unadjuvanted form, elicits anti-hemagglutinin antibodies which do not cross-react with A/California/04/2009 hemagglutinin (SEQ ID NO: 1; GI:227809830); in these embodiments the vaccine's adjuvant enhances the immune response such that a human subject produces antibodies which do cross-react with A/California/04/2009 hemagglutinin. In other embodiments the hemagglutinin is one which, when administered to a human subject in unadjuvanted form, can elicit anti-hemagglutinin antibodies which do cross-react with A/California/04/2009 hemagglutinin (SEQ ID NO: 1); in these embodiments the vaccine's adjuvant enhances the immune response such that a human subject produces a broader spectrum of antibodies, which can help to protect against drift strains of A/California/04/2009. In other embodiments the hemagglutinin is from A/California/04/2009 (SEQ ID NO: 1). In other embodiments the hemagglutinin comprises an HAl amino acid sequence having at least i% sequence identity to SEQ ID NO: 2, where / is 85 or more e.g. 85, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more {e.g. 100). Many such sequences are available e.g. from any of the following known strains:

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/Ontario/ 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, etc.

Further HlNl strains with suitable HA antigens include A/California/04/2009 itself, A/California/7/2009, A/Texas/5/2009, A/England/ 195/2009, and A/New York/18/2009.

Preferred embodiments comprise a hemagglutinin which, when administered to a human subject in unadjuvanted form, can elicit anti-hemagglutinin antibodies which cross-react with A/California/04/2009 hemagglutinin (SEQ ID NO: 1), such as hemagglutinins comprising an amino acid sequence having at least i% sequence identity to SEQ ID NO: 2 as discussed above. In some embodiments, the hemagglutinin is more closely related to SEQ ID NO: 1 (A/California/04/2009) than to SEQ ID NO: 3 (A/Chile/1/1983); in other embodiments, the hemagglutinin is more closely related to SEQ ID NO: 3 than to SEQ ID NO: 1. A hemagglutinin which is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3 (i.e. has a higher degree sequence identity when compared to SEQ ID NO: 1 than to SEQ ID NO: 3 using the same algorithm and parameters) is referred to hereafter as a 'Hl*' hemagglutinin. SEQ ID NOs: 1 and 3 are 80.4% identical.

Useful full-length Hl hemagglutinin sequences for use with the invention include SEQ ID NO: 1 and SEQ ID NO: 6, as well as those comprising an amino acid sequence having at least i% sequence identity to SEQ ID NO: 2 as discussed above, or having at least i% sequence identity to SEQ ID NO: 7. Ideally the hemagglutinin does not include a hyper-basic regions around the HA1/HA2 cleavage site. Preferred hemagglutinins have a binding preference for oligosaccharides with a Sia(α2,6)Gal terminal disaccharide compared to oligosaccharides with a Sia(α2,3)Gal terminal disaccharide (see below).

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

As well as including a Hl hemagglutinin (such as a Hl* hemagglutinin) and an oil-in-water emulsion adjuvant, compositions of the invention may include antigen(s) from one or more (e.g. 1, 2, 3, 4 or more) additional influenza virus strains, including influenza A virus and/or influenza B virus. Thus a composition may include antigen from one or more strains characteristics of a normal seasonal vaccine, and an oil-in-water emulsion adjuvant, plus at least one Hl* hemagglutinin e.g. a 4-valent vaccine with two Hl strains (one a Hl* hemagglutinin, one not a Hl* hemagglutinin), a H3N2 strain, and one influenza B strain, or a 5-valent vaccine with two Hl strains (one a Hl* hemagglutinin, one not a Hl * hemagglutinin), a H3N2 strain, and two influenza B virus strains (a B/Victoria/2/87-like strain and a B/Yamagata/16/88-like strain). The invention also provides a 2-valent vaccine comprising a Hl* hemagglutinin and a H5 hemagglutinin and an oil-in-water emulsion adjuvant. Where a vaccine includes more than one strain of influenza, the different strains are typically grown separately and are mixed after the viruses have been harvested and antigens have been prepared. Thus a process of the invention may include the step of mixing antigens from more than one influenza strain.

Where a vaccine of the invention includes two influenza B strains, one B/Victoria/2/87-like strain and one B/Yamagata/16/88-like strain will be included. These strains are usually distinguished antigenically, but differences in amino acid sequences have also been described for distinguishing the two lineages e.g. B/Yamagata/16/88-like strains often (but not always) have HA proteins with deletions at amino acid residue 164, numbered relative to the 'Lee40' HA sequence [14]. In some embodiments of the invention where antigens are present from two or more influenza B virus strains, at least two of the influenza B virus strains may have distinct hemagglutinins but related neuraminidases. For instance, they may both have a B/Victoria/2/87-like neuraminidase [15] or may both have a B/Yamagata/16/88-like neuraminidase. For instance, two B/Victoria/2/87-like neuraminidases may both have one or more of the following sequence characteristics: (1) not a serine at residue 27, but preferably a leucine; (2) not a glutamate at residue 44, but preferably a lysine; (3) not a threonine at residue 46, but preferably an isoleucine; (4) not a proline at residue 51, but preferably a serine; (5) not an arginine at residue 65, but preferably a histidine; (6) not a glycine at residue 70, but preferably a glutamate; (7) not a leucine at residue 73, but preferably a phenylalanine; and/or (8) not a proline at residue 88, but preferably a glutamine. Similarly, in some embodiments the neuraminidase may have a deletion at residue 43, or it may have a threonine; a deletion at residue 43, arising from a trinucleotide deletion in the NA gene, has been reported as a characteristic of B/Victoria/2/87-like strains, although recent strains have regained Thr-43 [15]. Conversely, of course, the opposite characteristics may be shared by two B/Yamagata/16/88-like neuraminidases e.g. S27, E44, T46, P51, R65, G70, L73, and/or P88. These amino acids are numbered relative to the 'Lee40' neuraminidase sequence [16].

An influenza virus from which hemagglutinin protein is purified may be resistant to antiviral therapy (e.g. resistant to oseltamivir [17] and/or zanamivir). In some embodiments, strains used with the invention will thus have hemagglutinin with a binding preference for oligosaccharides with a Sia(α2,6)Gal terminal disaccharide compared to oligosaccharides with a Sia(α2,3)Gal terminal disaccharide. Human influenza viruses bind to receptor oligosaccharides having a Sia(α2,6)Gal terminal disaccharide (sialic acid linked α-2,6 to galactose), but eggs and Vero cells have receptor oligosaccharides with a Sia(α2,3)Gal terminal disaccharide. Growth of human influenza viruses in cells such as MDCK provides selection pressure on hemagglutinin to maintain the native Sia(α2,6)Gal binding, unlike egg passaging. To determine if a virus has a binding preference for oligosaccharides with a Sia(α2,6)Gal terminal disaccharide compared to oligosaccharides with a Sia(α2,3)Gal terminal disaccharide, various assays can be used. For instance, reference 18 describes a solid-phase enzyme-linked assay for influenza virus receptor- binding activity which gives sensitive and quantitative measurements of affinity constants. Reference 19 used a solid-phase assay in which binding of viruses to two different sialylglycoproteins was assessed (ovomucoid, with Sia(α2,3)Gal determinants; and pig α₂-macroglobulin, which Sia(α2,6)Gal determinants), and also describes an assay in which the binding of virus was assessed against two receptor analogs: free sialic acid (Neu5Ac) and 3'-sialyllactose (Neu5Acα2-3Galβl- 4GIc). Reference 20 reports an assay using a glycan array which was able to clearly differentiate receptor preferences for α2,3 or α2,6 linkages. Reference 21 reports an assay based on agglutination of human erythrocytes enzymatically modified to contain either Sia(α2,6)Gal or Sia(α2,3)Gal. Depending on the type of assay, it may be performed directly with the virus itself, or can be performed indirectly with hemagglutinin purified from the virus.

In some embodiments the Hl hemagglutinin has a different glycosylation pattern from the patterns seen in egg-derived viruses. Thus the HA (and other glycoproteins) may include glycoforms that are not seen in chicken eggs. Useful HA includes canine glycoforms.

In addition to including hemagglutinin antigen, vaccines of the invention typically also include a neuraminidase protein e.g. the vaccine will include viral neuraminidase. The invention may protect against one or more of influenza A virus NA subtypes Nl, N2, N3, N4, N5, N6, N7, N8 or N9, but it will usually be against Nl (e.g. a HlNl virus) or N2 (e.g. a H1N2 virus). Whole virions, split virions and subunit vaccines all include both hemagglutinin and neuraminidase. When a vaccine includes a neuraminidase antigen, the neuraminidase may have at least j% sequence identity to SEQ ID NO: 4, where y is 75 or more e.g. 75, 80, 85, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more (e.g. 100). Many such sequences are available. In some embodiments, the neuraminidase is more closely related to SEQ ID

NO: 4 than to SEQ ID NO: 5. SEQ ID NOs: 4 and 5 are 82% identical.

Vaccines may also include a matrix protein, such as Ml and/or M2 (or a fragment thereof), and/or nucleoprotein. A pig model has shown that addition of M2 to inactivated HlNl swine influenza virus vaccine (adjuvanted with an oil-in-water emulsion) can enhance the vaccine's efficacy [22].

The influenza virus may be a reassortant strain, and may have been obtained by reverse genetics techniques. Reverse genetics techniques [e.g. 23-27] allow influenza viruses with desired genome segments to be prepared in vitro using plasmids, or by plasmid-free systems. Typically, the technique involves expressing (a) DNA molecules that encode desired viral RNA molecules e.g. from poll promoters, and (b) DNA molecules that encode viral proteins e.g. from polll promoters, such that expression of both types of DNA in a cell leads to assembly of a complete intact infectious virion. The DNA preferably provides all of the viral RNA and proteins, but it is also possible to use a helper virus to provide some of the RNA and proteins. Plasmid-based methods using separate plasmids for producing each viral RNA are preferred [28-30], and these methods will also involve the use of plasmids to express all or some (e.g. just the PBl, PB2, PA and NP proteins) of the viral proteins, with up to 12 plasmids being used in some methods. If canine cells are used, a canine poll promoter may be used [31]. To reduce the number of plasmids needed, one approach [32] combines a plurality of RNA polymerase I transcription cassettes (for viral RNA synthesis) on the same plasmid (e.g. sequences encoding 1, 2, 3, 4, 5, 6, 7 or all 8 influenza A vRNA segments), and a plurality of protein-coding regions with RNA polymerase II promoters on another plasmid (e.g. sequences encoding 1, 2, 3, 4, 5, 6, 7 or all 8 influenza A mRNA transcripts). The method may involve: (a) PBl, PB2 and PA mRNA-encoding regions on a single plasmid; and (b) all 8 vRNA-encoding segments on a single plasmid. Including the NA and HA segments on one plasmid and the six other segments on another plasmid can also facilitate matters.

As an alternative to using poll promoters to encode the viral RNA segments, it is possible to use bacteriophage polymerase promoters [33]. For instance, promoters for the SP6, T3 or T7 polymerases can conveniently be used. Because of the species-specificity of poll promoters, bacteriophage polymerase promoters can be more convenient for many cell types (e.g. MDCK), although a cell must also be transfected with a plasmid encoding the exogenous polymerase enzyme.

In other techniques it is possible to use dual poll and polll promoters to simultaneously code for the viral RNAs and for expressible mRNAs from a single template [34,35].

An influenza A virus used with the invention may include one or more RNA segments from a A/PR/8/34 virus (typically 6 segments from A/PR/8/34, with the HA and N segments being from a vaccine strain, i.e. a 6:2 reassortant), particularly when viruses are grown in eggs. It may also include one or more RNA segments from a A/WSN/33 virus, or from any other virus strain useful for generating reassortant viruses for vaccine preparation. Typically, the invention protects against a strain that is capable of human-to-human transmission, and so the strain's genome will usually include at least one RNA segment that originated in a mammalian (e.g. in a human) influenza virus.

The viruses used as the source of the antigens can be grown either on eggs or on cell culture. The current standard method for influenza virus growth uses specific pathogen-free (SPF) embryonated hen eggs, with virus being purified from the egg contents (allantoic fluid). More recently, however, viruses have been grown in animal cell culture and, for reasons of speed and patient allergies, this growth method is preferred. If egg-based viral growth is used then one or more amino acids may be introduced into the allantoid fluid of the egg together with the virus [12].

When cell culture is used, the viral growth substrate will typically be a cell line of mammalian origin. Suitable mammalian cells of origin include, but are not limited to, hamster, cattle, primate (including humans and monkeys) and dog cells. Various cell types may be used, such as kidney cells, fibroblasts, retinal cells, lung cells, etc. Examples of suitable hamster cells are the cell lines having the names BHK21 or HKCC. Suitable monkey cells are e.g. African green monkey cells, such as kidney cells as in the Vero cell line. Suitable dog cells are e.g. kidney cells, as in the MDCK cell line. Thus suitable cell lines include, but are not limited to: MDCK; CHO; 293T; BHK; Vero; MRC-5; PER.C6; WI-38; etc.. Preferred mammalian cell lines for growing influenza viruses include: MDCK cells [36-39], derived from Madin Darby canine kidney; Vero cells [40-42], derived from African green monkey (Cercopithecus aethiops) kidney; or PER.C6 cells [43], derived from human embryonic retinoblasts. These cell lines are widely available e.g. from the American Type Cell Culture (ATCC) collection, from the Coriell Cell Repositories, or from the European Collection of Cell Cultures (ECACC). For example, the ATCC supplies various different Vero cells under catalog numbers CCL-81, CCL-81.2, CRL- 1586 and CRL- 1587, and it supplies MDCK cells under catalog number CCL-34. PER.C6 is available from the ECACC under deposit number 96022940. As a less-preferred alternative to mammalian cell lines, virus can be grown on avian cell lines [e.g. refs. 44-46], including cell lines derived from ducks (e.g. duck retina) or hens. Examples of avian cell lines include avian embryonic stem cells [44,47] and duck retina cells [45]. Suitable avian embryonic stem cells, include the EBx cell line derived from chicken embryonic stem cells, EB45, EB 14, and EB 14-074 [48] . Chicken embryo fibroblasts (CEF) may also be used.

The most preferred cell lines for growing influenza viruses are MDCK cell lines. The original MDCK cell line is available from the ATCC as CCL-34, but derivatives of this cell line may also be used. For instance, reference 36 discloses a MDCK cell line that was adapted for growth in suspension culture ('MDCK 33016', deposited as DSM ACC 2219). Similarly, reference 49 discloses a MDCK-derived cell line that grows in suspension in serum-free culture ('B-702', deposited as FERM BP-7449). Reference 50 discloses non-tumorigenic MDCK cells, including 'MDCK-S' (ATCC PTA-6500), 'MDCK-SFlOl' (ATCC PTA-6501), 'MDCK-SF102' (ATCC PTA-6502) and 'MDCK-SF103' (PTA-6503). Reference 51 discloses MDCK cell lines with high susceptibility to infection, including 'MDCK.5F1' cells (ATCC CRL-12042). Any of these MDCK cell lines can be used.

Where virus has been grown on a mammalian cell line then the composition will advantageously be free from egg proteins (e.g. ovalbumin and ovomucoid) and from chicken DNA, thereby reducing allergenicity.

Where virus has been grown on a cell line then the culture for growth, and also the viral inoculum used to start the culture, will preferably be free from (i.e. will have been tested for and given a negative result for contamination by) herpes simplex virus, respiratory syncytial virus, parainfluenza virus 3, SARS coronavirus, adenovirus, rhinovirus, reoviruses, polyomaviruses, birnaviruses, circoviruses, and/or parvoviruses [52]. Absence of herpes simplex viruses is particularly preferred.

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

Cell lines supporting influenza virus replication are preferably grown in serum-free culture media and/or protein free media. A medium is referred to as a serum-free medium in the context of the present invention in which there are no additives from serum of human or animal origin. Protein-free is understood to mean cultures in which multiplication of the cells occurs with exclusion of proteins, growth factors, other protein additives and non-serum proteins, but can optionally include proteins such as trypsin or other proteases that may be necessary for viral growth. The cells growing in such cultures naturally contain proteins themselves. Cell lines supporting influenza virus replication are preferably grown below 37°C [55] during viral replication e.g. 30-36°C, at 31-35°C, or at 33±1°C.

The method for propagating virus in cultured cells generally includes the steps of inoculating the cultured cells with the strain to be cultured, cultivating the infected cells for a desired time period for virus propagation, such as for example as determined by virus titer or antigen expression (e.g. between 24 and 168 hours after inoculation) and collecting the propagated virus. The cultured cells are inoculated with a virus (measured by PFU or TCID₅₀) to cell ratio of 1:500 to 1:1, preferably 1:100 to 1:5, more preferably 1 :50 to 1 :10. The virus is added to a suspension of the cells or is applied to a monolayer of the cells, and the virus is absorbed on the cells for at least 60 minutes but usually less than 300 minutes, preferably between 90 and 240 minutes at 25°C to 40⁰C, preferably 28°C to 37°C. The infected cell culture (e.g. monolayers) may be removed either by freeze-thawing or by enzymatic action to increase the viral content of the harvested culture supernatants. The harvested fluids are then either inactivated or stored frozen. Cultured cells may be infected at a multiplicity of infection ("m.o.i.") of about 0.0001 to 10, preferably 0.002 to 5, more preferably to 0.001 to 2. Still more preferably, the cells are infected at a m.o.i of about 0.01. Infected cells may be harvested 30 to 60 hours post infection. Preferably, the cells are harvested 34 to 48 hours post infection. Still more preferably, the cells are harvested 38 to 40 hours post infection. Proteases (typically trypsin) are generally added during cell culture to allow viral release, and the proteases can be added at any suitable stage during the culture. Haemagglutinin (HA) is the main immunogen in inactivated influenza vaccines, and vaccine doses are standardised by reference to HA levels, typically as measured by a single radial immunodiffusion (SRID) assay. Current vaccines typically contain about 15μg of HA per strain, although lower doses are also used e.g. for children, or in emergency situations. Fractional doses such as ¹A (i.e. 7.5μg HA per strain, as in FOCETRIA™), ¹A (i.e. 3.75μg per strain, as in PREPANDRIX™) and V₈ have been used [56,57], as have higher doses (e.g. 3x or 9x doses [58,59]).Thus vaccines may include between 0.1 and 150μg of HA per influenza strain, preferably between 0.1 and 50μg e.g. 0.1-20μg, 0.1-15μg, 0.1-10μg, 0.1-7.5μg, 0.5-5μg, 3.75-15μg etc. Particular doses include e.g. about 45, about 30, about 15, about 10, about 7.5, about 5, about 3.8, about 3.75, about 1.9, about 1.5, etc. μg per strain. An equal HA mass per strain is typical. Lower doses (i.e. <15μg/dose) are most useful when an adjuvant is present in the vaccine, as with the invention. Although doses as high as 90μg have been used in some studies (e.g. reference 60), compositions of the invention will usually include 15μg/dose/strain or less.

HA used with the invention may be a natural HA as found in a virus, or may have been modified.

Compositions of the invention may include detergent e.g. a polyoxyethylene sorbitan ester surfactant (known as 'Tweens' e.g. polysorbate 80), an octoxynol (such as octoxynol-9 (Triton X-100) or 10, or t-octylphenoxypolyethoxyethanol), a cetyl trimethyl ammonium bromide ('CTAB'), or sodium deoxycholate, particularly for a split or surface antigen vaccine. The detergent may be present only at trace amounts. Thus the vaccine may include less than lmg/ml of each of octoxynol- 10, α-tocopheryl hydrogen succinate and polysorbate 80. Other residual components in trace amounts could be antibiotics (e.g. neomycin, kanamycin, polymyxin B).

Host cell DNA

Where virus has been grown on a cell line then it is standard practice to minimize the amount of residual cell line DNA in the final vaccine, in order to minimize any oncogenic activity of the DNA. Thus the composition preferably contains less than IOng (preferably less than Ing, and more preferably less than lOOpg) of residual host cell DNA per dose, although trace amounts of host cell DNA may be present. In general, the host cell DNA that it is desirable to exclude from compositions of the invention is DNA that is longer than lOObp. Measurement of residual host cell DNA is now a routine regulatory requirement for biologicals and is within the normal capabilities of the skilled person. The assay used to measure DNA will typically be a validated assay [61,62]. The performance characteristics of a validated assay can be described in mathematical and quantifiable terms, and its possible sources of error will have been identified. The assay will generally have been tested for characteristics such as accuracy, precision, specificity. Once an assay has been calibrated (e.g. against known standard quantities of host cell DNA) and tested then quantitative DNA measurements can be routinely performed. Three principle techniques for DNA quantification can be used: hybridization methods, such as Southern blots or slot blots [63]; immunoassay methods, such as the Threshold™ System [64]; and quantitative PCR [65]. These methods are all familiar to the skilled person, although the precise characteristics of each method may depend on the host cell in question e.g. the choice of probes for hybridization, the choice of primers and/or probes for amplification, etc. The Threshold™ system from Molecular Devices is a quantitative assay for picogram levels of total DNA, and has been used for monitoring levels of contaminating DNA in biopharmaceuticals [64]. A typical assay involves non-sequence-specific formation of a reaction complex between a biotinylated ssDNA binding protein, a urease-conjugated anti-ssDNA antibody, and DNA. All assay components are included in the complete Total DNA Assay Kit available from the manufacturer. Various commercial manufacturers offer quantitative PCR assays for detecting residual host cell DNA e.g. AppTec™ Laboratory Services, BioReliance™, Althea Technologies, etc. A comparison of a chemiluminescent hybridisation assay and the total DNA Threshold™ system for measuring host cell DNA contamination of a human viral vaccine can be found in reference 66.

Contaminating DNA can be removed during vaccine preparation using standard purification procedures e.g. chromatography, etc. Removal of residual host cell DNA can be enhanced by nuclease treatment e.g. by using a DNase. A convenient method for reducing host cell DNA contamination is disclosed in references 67 & 68, involving a two-step treatment, first using a DNase (e.g. Benzonase), which may be used during viral growth, and then a cationic detergent (e.g. CTAB), which may be used during virion disruption. Treatment with an alkylating agent, such as β-propiolactone, can also be used to remove host cell DNA, and advantageously may also be used to inactivate virions [69] while avoiding use of formaldehyde. Vaccines containing <10ng (e.g. <lng, <100pg) host cell DNA per 15μg of haemagglutinin are preferred, as are vaccines containing <10ng (e.g. <lng, <100pg) host cell DNA per 0.25ml volume. Vaccines containing <10ng (e.g. <lng, <100pg) host cell DNA per 50μg of haemagglutinin are more preferred, as are vaccines containing <10ng (e.g. <lng, <100pg) host cell DNA per 0.5ml volume. Oil-in-water emulsion adjuvants

Compositions of the invention include an oil-in-water emulsion adjuvant which can function to enhance the immune responses (humoral and/or cellular) elicited in a patient who receives the composition. The FLU AD™ product from Novartis Vaccines includes an oil-in-water emulsion.

Various suitable emulsions are known, and they typically include at least one oil and at least one surfactant, with the oil(s) and surfactant(s) being biodegradable (metabolisable) and biocompatible. The oil droplets in the emulsion are generally less than 5μm in diameter, and advantageously the emulsion comprises oil droplets with a sub-micron diameter, with these small sizes being achieved with a microfluidiser to provide stable emulsions. Droplets with a size less than 220nm are preferred as they can be subjected to filter sterilization. The invention can be used with oils such as those from an animal (such as fish) or vegetable source. Sources for vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, exemplify the nut oils. Jojoba oil can be used e.g. obtained from the jojoba bean. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the grain group, corn oil is the most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale and the like may also be used. 6-10 carbon fatty acid esters of glycerol and 1,2-propanediol, while not occurring naturally in seed oils, may be prepared by hydrolysis, separation and esterification of the appropriate materials starting from the nut and seed oils. Fats and oils from mammalian milk are metabolizable and may therefore be used in the practice of this invention. The procedures for separation, purification, saponification and other means necessary for obtaining pure oils from animal sources are well known in the art. Most fish contain metabolizable oils which may be readily recovered. For example, cod liver oil, shark liver oils, and whale oil such as spermaceti exemplify several of the fish oils which may be used herein. A number of branched chain oils are synthesized biochemically in 5-carbon isoprene units and are generally referred to as terpenoids. Shark liver oil contains a branched, unsaturated terpenoid known as squalene, 2,6,10,15, 19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene. Other preferred oils are the tocopherols (see below). Oil-in-water emulsions comprising squalene are particularly preferred. Mixtures of oils can be used.

Surfactants can be classified by their 'HLB' (hydrophile/lipophile balance). Preferred surfactants of the invention have a HLB of at least 10, preferably at least 15, and more preferably at least 16. The invention can be used with surfactants including, but not limited to: the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWF AX™ tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-l,2-ethanediyl) groups, with octoxynol-9 (Triton X-IOO, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin); polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as the SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Preferred surfactants for including in the emulsion are Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-100. As mentioned above, detergents such as Tween 80 may contribute to the thermal stability seen in the examples below. Mixtures of surfactants can be used e.g. Tween 80/Span 85 mixtures. A combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylene sorbitan esters (such as Tween 80) 0.01 to 1%, in particular about 0.1 %; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or other detergents in the Triton series) 0.001 to 0.1 %, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20 %, preferably 0.1 to 10 % and in particular 0.1 to 1 % or about 0.5%.

Specific oil-in-water emulsion adjuvants useful with the invention include, but are not limited to: • A submicron emulsion of squalene, Tween 80, and Span 85. The composition of the emulsion by volume can be about 5% squalene, about 0.5% polysorbate 80 and about 0.5% Span 85. In weight terms, these ratios become 4.3% squalene, 0.5% polysorbate 80 and 0.48% Span 85. This adjuvant is known as 'MF59' [70-72], as described in more detail in Chapter 10 of ref. 73 and chapter 12 of ref. 74. The MF59 emulsion advantageously includes citrate ions e.g. 1OmM sodium citrate buffer.

• An emulsion comprising squalene, an α-tocopherol, and polysorbate 80. These emulsions may have from 2 to 10% squalene, from 2 to 10% tocopherol and from 0.3 to 3% Tween 80, and the weight ratio of squalene:tocopherol is preferably <1 (e.g. 0.90) as this provides a more stable emulsion. Squalene and Tween 80 may be present volume ratio of about 5:2, or at a weight ratio of about 11:5. One such emulsion can be made by dissolving Tween 80 in PBS to give a

2% solution, then mixing 90ml of this solution with a mixture of (5g of DL-α-tocopherol and 5ml squalene), then microfluidising the mixture. The resulting emulsion may have submicron oil droplets e.g. with an average diameter of between 100 and 250nm, preferably about 180nm.

• An emulsion of squalene, a tocopherol, and a Triton detergent (e.g. Triton X-100). The emulsion may also include a 3d-MPL (see below). The emulsion may contain a phosphate buffer.

• An emulsion comprising a polysorbate (e.g. polysorbate 80), a Triton detergent (e.g. Triton X-100) and a tocopherol (e.g. an α-tocopherol succinate). The emulsion may include these three components at a mass ratio of about 75:11 :10 {e.g. 750μg/ml polysorbate 80, HOμg/ml Triton X-100 and lOOμg/ml α-tocopherol succinate), and these concentrations should include any contribution of these components from antigens. The emulsion may also include squalene. The emulsion may also include a 3d-MPL (see below). The aqueous phase may contain a phosphate buffer.

• An emulsion of squalane, polysorbate 80 and poloxamer 401 ("Pluronic™ L121"). The emulsion can be formulated in phosphate buffered saline, 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 [75] (0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can also be used without the Thr-MDP, as in the "AF" adjuvant [76] (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80). Microfluidisation is preferred.

• An emulsion comprising squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant {e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic nonionic surfactant {e.g. a sorbitan ester or mannide ester, such as sorbitan monoleate or 'Span 80'). The emulsion is preferably thermoreversible and/or has at least 90% of the oil droplets (by volume) with a size less than 200 nm [77]. The emulsion may also include one or more of: alditol {e.g. mannitol); a cryoprotective agent {e.g. a sugar, such as dodecylmaltoside and/or sucrose); and/or an alkylpolyglycoside. Such emulsions may be lyophilized. The emulsion may include squalene : polyoxyethylene cetostearyl ether : sorbitan oleate : mannitol at a mass ratio of 330 : 63 : 49 : 61.

• An emulsion having from 0.5-50% of an oil, 0.1-10% of a phospholipid, and 0.05-5% of a non-ionic surfactant. As described in reference 78, preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin. Submicron droplet sizes are advantageous.

• A submicron oil-in-water emulsion of a non-metabolisable oil (such as light mineral oil) and at least one surfactant (such as lecithin, Tween 80 or Span 80). Additives may be included, such as QuilA saponin, cholesterol, a saponin-lipophile conjugate (such as GPI-0100, described in reference 79, produced by addition of aliphatic amine to desacylsaponin via the carboxyl group of glucuronic acid), dimethyidioctadecylammonium bromide and/or N,N-dioctadecyl-N,N-bis

(2-hydroxyethyl)propanediamine.

• An emulsion comprising a mineral oil, a non-ionic lipophilic ethoxylated fatty alcohol, and a non-ionic hydrophilic surfactant {e.g. an ethoxylated fatty alcohol and/or polyoxyethylene- polyoxypropylene block copolymer) [80]. • An emulsion comprising a mineral oil, a non-ionic hydrophilic ethoxylated fatty alcohol, and a non-ionic lipophilic surfactant {e.g. an ethoxylated fatty alcohol and/or polyoxyethylene- polyoxypropylene block copolymer) [80]. • An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol (e.g. a cholesterol) are associated as helical micelles [81].

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

After the antigen and adjuvant have been mixed, haemagglutinin antigen will generally remain in aqueous solution but may distribute itself around the oil/water interface. In general, little if any haemagglutinin will enter the oil phase of the emulsion.

Where a composition includes a tocopherol, any of the α, β, γ, δ, ε or ξ tocopherols can be used, but α-tocopherols are preferred. The tocopherol can take several forms e.g. different salts and/or isomers. Salts include organic salts, such as succinate, acetate, nicotinate, etc. D-α-tocopherol and DL-α-tocopherol can both be used. Tocopherols are advantageously included in vaccines for use in elderly patients (e.g. aged 60 years or older) because vitamin E has been reported to have a positive effect on the immune response in this patient group [82]. They also have antioxidant properties that may help to stabilize the emulsions [83]. A preferred α-tocopherol is DL-α-tocopherol, and the preferred salt of this tocopherol is the succinate. The succinate salt has been found to cooperate with TNF-related ligands in vivo. Moreover, α-tocopherol succinate is known to be compatible with influenza vaccines and to be a useful preservative as an alternative to mercurial compounds.

As mentioned above, oil-in-water emulsions comprising squalene are particularly preferred. In some embodiments, the squalene concentration in a vaccine dose may be in the range of 5-15mg (i.e. a concentration of 10-30mg/ml, assuming a 0.5ml dose volume). It is possible, though, to reduce the concentration of squalene [84,85] e.g. to include <5mg per dose, or even <l.lmg per dose. For example, a human dose may include 9.75mg squalene per dose (as in the FLU AD™ product: 9.75mg squalene, 1.175mg polysorbate 80, 1.175mg sorbitan trioleate, in a 0.5ml dose volume), or it may include a fractional amount thereof e.g. 3/4, 2/3, 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9, or 1/10. For example, a composition may include 7.3 lmg squalene per dose (and thus 0.88mg each of polysorbate 80 and sorbitan trioleate), 4.875mg squalene/dose (and thus 0.588mg each of polysorbate 80 and sorbitan trioleate), 3.25mg squalene/dose, 2.438mg/dose, 1.95mg/dose, 0.975mg/dose, etc. Any of these fractional dilutions of the FLUAD™-strength MF59 can be used with the invention.

As mentioned above, antigen/emulsion mixing may be performed extemporaneously, at the time of delivery. Thus the invention provides kits including the antigen and adjuvant components ready for mixing. The kit allows the adjuvant and the antigen to be kept separately until the time of use. The components are physically separate from each other within the kit, and this separation can be achieved in various ways. For instance, the two components may be in two separate containers, such as vials. The contents of the two vials can then be mixed e.g. by removing the contents of one vial and adding them to the other vial, or by separately removing the contents of both vials and mixing them in a third container. In a preferred arrangement, one of the kit components is in a syringe and the other is in a container such as a vial. The syringe can be used (e.g. with a needle) to insert its contents into the second container for mixing, and the mixture can then be withdrawn into the syringe. The mixed contents of the syringe can then be administered to a patient, typically through a new sterile needle. Packing one component in a syringe eliminates the need for using a separate syringe for patient administration. In another preferred arrangement, the two kit components are held together but separately in the same syringe e.g. a dual-chamber syringe, such as those disclosed in references 86-93 etc. When the syringe is actuated (e.g. during administration to a patient) then the contents of the two chambers are mixed. This arrangement avoids the need for a separate mixing step at the time of use.

Pharmaceutical compositions

Compositions of the invention are pharmaceutically acceptable. They usually include components in addition to the antigens and adjuvants e.g. they typically include one or more pharmaceutical carrier(s) and/or excipient(s). A thorough discussion of such components is available in reference 94.

Compositions will generally be in aqueous form.

The composition may include preservatives such as thiomersal (e.g at lOμg/ml) or 2-phenoxyethanol. It is preferred, however, that the vaccine should be substantially free from (i.e. less than 5μg/ml) mercurial material e.g. thiomersal-free [95]. Vaccines containing no mercury are more preferred. Preservative-free vaccines are particularly preferred.

To control tonicity, it is preferred to include a physiological salt, such as a sodium salt. Sodium chloride (NaCl) is preferred, which may be present at between 1 and 20 mg/ml. Other salts that may be present include potassium chloride, potassium dihydrogen phosphate, disodium phosphate dehydrate, magnesium chloride, calcium chloride, etc. Compositions will generally have an osmolality of between 200 mθsm/kg and 400 mθsm/kg, preferably between 240-360 mθsm/kg, and will more preferably fall within the range of 290-310 mθsm/kg. Osmolality has previously been reported not to have an impact on pain caused by vaccination [96], but keeping osmolality in this range is nevertheless preferred.

Compositions may include one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. Buffers will typically be included in the 5-2OmM range. The buffer may be in the emulsion's aqueous phase.

The pH of a composition will generally be between 5.0 and 8.1, and more typically between 6.0 and 8.0 e.g. 6.5 and 7.5, or between 7.0 and 7.8. A process of the invention may therefore include a step of adjusting the pH of the bulk vaccine prior to packaging. The composition is preferably sterile. The composition is preferably gluten free. Preferred vaccines have a low endotoxin content e.g. less than 1 IU/ml, and preferably less than 0.5 IU/ml. The international unit for endotoxin measurement is well known and can be calculated for a sample by, for instance, comparison to an international standard [97,98], such as the 2nd International Standard (Code 94/580 - IS) available from the NIBSC. Current vaccines prepared from virus grown in eggs have endotoxin levels in the region of 0.5-5 IU/ml.

The vaccine is preferably free from antibiotics (e.g. neomycin, kanamycin, polymyxin B).

The composition may include material for a single immunisation, or may include material for multiple immunisations (i.e. a 'multidose' composition). Multidose arrangements usually include a preservative in the vaccine. To avoid this need, a vaccine may be contained in a container having an aseptic adaptor for removal of material.

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

Packaging of compositions or kit components Processes of the invention can include a step in which vaccine is placed into a container, and in particular into a container for distribution for use by physicians.

Suitable containers for the vaccines include vials, nasal sprays and disposable syringes, which should be sterile.

Where a composition/component is located in a vial, the vial is preferably made of a glass or plastic material. The vial is preferably sterilized before the composition is added to it. To avoid problems with latex-sensitive patients, vials are preferably sealed with a latex-free stopper, and the absence of latex in all packaging material is preferred. The vial may include a single dose of vaccine, or it may include more than one dose (a 'multidose' vial) e.g. 10 doses. Preferred vials are made of colorless glass. A vial can have a cap (e.g. a Luer lock) adapted such that a pre-filled syringe can be inserted into the cap, the contents of the syringe can be expelled into the vial, and the contents of the vial can be removed back into the syringe. After removal of the syringe from the vial, a needle can then be attached and the composition can be administered to a patient. The cap is preferably located inside a seal or cover, such that the seal or cover has to be removed before the cap can be accessed. A vial may have a cap that permits aseptic removal of its contents, particularly for multidose vials.

Where a composition/component is packaged into a syringe, the syringe may have a needle attached to it. If a needle is not attached, a separate needle may be supplied with the syringe for assembly and use. Such a needle may be sheathed. Safety needles are preferred. 1-inch 23-gauge, 1-inch 25-gauge and 5/8-inch 25-gauge needles are typical. Syringes may be provided with peel-off labels on which the lot number, influenza season and expiration date of the contents may be printed, to facilitate record keeping. The plunger in the syringe preferably has a stopper to prevent the plunger from being accidentally removed during aspiration. The syringes may have a latex rubber cap and/or plunger. Disposable syringes contain a single dose of vaccine. The syringe will generally have a tip cap to seal the tip prior to attachment of a needle, and the tip cap is preferably made of a butyl rubber. If the syringe and needle are packaged separately then the needle is preferably fitted with a butyl rubber shield. Preferred syringes are those marketed under the trade name "Tip-Lok"™. Containers may be marked to show a half-dose volume e.g. to facilitate delivery to children. For instance, a syringe containing a 0.5ml dose may have a mark showing a 0.25ml volume.

Where a glass container (e.g. a syringe or a vial) is used, then it is preferred to use a container made from a borosilicate glass rather than from a soda lime glass.

A composition may be combined (e.g. in the same box) with a leaflet including details of the vaccine e.g. instructions for administration, details of the antigens within the vaccine, etc. The instructions may also contain warnings e.g. to keep a solution of adrenaline readily available in case of anaphylactic reaction following vaccination, etc.

Methods of treatment, and administration of the vaccine

Compositions of the invention are suitable for administration to human patients, and the invention provides a method of raising an immune response in a patient, comprising the step of administering a composition of the invention to the patient.

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

The immune response raised by the methods and uses of the invention will generally include an antibody response, preferably a protective antibody response. Methods for assessing antibody responses, neutralising capability and protection after influenza virus vaccination are well known in the art. Human studies have shown that antibody titers against hemagglutinin of human influenza virus are correlated with protection (a serum sample hemagglutination-inhibition titer of about 30-40 gives around 50% protection from infection by a homologous virus) [99]. Antibody responses are typically measured by hemagglutination inhibition, by microneutralisation, by single radial immunodiffusion (SRID), and/or by single radial hemolysis (SRH). These assay techniques are well known in the art.

Compositions of the invention can be administered in various ways. The most preferred immunisation route is by intramuscular injection (e.g. into the arm or leg), but other available routes include subcutaneous injection, intranasal [100-102], intradermal [103,104], oral [105], transcutaneous, transdermal [106], etc. Intradermal and intranasal routes are attractive. Intradermal administration may involve a microinjection device e.g. with a needle about 1.5mm long.

Vaccines prepared according to the invention may be used to treat both children and adults. Influenza vaccines are currently recommended for use in pediatric and adult immunisation, from the age of 6 months. Thus the patient may be less than 1 year old (e.g. <6 months old), 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. Preferred patients for receiving the vaccines are the elderly (e.g. >50 years old, >60 years old, and preferably >65 years), the young (e.g. <5 years old, or those aged between 6 months and 24 years, or between 6 months and 4 years, or between 5-18 years), middle aged (25-64 years old), hospitalised patients, healthcare workers, armed service and military personnel, pregnant women, the chronically ill, immunodeficient patients, patients who have taken an antiviral compound (e.g. an oseltamivir or zanamivir compound; see below) in the 7 days prior to receiving the vaccine, people with egg allergies and people travelling abroad. The vaccines are not suitable solely for these groups, however, and may be used more generally in a population.

Some older adults (about a third of those older than 60 years) but few young adults and essentially no children have pre-existing serum antibody against the pandemic A/CA/04/09 strain. Seasonal immunization of young people does not elicit antibodies against this strain [107]. A useful group of subjects to receive immunogenic compositions of the invention comprising an oil-in-water adjuvant is those subjects who have no existing serum antibody against the pandemic A/CA/04/09 strain e.g. patients born after 1960, after 1970, after 1980, after 1990, or after 2000.

Preferred compositions of the invention satisfy 1, 2 or 3 of the CPMP criteria for efficacy. In adults (18-60 years), these criteria are: (1) >70% seroprotection; (2) >40% seroconversion; and/or (3) a GMT increase of >2.5-fold. In elderly (>60 years), these criteria are: (1) >60% seroprotection; (2) >30% seroconversion; and/or (3) a GMT increase of >2-fold. These criteria are based on open label studies with at least 50 patients. The criteria apply for each strain in a vaccine.

Treatment can be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. In a multiple dose schedule the various doses may be given by the same or different routes e.g. a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc. Administration of more than one dose (typically two doses) is particularly useful in immunologically naϊve patients e.g. for people who have never received an influenza vaccine before, or for vaccinating against a new HA subtype. Multiple doses will typically be administered at least 1 week apart (e.g. about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 12 weeks, about 16 weeks, etc.). Vaccines produced by the invention may be administered to patients at substantially the same time as (e.g. during the same medical consultation or visit to a healthcare professional or vaccination centre) other vaccines e.g. at substantially the same time as a measles vaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine, a varicella vaccine, a MMRV vaccine, a diphtheria vaccine, a tetanus vaccine, a pertussis vaccine, a DTP vaccine, a conjugated H.influenzae type b vaccine, an inactivated poliovirus vaccine, a hepatitis B virus vaccine, a meningococcal conjugate vaccine (such as a tetravalent A-C-Wl 35-Y vaccine), a respiratory syncytial virus vaccine, a pneumococcal conjugate vaccine, etc. Administration at substantially the same time as a pneumococcal vaccine and/or a meningococcal vaccine is particularly useful in elderly patients.

Similarly, vaccines of the invention may be administered to patients at substantially the same time as (e.g. during the same medical consultation or visit to a healthcare professional) an antiviral compound, and in particular an antiviral compound active against influenza virus (e.g. oseltamivir and/or zanamivir). These antivirals include neuraminidase inhibitors, such as a (3R,4R,5S)-4- acetylamino-5-amino-3(l-ethylpropoxy)-l-cyclohexene-l-carboxylic acid or 5-(acetylamino)-4- [(aminoiminomethyO-aminoJ-ljβ-anhydro-S^^-trideoxy-D-glycero-D-galactonon-l-enonic acid, including esters thereof (e.g. the ethyl esters) and salts thereof (e.g. the phosphate salts). A preferred antiviral is (3R,4R,5S)-4-acetylamino-5-amino-3(l-ethylpropoxy)-l-cyclohexene-l-carboxylic acid, ethyl ester, phosphate (1:1), also known as oseltamivir phosphate (TAMIFLU™). Another antiviral which can be administered is thymosin alpha 1 (e.g. thymalfasin, a 28 amino acid synthetic peptide, available as ZADAXIN™) [108]. In one specific embodiment, a patient receives a neuraminidase inhibitor, such as oseltamivir phosphate, at substantially the same time as receiving an inactivated whole virion vaccine (e.g. monovalent, Hl*).

Vaccine products and kits As mentioned above, the invention provides a vaccine comprising (i) a Hl subtype influenza A virus hemagglutinin which is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3 and (ii) an oil- in-water emulsion adjuvant. In a useful embodiment, this composition is a monovalent inactivated surface antigen vaccine. The inactivated viruses may have been grown on eggs or in cell culture (e.g. in MDCK cells [36, 118]). The vaccine may be presented in a syringe (e.g. borosilicate glass) containing a 0.5ml unit dose, with each unit dose including about 7.5μg of the Hl hemagglutinin (e.g. a A/California/7/2009-like strain, such as from reassortant strain X-179A or X-181). The syringe may have a bromo-butyl rubber plunger-stopper The adjuvant comprises squalene, polysorbate 80 and sorbitan trioleate e.g. about 9.75mg of squalene, about 1.18mg polysorbate 80 and about 1.18mg sorbitan trioleate per 7.5μg of HA. The composition may include a citrate buffer. The composition is ideally mercury-free, although a low dose of thimerosal may sometimes be included. In some embodiments an adjuvanted vaccine has 3.75μg HA, particularly when a 0.25ml dosage volume is used. Adjuvanted vaccine may be administered intramuscularly e.g. to the deltoid or anterolateral thigh. A subject may receive a single dose of the adjuvanted vaccine or may receive two doses (e.g. separated by between 2 weeks and 6 months e.g. 3 weeks apart). Syringes can be packaged in a carton e.g. 10 per carton, each in a blister pack.

The invention also provides a vaccine comprising (i) a Hl subtype influenza A virus hemagglutinin which is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3 and (ii) an oil-in-water emulsion adjuvant. In a useful embodiment, this composition is a monovalent inactivated surface antigen vaccine. The inactivated viruses may have been grown on eggs. The vaccine may be presented in a vial containing multiple 0.5ml unit doses e.g. a 10-dose vial including thimerosal, with each unit dose including about 7.5μg or 15μg or 30μg of the Hl hemagglutinin (e.g. a A/California/7/2009-like strain, such as from reassortant strain X- 179A). The adjuvant comprises squalene, polysorbate 80 and sorbitan trioleate e.g. about 9.75mg of squalene, about 1.18mg polysorbate 80 and about 1.18mg sorbitan trioleate per 7.5μg of HA. The composition may include a citrate buffer. The adjuvanted vaccine may be administered intramuscularly e.g. to the deltoid or anterolateral thigh. A subject may receive a single dose of the adjuvanted vaccine or may receive two doses (e.g. separated by between 2 weeks and 6 months e.g. 3 weeks apart). The invention also provides a kit comprising (i) a first kit component comprising an unadjuvanted Hl subtype influenza A virus hemagglutinin which is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3 and (ii) a second kit component comprising an oil-in-water emulsion adjuvant. The two kit components can be mixed at the time of use to give a monovalent vaccine of the invention. In a useful embodiment, the first kit component is a monovalent split virion inactivated vaccine. The inactivated viruses may have been grown on eggs. The kit may be presented as a two-vial composition (e.g. borosilicate glass, optionally with butyl rubber stoppers), with each vial including an equal volume of liquid for mixing at a 1:1 volume ratio e.g. to mix 2.5ml antigen with 2.5ml adjuvant. A 0.5ml unit dose of the monovalent adjuvanted vaccine can include about 7.5μg, 3.75μg or 1.9μg of the Hl hemagglutinin (e.g. a A/California/7/2009-like strain, such as from reassortant strain X- 179A). The adjuvant comprises squalene, DL-α-tocopherol and polysorbate 80 e.g. in a 0.5ml unit dose: about 10.7mg of squalene, about 11.9mg tocopherol and about 4.9mg polysorbate 80 (or a fractional amount thereof e.g. 3/4, 2/3, 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9, or 1/10 of these amounts of squalene, tocopherol and polysorbate 80). Thus the adjuvant components may be present at a mass ratio (squalene tocopherol :polysorbate 80) of 2.20:2.44:1. The adjuvant components may be present at 2.85μg squalene, 3.16μg tocopherol and 1.30μg polysorbate 80 per μg of Hl hemagglutinin. The vaccine may include thiomersal preservative e.g. at about lOμg/ml i.e. about 5μg in a 0.5ml dose. A subject may receive a single dose of the adjuvanted vaccine or may receive two doses (e.g. separated by 1, 2 or 3 weeks, or by more than 3 weeks e.g. 3-26 weeks). Adults aged 18- 60 years may usefully receive a single dose, whereas elderly >60 years may receive two doses. Children aged 3-9 years may receive a half dose e.g. 0.25ml volume with 1.875μg HA. The antigen and adjuvant components may both include a phosphate buffer. The antigen component may include polysorbate 80, octoxynol 10, potassium chloride, and/or magnesium chloride. A kit of the invention may include 50 vials of antigen (2.5ml suspension in each) and 50 vials of adjuvant (2.5ml of emulsion in each). The antigen vials may be in a single pack; the adjuvant vials may be in two packs. Adjuvanted vaccine may be administered intramuscularly e.g. to the deltoid or anterolateral thigh.

The invention also provides a vaccine comprising (i) a Hl subtype influenza A virus hemagglutinin which is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3 and (ii) an oil-in-water emulsion adjuvant. In a useful embodiment, this composition is a monovalent inactivated surface antigen vaccine. The inactivated viruses were grown in MDCK cells [36, 118]. The vaccine is presented with a unit dose containing 3.75μg of the Hl hemagglutinin (e.g. a A/California/7/2009- like strain, such as from reassortant strain X- 179A). The adjuvant comprises squalene, polysorbate 80 and sorbitan trioleate e.g. about 4.875mg of squalene, about 0.59mg polysorbate 80 and about 0.59mg sorbitan trioleate. The composition may include a citrate buffer. Adjuvanted vaccine may be administered intramuscularly e.g. to the deltoid or anterolateral thigh. A subject may receive a single dose of the adjuvanted vaccine or may receive two doses (e.g. separated by between 2 weeks and 6 months e.g. 3 weeks apart). A unit dose may have a volume of 0.25ml, and patients can receive one unit dose (e.g. for patients 3-17 or 18-40 years old) or two unit doses (e.g. for patients >40 years old) in a single immunisation. The vaccine may be distributed as a pack of 10 x 0.25ml doses. The invention also provides a kit comprising (i) a first kit component comprising an unadjuvanted Hl subtype influenza A virus hemagglutinin which is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3 and (ii) a second kit component comprising an oil-in-water emulsion adjuvant. The two kit components can be mixed at the time of use to give a monovalent vaccine of the invention. In a useful embodiment, the first kit component is a monovalent inactivated split virion. The inactivated viruses may have been grown on eggs. The kit may be presented as a two-vial composition (e.g. borosilicate glass, optionally with chlorobutyl stoppers), with the first vial containing a unit volume of antigen and the second vial containing 3x that unit volume of emulsion e.g. for mixing to give 4x the unit volume of final vaccine. Thus 1.5ml of antigen can be combined with 4.5ml of emulsion to give 6ml of vaccine. A 0.5ml unit dose of the monovalent adjuvanted vaccine can include about 3.8μg of the Hl hemagglutinin (e.g. a A/California/7/2009-like strain, such as from reassortant strain X- 179A). The adjuvant comprises squalene, sorbitan oleate, polyoxyethylene cetostearyl ether and mannitol e.g. in a 0.5ml unit dose: about 12.4mg squalene, about 1.9mg sorbitan oleate, about 2.4mg polyoxyethylene cetostearyl ether, and about 2.3mg mannitol (or a fractional amount thereof e.g. 3/4, 2/3, 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9, or 1/10 of these amounts of squalene, sorbitan oleate, polyoxyethylene cetostearyl ether and mannitol). Thus the adjuvant components may be present at a mass ratio (squalene : sorbitan oleate : polyoxyethylene cetostearyl ether : mannitol) of 124:19:24:23. The vaccine may include thiomersal preservative e.g. at about 11.3μg per 0.5ml, or at about 3μg of thiomersal per μg of hemagglutinin. The antigen and adjuvant components may both include a phosphate buffer. A subject may receive a single dose of the adjuvanted vaccine or, more typically, may receive two doses (e.g. separated by more than 3 weeks e.g. 3-26 weeks). Subjects aged 3-60 years may usefully receive a single dose, whereas elderly >60 years may receive two doses. Children aged 6 months to less than 3 years may receive a half dose e.g. 0.25ml volume with about 1.9μg HA. Adjuvanted vaccine may be administered intramuscularly e.g. to the deltoid or anterolateral thigh. The invention also provides a method for preparing an influenza vaccine, comprising a step of mixing a first kit component as defined in the preceding paragraphs with a second kit component as defined in the preceding paragraphs.

Vaccines mentioned in this section can usefully include a hemagglutinin comprising SEQ ID NO: 7.

Mixed-source vaccines Some embodiments of the invention mentioned above are multivalent i.e. they include HA antigen from more than one strain of influenza virus. The viruses used to prepare a multivalent vaccine may all be grown using the same substrate (e.g. all grown in eggs, or all grown in MDCK culture, etc.) or they may be grown in different substrates (e.g. one strain grown in eggs, another strain grown in cell culture; or one strain grown in MDCK culture or another strain grown in Vero culture). For example, growth substrates can be chosen according to the growth preferences of a particular strain e.g. if a HlNl strain grows better in cell culture than in eggs, but an influenza B virus shows the opposite preference, they may be grown on the different substrates and then mixed. In one embodiment, a Hl* strain (e.g. HlNl) is grown in cell culture (e.g. in MDCK culture, such as a suspension culture [36,118]) and another strain (e.g. a H3N2 strain, an influenza B strain, etc.) is grown in eggs. Antigens prepared from the strains are then mixed to provide a multivalent influenza vaccine. This process is particularly suitable for preparing a 4-valent vaccine with two Hl strains (one a Hl * hemagglutinin, one not a Hl * hemagglutinin), a H3N2 strain, and one influenza B strain.

Thus the invention provides a vaccine comprising hemagglutinin obtained from at least two different strains of influenza virus, wherein a first hemagglutinin is prepared from influenza viruses grown in eggs and a second hemagglutinin is prepared from influenza viruses grown in cell culture. Thus two different strains of influenza virus are grown, one in cell culture and one in eggs. Virus is purified from both sources and then mixed to give a vaccine.

The first and second hemagglutinins may both be from an influenza A virus, both from an influenza B virus, or one may be from an influenza A virus and the other from an influenza B virus. Preferably at least one of the first and second hemagglutinins is from an influenza A virus. Where both the first and second hemagglutinins is from an influenza A virus, this will typically be a Hl hemagglutinin and a H3 hemagglutinin e.g. from a HlNl strain and from a H3N2 strain.

Where the first and second hemagglutinins include an influenza A virus hemagglutinin, one of these can be a Hl * hemagglutinin. It is preferred that the two influenza A hemagglutinins are not both Hl * hemagglutinins, and it is more preferred that the two influenza A hemagglutinins are not both Hl hemagglutinins. Where a vaccine includes a Hl* hemagglutinin this is preferably the second hemagglutinin i.e. the Hl* strain is grown in cell culture and Hl* vaccine antigen is then combined with a non-Hl* vaccine antigen prepared from eggs. In other embodiments, the Hl* hemagglutinin is the first hemagglutinin i.e. the Hl* strain is grown in eggs and a Hl* vaccine antigen is then combined with a non-Hl* vaccine antigen prepared from cell culture.

Suitable cell culture hosts are described above and include MDCK cells e.g. MDCK 33016, which can be grown in suspension and is useful for preparing virus having a Hl* hemagglutinin.

This mixed-source approach is particularly useful for making a vaccine comprising a Hl* strain, a non-Hl* Hl strain, a H3 strain and an influenza B strain. The Hl* strain can be grown in cell culture, and the other three strains (i.e. the usual trivalent mixture for recent seasonal vaccines) can be grown in eggs in the usual manner. General

The term "comprising" encompasses "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y.

The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.

The term "about" in relation to a numerical value x is optional and means, for example, x+10%. "GI" numbering is used above. A GI number, or "Genlnfo Identifier", is a series of digits assigned consecutively to each sequence record processed by NCBI when sequences are added to its databases. The GI number bears no resemblance to the accession number of the sequence record. When a sequence is updated (e.g. for correction, or to add more annotation or information) then it receives a new GI number. Thus the sequence associated with a given GI number is never changed.

Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc. Where animal (and particularly bovine) materials are used in the culture of cells, they should be obtained from sources that are free from transmissible spongiform encaphalopathies (TSEs), and in particular free from bovine spongiform encephalopathy (BSE). Overall, it is preferred to culture cells in the total absence of animal-derived materials.

Where a compound is administered to the body as part of a composition then that compound may alternatively be replaced by a suitable prodrug.

Where a cell substrate is used for reassortment or reverse genetics procedures, it is preferably one that has been approved for use in human vaccine production e.g. as in Ph Eur general chapter 5.2.3.

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

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows HI titers obtained after immunization with HlNl sw antigen either unadjuvanted (0.5 or lμg HA dose) or adjuvanted with MF59 (0.5μg). A PBS control was also used. The black bars show titers after one immunization; the grey bars show titers after two immunizations. Figure 2 shows lung viral load in ferrets immunized with various prime/boost regimens. Animal groups A to H are described below. The y-axis shows LogioTCID₅o/gr. Figure 3 shows nasal viral load in the same ferrets and the y-axis shows logio CDU. Figure 4 shows HI titers in the same ferrets.

Figure 5 shows IgG serum antibody titers (ELISA) after two HlNlsw boosting doses in mice primed with seasonal HlNl (Brisbane). The priming and boosting strains and adjuvanting are indicated. MODES FOR CARRYING OUT THE INVENTION

Ferret study

Reference 109 reports a ferret model for investigating influenza vaccines. Ferrets were primed with an adjuvanted (squalene-containing oil-in-water emulsion, MF59™) or unadjuvanted seasonal vaccine, or with PBS. Three weeks later (day 21) these ferrets received a booster dose of adjuvanted or unadjuvanted trivalent seasonal or a monovalent pandemic ('HlNlsw') vaccine, or PBS. Eight animal groups A to H were used in total:

S = seasonal, sw = swine, A = adjuvanted

At day 49 ferrets were then challenged with a HlNlsw strain (10⁶ TCID₅₀) and lung pathology was assessed in each group. Unlike seasonal HlNl, which infects only nose and trachea, the HlNlsw virus also infects the lungs. The HlNlsw virus is not lethal for the ferrets.

The average % of affected lung parenchyma were:

Thus, compared to the PBS group, all ferrets previously primed with either adjuvanted or unadjuvanted seasonal vaccine and then boosted with either the homologous seasonal or with HlNlsw had an important reduction in the lung pathology. The best effect was observed when the boosting dose was HlNlsw, and the priming and boosting vaccines were both adjuvanted (group C). A single dose of adjuvanted HlNlsw, even in the absence of priming, gave similar protection.

Lung viral load was also assessed and results are shown in Figure 2. As compared to PBS, one dose of adjuvanted HlNlsw vaccine reduced lung viral load by 2 to 3 logs (compare groups G & H). The viral load in the lungs was reduced to almost undetectable levels (group F) if the HlNlsw vaccination was preceded by administration of an unadjuvanted seasonal influenza vaccine, and the viral load was undetectable levels if the prior seasonal vaccine was adjuvanted (group C).

Viral load was also assessed from nasal swabs (Figure 3). As compared to PBS, one dose of adjuvanted HlNlsw vaccine, but not of unadjuvanted vaccine, reduced the viral load in the nasal swabs by 1 log. The nasal viral load was further reduced if the HlNlsw vaccination was preceded by vaccination with unadjuvanted seasonal vaccine (group F). The nasal viral load was undetectable if the HlNlsw vaccination was preceded by vaccination with an adjuvanted seasonal vaccine (group C). Similar results were found in throat swabs.

HI antibody responses were also measured at day 49 (Figure 4). One dose of adjuvanted HlNlsw vaccine was more immunogenic than unadjuvanted HlNlsw vaccine. HI titers against HlNlsw virus increased by at least 1 log in ferrets previously immunized with adjuvanted seasonal vaccine.

HI antibody responses against seasonal HlNl and H3N2 seasonal strains were also assessed. Antibodies which cross-react between seasonal HlNl and HlNlsw were not detected by HI.

Thus one dose of adjuvanted HlNlsw vaccine was more immunogenic and more efficacious than unadjuvanted vaccine, measured by viral loads in lungs, nose, and throat. Both immunogenicity and efficacy were enhanced by previous immunization with seasonal influenza vaccine, and this effect was better if the seasonal vaccine was adjuvanted. This enhancement of immunogenicity and efficacy does not appear to be due to antibodies cross-reacting (by HI) between seasonal HlNl and HlNlsw. These results can explain why elderly people might be better protected against HlNlsw virus despite little cross-reactivity of antibodies. They can also explain the preliminary results of clinical trials showing good response after one single dose in healthy adults, as this effect could be due to previous immunological experience with seasonal viruses (via natural infection or vaccination), despite little or no cross-reactivity of antibodies. The results also imply that better HlNlsw protection is achieved in the presence of an adjuvant and if a patient has previously been immunized with adjuvanted seasonal vaccine. The data suggest that immunologically naive individuals (e.g. children) and immunologically frail individuals may require more than one dose of adjuvanted HlNlsw vaccine for optimal and sustained protection even though a single dose can still be clinically useful. Further details of this ferret study are in reference 110.

Mouse study I

The benefits of using an oil-in-water emulsion adjuvant with a HlNlsw vaccine are apparent from an immunogenicity study performed in mice. In unprimed mice, without any prior exposures to flu antigens, a single dose of emulsion-adjuvanted HlNlsw vaccine gives HI titers associated with protection in humans. Without the adjuvant, however, two doses were required to reach this titer. Thus human protection may be achieved, even in children, with a single adjuvanted dose. In contrast, the 1976 swine flu vaccine required two doses for children and young adults. Furthermore, as the adjuvant can facilitate a single dose immunization even in unprimed subjects, subjects who have already been exposed to influenza (e.g. the general adult population) should also require only a single adjuvanted dose of vaccine to achieve a robust response.

Vaccines were prepared from HlNlsw A/California/07/2009 HlNl-like viruses grown in eggs. Vaccines were either unadjuvanted or were adjuvanted with an oil-in-water emulsion comprising squalene (MF59™). Vaccines were standardized by SRID with a HA dose of either 0.5μg or lμg. Balb/c mice aged 6-7 weeks were immunized intramuscularly on day 0 with phosphate buffered saline, with 0.5 or 1.0 μg (HA content) of antigen alone, or with 0.5 μg of antigen with 50 μl of adjuvant. Dose volume was 100 μl. Sera were obtained on day 13. Mice were boosted with a second dose, matching the first, on day 14. Sera were again collected on day 21. Sera were assayed by hemagglutination inhibition (HI) using inactivated whole virus for antigen and turkey red blood cells.

A single immunization with 0.5 μg adjuvanted antigen elicited an average functional antibody (HI) titer of 1 :63 in serum obtained two weeks after immunization (Figure 1). A HI titer of 1 :40 or more is associated with protection of humans from seasonal influenza [111]. A second immunization with adjuvanted vaccine two weeks later increased the average HI titer to 1:1280 in serum obtained one week after the boost. A single immunization with antigen without adjuvant did not elicit significant

HI titers, but a second immunization two weeks later elicited a HI titer of 1 :160. There was no significant difference in titers elicited by immunization with 0.5 or 1.0 μg of unadjuvanted antigen.

These data are consistent with results of human immunization with vaccines against other potential pandemic influenza strains. Without adjuvant, vaccines against H5 avian influenza strains elicit low antibody titers; MF59 greatly increases the rapidity, titer, and breadth of the elicited antibodies [112,113]. During a much smaller human outbreak of swine origin influenza in 1976, adjuvanted vaccines were not available. A single dose of the 1976 vaccines elicited low antibody titers in young people, but significantly higher titers in older individuals, probably because older subjects had experienced more priming influenza infections or immunizations [114]. The mouse immunization data support including adjuvants such as MF59 in HlNl sw pandemic immunization campaigns, particularly for children and young adults with little or no previous exposure to influenza infection or immunization. These individuals are particularly vulnerable to morbidity and mortality in the current pandemic [115]. With MF59-adjuvanted pandemic antigen, a single dose given to an immunologically naive mouse produces an antibody response that is associated with protection from seasonal influenza in humans; without adjuvant, two doses are required. In this study, no dose response was observed between 0.5 and 1 μg of unadjuvanted antigen. This finding in mice increases the likelihood that dose-sparing regimens that can increase the number of available doses may prove effective in human clinical trials.

Mouse study II Mice primed with seasonal HlNl (A/Brisbane/59/2007; 0.2μg HA dose) monovalent vaccine (with or without MF59 adjuvant) were boosted twice (days 36 and 66) with the same vaccine or with equivalent monovalent vaccines (again, with or without MF59 adjuvant) prepared from pandemic H INl sw strains (A/California/04/2009 hemagglutinins).

ELISA analysis of the immune responses (Figure 5) suggests that prior seasonal adjuvanted vaccination effectively primed the mice for a higher titer response to the HlNlsw vaccine, and this priming was especially important if the HlNlsw vaccine was unadjuvanted. In unprimed mice or mice primed with unadjuvanted seasonal HlNl, a high titer response was seen only if the HlNlsw vaccine was adjuvanted.

Thus adjuvanting of the HlNlsw vaccine seems to be important for a robust immune response. Moreover, adjuvanting seems to be important for allowing the seasonal vaccine to prime for a robust antibody response to the pandemic vaccine.

In summary, immunization with two doses of unadjuvanted pandemic vaccine elicited little functional antibody in un-primed mice or in mice primed with unadjuvanted seasonal vaccine. In mice primed with adjuvanted seasonal vaccine, however, two doses of unadjuvanted pandemic vaccine gave a good response. Mice responded robustly to two doses of adjuvanted pandemic vaccine regardless of whether they had been primed. Although adjuvanted seasonal vaccines may not efficiently elicit antibodies against the pandemic strain, therefore, they may prime for a higher titer response to pandemic vaccines. These data support the use of oil-in-water adjuvants for pandemic immunization, and also in seasonal campaigns to ready the population for pandemic immunization. Mouse study HI

Three groups of 40 6-week-old female BALB/c mice received a single i.m. injection of a trivalent seasonal vaccine, from either the 2005/06 season or the 2009/10 season (both northern hemisphere). Influenza-naive control mice received PBS. The vaccines were administered at 1/lOth the human dose (1.5μg HA per strain) on day 0. On day 40 mice were divided into four subgroups of 10 animals each and were re-vaccinated with a monovalent inactivated HlNlsw vaccine. The four groups received a high or low dose (3μg HA or 0.3 μg HA), with or without a submicron oil-in- water emulsion adjuvant comprising squalene in combination with sorbitan oleate, polyoxyethylene cetostearyl ether and mannitol. All animals then received a second HlNlsw dose at day 61. The presence of HI antibodies against the seasonal and pandemic HlNl strains was assessed at days 40, 61, 75 and 102. Full details of this mouse study are given in reference 116.

The results confirmed that a single injection of the HlNlsw vaccine was sufficient to induce HI antibody responses to protective levels, with or without adjuvant. The HI antibody titer (GMT) against the HlNlsw strain was >40 in all groups except for the group of naϊve mice immunized with 0.3 μg HA of unadjuvanted vaccine.

Antibodies elicited by previous seasonal influenza vaccination did not cross-react with the HlNlsw strain, but priming with seasonal influenza vaccines did result in higher antibody responses to non- adjuvanted HlNlsw vaccine. In contrast, previous seasonal immunization did not appear to influence the immunogenicity of the adjuvanted HlNlsw vaccine in mice, likely due to a strong primary response induced by the adjuvanted vaccine in these groups.

In conclusion, mouse study III supports the use in humans of a split-virion inactivated HlNlsw vaccine formulated with the squalene-in-water emulsion. Focetria™ and Celtura™ products

Either SPF eggs (for Focetria™) or a suspension culture of MDCK cells (for Celtura™) have been infected with a reassortant HlNl influenza A virus strain with a hemagglutinin that is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3. The viruses have been grown using known techniques, then collected and inactivated, and monovalent surface antigen vaccines have been prepared from the purified viruses. The purified antigens have been diluted and then combined with an oil-in-water emulsion comprising submicron squalene droplets (MF59™) to provide bulk vaccine for the Focetria™ product (having 7.5μg of hemagglutinin per 0.5ml unit dose) and the Celtura™ product (having 3.75μg of hemagglutinin per 0.25ml unit dose). The products are filled into syringes. Thus the products are distributed in pre-filled syringes for injection. These two monovalent adjuvanted products have been authorised for human use in various territories.

Human study I (Leicester, UK)

As reported in reference 117 monovalent surface antigen vaccines were prepared from an A/California/7/2009 HlNlsw strain. The vaccine strain had HA, NA and PBl gene segments from A/California/7/2001 HlNlsw and the other five segments were from A/PR8/8/34. Virus was grown in MDCK cells. Viruses and antigens were prepared using the process used to make the trivalent OPTAFLU™ product [118]. Two vaccines were prepared: an adjuvanted vaccine with 7.5μg HA and the MF59 oil-in-water emulsion comprising submicron squalene droplets; and an unadjuvanted vaccine with 15μg HA in buffer. All vaccines had a 0.5ml volume. A half-dose of the adjuvanted vaccine was used for some subjects (i.e. with a 0.25ml volume). HA content in the final vaccine was determined by means of reverse-phase HPLC because SRID reagents were unavailable.

175 subjects were split into seven groups. Subjects received either one dose (day 0) or two identical doses (day 0; day 7, 14 or 21). The seven groups A to G were as follows (doses; adj = adjuvanted):

Immunogenic ity was assessed at days 0, 14 and 21. An interim assessment measured immunogenicity immediately prior to administration of the day 21 dose. Thus groups A to C had completed their regimens whereas group D had received only a single 7.5μg adjuvanted dose. Groups E to G were not assessed at this interim stage. Antibody responses by were assessed by hemagglutination (HI) assay, as geometric mean titers (GMT), geometric mean ratios, seroconversion (%) and seroprotection (%).. Antibody responses were also assessed by microneutralization (MN) as GMTs, proportion of subjects with a titer >40 (%) or seroconversion. Antibody responses by HI in the interim assessment were as follows:

Antibody responses by MN in the interim assessment were as follows:

Pre-immunization antibodies were detected by HI assay (titer >1:8) and MN assay (titer >l:10) in 14% and 39% of subjects, respectively, with this frequency unrelated to age or to previous receipt of seasonal vaccine. On day 14, geometric mean titers (GMTs), as measured with the use of HI and MN assays were higher in subjects who received two 7.5μg adjuvanted doses as compared to those who had received only one dose (compare groups A to C against group D) but there was no significant difference in titer among the groups. On day 21, there was no significant difference in titer among subjects who had received one dose or two doses. All subjects had MN antibody at a titer exceeding 1:40 by day 21. Thus immune responses at the interim stage were consistent, with seroprotection against the 2009 HlNlsw virus within 2 weeks after administration of a single dose of the adjuvanted vaccine. One or two doses of the adjuvanted vaccine containing 7.5 μg of HA administered on various schedules elicited robust antibody titers. Although a double dose (15 μg HA) gave higher antibody levels than one dose, the seroprotective titer was attained in at least 80% of subjects in every group. Human study II

A monovalent inactivated surface antigen vaccine was prepared from an A/California/7/2009 HlNlsw virus grown in eggs. The antigen was diluted to a HA concentration of 30μg/ml and was mixed with MF59 oil-in-water emulsion (comprising submicron squalene droplets in a citrate buffer) to give an adjuvanted bulk with HA concentration of 15μg/ml. The adjuvanted vaccine was packaged into syringes as individual 0.5ml doses, to provide a vaccine with 7.5μg HA per 0.5ml dose. The adjuvanted vaccine (e.g. the FOCETRIA™ product) is administered intramuscularly e.g. to the deltoid or anterolateral thigh.

Human study III

A monovalent inactivated split vaccine from a HlNlsw strain was given to human adult volunteers (18-60 years old). Patients received either an adjuvanted or unadjuvanted vaccine. The adjuvanted vaccine had 5.25μg HA with a submicron oil-in-water emulsion comprising squalene (AS03); the unadjuvanted vaccine had 21μg HA. Vaccines were administered on days 0 and 21. HI titers against A/California/7/2009 were assessed on these days, as well as seroconversion and seroprotection. Full details of this human study are given in reference 119. The vaccine was well tolerated, and immunogenicity results were as follows:

Thus the adjuvanted and unadjuvanted vaccines were both immunogenic in adults, and a single dose of either 5.25μg HA (adjuvanted) or 21μg HA (non-adjuvanted) was enough to satisfy licensure criteria. The adjuvanted vaccine with fourfold less antigen induced a comparable immune response to the unadjuvanted vaccine.

Human study IV

An adjuvanted monovalent HlNlsw vaccine (7.5μg HA; FOCETRJA™) was given to human subjects (adults and elderly) either at the same time as, or three months after, trivalent (3x15 μg HA) 2009/10 seasonal vaccine (adjuvanted or unadjuvanted). All vaccines were inactivated surface antigen vaccines, and the adjuvant was a squalene-containing oil-in-water emulsion (MF59™). Immunogenicity of all vaccines was assessed by haemagglutination inhibition on Days 1 and 22, and safety and reactogenicity were monitored using patient diaries. Full details of this human study are given in reference 120.

One dose of adjuvanted HlNlsw vaccine met the licensure criteria for adult and elderly subjects 3 months after seasonal vaccination, or concomitantly with seasonal vaccine in adults, without impacting the tolerability or immunogenicity of either vaccine.

Human study V: combination therapy

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

Two different doses of thymalfasin were tested, to give a randomized, three-arm open label study.

Thymalfasin was given twice, the first injection seven days prior to vaccination and the second on the day of vaccination. All subjects who did not achieve an antibody titer of at least 1:40 on day 21 received a second vaccination on that day.

Compared to subjects who received the FOCETRJA™ vaccine alone, the addition of thymalfasin at both doses led to a statistically significant increase in the percentage of subjects who seroconverted at both 21 and 42 days after vaccination. 93% of patients receiving a low dose of thymalfasin (3.2mg), and 94% of patients receiving a high dose (6.4mg), achieved seroconversion against the HlNl* virus after 42 days, compared to 77% of patients who received vaccine alone. Human study VJ

Two multicenter randomized dose-ranging studies evaluated adjuvanted (with MF59) and non-adjuvanted egg-derived and cell culture-derived monovalent HlNl sw vaccines in healthy children 6 months to 17 years of age. The aim was to identify the preferred vaccine formulation (with or without adjuvant), dosage and schedule (one or two administrations) in healthy children and adolescents.

At enrolment, subjects were (i) stratified into four age cohorts i.e. 9-17 yr., 3-8 yr., 12-35 mo. and 6- 11 mo; and (ii) randomized into three vaccine groups given 3.75μg HA + Vi dose MF59, 7.5μg HA + full dose MF59 or 15μg HA unadjuvanted. Children aged 9-17 yr and infants aged 6-11 mo received only the adjuvanted vaccines. Subjects received two vaccinations 21 days apart. Vaccines were prepared either in eggs or in MDCK cell culture (suspension culture).

Immunogenicity was determined 21 days after each vaccination by hemagglutination inhibition (HI). Geometric mean HI titer (GMT) and geometric mean ratio (GMR) of post-/pre-vaccination HI titers were calculated. Seroconversion rate was also assessed i.e. % of subjects with post-vaccination HI >l :40 and negative at baseline (HI <l :10), or a minimum 4-fold increase in HI titre for subjects positive at baseline (HI≥l :10). Seroprotection rate (SP) was also assessed i.e. ^Λ of subjects with a HI titer > 1:40

Interim presents were obtained from subjects 3-8 and 9-17 years of age (388 subjects who received cell-derived vaccine, and 403 subjects who received egg-derived vaccine). GMT and GMR values in the subjects receiving the cell-derived vaccine were as follows:

GMT and GMR values in the subjects receiving the egg-derived vaccine were as follows:

The adjuvanted vaccines in the two studies had SP rates >70% 3 weeks after the first and the second vaccination in the 9-17 and 3-8 year age cohorts. Unadjuvanted vaccines in the two studies achieved SP rates >70% in 3-8 year age cohorts 3 weeks after the second vaccine dose. All vaccines in both age cohorts (3-17 years) had SC rates >40% three weeks after the first and the second vaccination in both studies. GMTs increased strongly three weeks after each dose, and all vaccines in both cohorts had GMRs >2.5.

The adjuvanted egg-derived (FOCETRIA™) and cell culture-derived (CELTURA™) vaccines induced rapid, strong immune responses at a lower HA dose than unadjuvanted vaccine. The immunogenicity of all adjuvanted vaccines met European regulatory pandemic influenza vaccine criteria (>70% subjects with HI titre >l:40; seroconversion >40% and GMR >2.5) with a single dose.

Human study VJI (Costa Rica)

This study aimed to determine the safety and antibody responses after administration of adjuvanted (with MF59) or unadjuvanted HlNlsw vaccines in a pediatric population. The vaccines were prepared from egg-grown virus. Subjects were divided in two age groups (children ages 3-8 yrs and adolescents ages 9 to 17 yrs) and were randomized to (a) one 7.5μg dose of adjuvanted vaccine, (b) one 15μg unadjuvanted dose, or (c) 30μg unadjuvanted dose (2xl5μg doses). Three weeks later, subjects received an MF59-adjuvanted vaccine with 7.5μg of H5N1 hemagglutinin (surface antigen vaccine, egg-derived). Blood samples for serologic testing were collected on day 1 (immunization), day 22, day 29 and day 43. Antibody titers against the HlNl vaccine antigen were evaluated by haemagglutination inhibition (HI). Geometric mean titers (GMTs) of anti-haemagglutination inhibition antibody, seroconversion (SC) rates and percentage of subjects with HI titer >l:40 were calculated. SC rates and HI titer >l :40 were compared to available Center for Biologies Evaluation and Research (CBER) regulatory criteria. The lower bound of the 95% CI for SC rate should be >40%. The lower bound of the 95% CI for percentage with HI titer >1 :40 should be > 70%.

194 children and 196 adolescents were enrolled. After the first dose (day 22), 93% of children given the 7.5μg adjuvanted vaccine achieved HI titer >l:40, compared with 72-74% of those given unadjuvanted vaccines. The SC rate (day 22) for the adjuvanted vaccine in children ages 3-8 years (91%) was higher than for non-adjuvanted vaccines (71-72%). By day 29, all subjects given 7.5μg of adjuvanted vaccine achieved HI titer >l :40; all vaccines met the CBER criteria. SC rates following the second vaccine dose ranged from 83-95% across all study groups. GMTs rose after each vaccination, but more strongly in subjects given 7.5μg adjuvanted vaccine, particularly in children. All three HlNl vaccines generated high HI antibody responses in a pediatric population within 2 doses of vaccine, but after a single dose only the adjuvanted vaccine achieved HI antibody responses meeting CBER immunogenicity criteria. These criteria were met even with a lower total dose of antigen (7.5μg) in the adjuvanted as compared with the unadjuvanted vaccine.

Human study VIII(USA) A dose-ranging study was performed to evaluate the optimal dose of a monovalent HlNlsw vaccine with or without an oil-in-water adjuvant (MF59) in the pediatric population. A total of 1357 healthy children, 3 to < 9 years of age, were enrolled. Children were randomized equally to eight groups and given intramuscular vaccine injections on Day 1 and Day 22. Vaccines were formulated as 3.75, 7.5, 15 or 30 μg HA with or without a full or half dose of MF59. Immunogenicity (HI assay) according to CBER criteria [HI titre > 1 :40 (95% CI lower bound > 70%) and seroconversion rate (95% CI lower bound > 40%)] was evaluated on Day 22 and 43. Seroconversion was defined as a prevaccination HI titre < 1 : 10 and post- vaccination titre > 1 :40, or a pre-vaccination HI titre > 1 :10 and > 4-fold rise in post-vaccination titre. HI antibody responses were expressed as geometric mean titres (GMTs) and geometric mean ratio (GMRs) of the post- to pre- vaccination titre. Pairwise comparisons of GMT ratios between each group were performed and 95% CI were assessed against a non-inferiority margin of 0.5, and, subsequently, 0.67. Differences between vaccine groups were assumed to be statistically significant if the 2-sided 95% CI around the GMT ratio did not contain 1, showing either statistically significant superiority or inferiority.

GMT and GMR results were as follows:

Baseline seropositivity rates (HI titre > 10) in each group was comparable (18% - 27%). All adjuvanted groups satisfied the HI titre > 1 :40 criterion after one dose while unadjuvanted groups met seroprotection criteria only after two doses. Subjects in all vaccine groups (except the unadjuvanted 7.5μg group) satisfied the seroconversion criterion after dose 1, and all groups met this criterion after two doses. Pairwise group comparisons of GMTs at Day 22 using two-sided 95% CIs shows that all adjuvanted vaccines were superior to the non-adjuvanted vaccines. The adjuvanted groups met the licensure criteria after one dose and the vaccine dose with 7.5μg antigen and a half dose of MF59 adjuvant showed a clearly superior response.

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

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Claims

I . A method for immunizing a human, comprising a step of administering to the patient a vaccine comprising (i) a Hl subtype influenza A virus hemagglutinin which is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3 and (ii) an oil-in-water emulsion adjuvant. 2. An immunogenic composition comprising (i) a Hl subtype influenza A virus hemagglutinin which is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3 and (ii) an oil-in-water emulsion adjuvant.

3. The composition of claim 2, which is a monovalent vaccine.

4. The composition of claim 2 or claim 3, wherein hemagglutinin comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2.

5. The composition of any one of claims 2 to 4, wherein the oil-in-water emulsion adjuvant comprises squalene and has droplets with a diameter below 250nm.

6. The composition of any one of claims 2 to 5, wherein the composition has a hemagglutinin concentration of about 7.5μg/ml or about 15μg/ml. 7. The composition of claim 2, which is a trivalent vaccine also including a H3N2 influenza A virus hemagglutinin and an influenza B virus hemagglutinin.

8. An immunogenic composition comprising two different Hl subtype influenza A virus hemagglutinins and an oil-in-water emulsion adjuvant, wherein (i) the first Hl subtype influenza A virus hemagglutinin is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3 and (ii) the second Hl subtype influenza A virus hemagglutinin is more closely related to SEQ ID NO: 3 than to SEQ ID NO: 1.

9. The composition of claim 8, also including (iii) a H3N2 influenza A virus hemagglutinin and (iv) an influenza B virus hemagglutinin.

10. The composition of claim 8, also including (iii) a H3N2 influenza A virus hemagglutinin, (iv) a B/Victoria/2/87 like influenza B virus hemagglutinin; and (v) a B/Yamagata/ 16/88 like influenza

B virus hemagglutinin.

I 1. A method for immunizing a human against influenza viruses, comprising steps of (i) administering to the patient a monovalent vaccine comprising a Hl subtype influenza A virus hemagglutinin and (ii) administering to the patient a trivalent A/H1N1-A/H3N2-B seasonal influenza vaccine; wherein (a) the monovalent vaccine includes a Hl subtype influenza A virus hemagglutinin which is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3, (b) the trivalent vaccine includes a Hl subtype influenza A virus hemagglutinin which is more closely related to SEQ ID NO: 3 than to SEQ ID NO: 1, (c) the monovalent vaccine comprises an oil-in-water emulsion adjuvant, and (d) the trivalent vaccine comprises an oil-in-water emulsion adjuvant.

12. The method of claim 11, wherein the monovalent vaccine is administered at least 4 weeks before the trivalent vaccine.

13. The method of claim 11, wherein the monovalent vaccine is administered at least 4 weeks after the trivalent vaccine. 14. Use of a Hl subtype influenza A virus hemagglutinin in the manufacture of a monovalent vaccine for immunizing a human, wherein the vaccine includes an oil-in-water emulsion adjuvant, and wherein the monovalent vaccine includes a Hl subtype influenza A virus hemagglutinin which is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3.

15. Use of claim 14, wherein the human has previously received a trivalent A/H1N1-A/H3N2-B seasonal influenza vaccine, and wherein the trivalent vaccine includes a Hl subtype influenza A virus hemagglutinin which is more closely related to SEQ ID NO: 3 than to SEQ ID NO: 1.

16. The method, composition or use of any preceding claim, wherein the emulsion includes oil droplets having a submicron diameter and wherein the emulsion comprises squalene.

17. A vaccine comprising hemagglutinin obtained from at least two different strains of influenza virus, wherein a first hemagglutinin is prepared from influenza viruses grown in eggs and a second hemagglutinin is prepared from influenza viruses grown in cell culture, and wherein the second hemagglutinin is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3.

18. The vaccine of claim 17, wherein the cell culture is a MDCK cell culture.

19. An immunogenic composition comprising (i) a purified Hl subtype influenza A virus haemagglutinin expressed in a recombinant host, wherein the Hl hemagglutinin is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3, and (ii) an oil-in-water emulsion adjuvant.

20. The composition of claim 19, wherein the hemagglutinin is expressed in an insect cell line using a baculovirus vector.

21. The composition of claim 19 or claim 20, wherein the composition includes recombinant influenza virus neuraminidase.

22. A method for immunizing a subject, comprising administering two separate doses of influenza vaccine to the subject, wherein (a) the two doses are administered from 1-6 weeks apart, (b) each vaccine contains a Hl subtype influenza A virus hemagglutinin which is more closely related to SEQ ID NO: 1 than to SEQ ID NO: 3, and (c) the subject takes a neuraminidase inhibitor, such as oseltamivir phosphate, at least 3 times between receiving the two vaccine doses.

23. The method of claim 22, wherein one or both of the administered vaccines comprise an oil-in-water emulsion adjuvant.