US20080171063A1 - Novel Composition - Google Patents

Novel Composition Download PDF

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US20080171063A1
US20080171063A1 US11/909,388 US90938806A US2008171063A1 US 20080171063 A1 US20080171063 A1 US 20080171063A1 US 90938806 A US90938806 A US 90938806A US 2008171063 A1 US2008171063 A1 US 2008171063A1
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influenza
composition
oil
antigen
strain
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Emmanuel Jules Hanon
Jean Stephenne
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GlaxoSmithKline Biologicals SA
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GlaxoSmithKline Biologicals SA
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Priority claimed from GB0506004A external-priority patent/GB0506004D0/en
Priority claimed from GB0506000A external-priority patent/GB0506000D0/en
Priority claimed from GB0505989A external-priority patent/GB0505989D0/en
Priority claimed from GB0506001A external-priority patent/GB0506001D0/en
Priority claimed from GB0505998A external-priority patent/GB0505998D0/en
Priority claimed from GB0510598A external-priority patent/GB0510598D0/en
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Priority claimed from GB0510589A external-priority patent/GB0510589D0/en
Priority claimed from GB0510591A external-priority patent/GB0510591D0/en
Priority claimed from GB0603790A external-priority patent/GB0603790D0/en
Priority claimed from GB0603788A external-priority patent/GB0603788D0/en
Priority claimed from GB0603789A external-priority patent/GB0603789D0/en
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Assigned to GLAXOSMITHKLINE BIOLOGICALS SA reassignment GLAXOSMITHKLINE BIOLOGICALS SA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HANON, EMMANUEL JULES, STEPHENNE, JEAN
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    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • A—HUMAN NECESSITIES
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    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
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    • A—HUMAN NECESSITIES
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    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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    • A61P37/02—Immunomodulators
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/11—Orthomyxoviridae, e.g. influenza virus
    • C—CHEMISTRY; METALLURGY
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    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
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    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
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    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to influenza vaccine formulations and vaccination regimes for immunising against influenza disease, their use in medicine, in particular their use in augmenting immune responses to various antigens, and to methods of preparation.
  • the invention relates to multivalent influenza immunogenic compositions comprising an influenza antigen or antigenic preparation thereof from at least two influenza virus strains, at least one strain being associated with a pandemic outbreak or having the potential to be associated with a pandemic outbreak, in combination with an oil-in-water emulsion adjuvant.
  • Influenza viruses are one of the most ubiquitous viruses present in the world, affecting both humans and livestock. Influenza results in an economic burden, morbidity and even mortality, which are significant.
  • the influenza virus is an RNA enveloped virus with a particle size of about 125 nm in diameter. It consists basically of an internal nucleocapsid or core of ribonucleic acid (RNA) associated with nucleoprotein, surrounded by a viral envelope with a lipid bilayer structure and external glycoproteins. The inner layer of the viral envelope is composed predominantly of matrix proteins and the outer layer mostly of host-derived lipid material.
  • Influenza virus comprises two surface antigens, glycoproteins neuraminidase (NA) and haemagglutinin (HA), which appear as spikes, 10 to 12 nm long, at the surface of the particles. It is these surface proteins, particularly the haemagglutinin that determine the antigenic specificity of the influenza subtypes.
  • drifts and ‘shifts’ are unpredictable and may have a dramatic impact from an immunological point of view as they eventually lead to the emergence of new influenza strains and that enable the virus to escape the immune system causing the well known, almost annual, epidemics.
  • influenza virus strains to be incorporated into influenza vaccine each season are determined by the World Health Organisation in collaboration with national health authorities and vaccine manufacturers.
  • HA is the most important antigen in defining the serological specificity of the different influenza strains.
  • This 75-80 kD protein contains numerous antigenic determinants, several of which are in regions that undergo sequence changes in different strains (strain-specific determinants) and others in regions which are common to many HA molecules (common to determinants).
  • Influenza viruses cause epidemics almost every winter, with infection rates for type A or B virus as high as 40% over a six-week period. Influenza infection results in various disease states, from a sub-clinical infection through mild upper respiratory infection to a severe viral pneumonia. Typical influenza epidemics cause increases in incidence of pneumonia and lower respiratory disease as witnessed by increased rates of hospitalization or mortality. The severity of the disease is primarily determined by the age of the host, his immune status and the site of infection.
  • influenza vaccines are either inactivated or live attenuated influenza vaccine.
  • Inactivated flu vaccines are composed of three possible forms of antigen preparation: inactivated whole virus, sub-virions where purified virus particles are disrupted with detergents or other reagents to solubilise the lipid envelope (so-called “split” vaccine) or purified HA and NA (subunit vaccine). These inactivated vaccines are given intramuscularly (i. m.) or intranasaly (i.n.).
  • Influenza vaccines of all kinds, are usually trivalent vaccines. They generally contain antigens derived from two influenza A virus strains and one influenza B strain. A standard 0.5 ml injectable dose in most cases contains 15 ⁇ g of haemagglutinin antigen component from each strain, as measured by single radial immunodiffusion (SRD) (J. M. Wood et al.: An improved single radial immunodiffusion technique for the assay of influenza haemagglutinin antigen: adaptation for potency determination of inactivated whole virus and subunit vaccines. J. Biol. Stand. 5 (1977) 237-247; J. M. Wood et al., International collaborative study of single radial diffusion and immunoelectrophoresis techniques for the assay of haemagglutinin antigen of influenza virus. J. Biol. Stand. 9 (1981) 317-330).
  • SRD single radial immunodiffusion
  • Influenza vaccines currently available are considered safe in all age groups (De Donato et al. 1999, Vaccine, 17, 3094-3101). However, there is little evidence that current influenza vaccines work in small children under two years of age. Furthermore, reported rates of vaccine efficacy for prevention of typical confirmed influenza illness are 23-72% for the elderly, which are significantly lower than the 60-90% efficacy rates reported for younger adults (Govaert, 1994, J. Am. Med. Assoc., 21, 166-1665; Gross, 1995, Ann Intern. Med. 123, 523-527).
  • influenza vaccine has been shown to correlate with serum titres of hemagglutination inhibition (HI) antibodies to the viral strain, and several studies have found that older adults exhibit lower HI titres after influenza immunisation than do younger adults (Murasko, 2002, Experimental gerontology, 37, 427-439).
  • HI hemagglutination inhibition
  • a sub-unit influenza vaccine adjuvanted with the adjuvant MF59, in the form of an oil-in-water emulsion is commercially available, and has demonstrated its ability to induce a higher antibody titer than that obtained with the non-adjuvanted sub-unit vaccine (De Donato et al. 1999, Vaccine, 17, 3094-3101). However, in a later publication, the same vaccine has not demonstrated its improved profile compared to a non-adjuvanted split vaccine (Puig-Barbera et al., 2004, Vaccine 23, 283-289).
  • influenza viruses circulate that are related to those from the preceding epidemic.
  • the viruses spread among people with varying levels of immunity from infections earlier in life.
  • Such circulation over a period of usually 2-3 years, promotes the selection of new strains that have changed enough to cause an epidemic again among the general population; this process is termed ‘antigenic drift’.
  • drift variants may have different impacts in different communities, regions, countries or continents in any one year, although over several years their overall impact is often similar.
  • an influenza pandemics occurs when a new influenza virus appears against which the human population has no immunity.
  • Typical influenza epidemics cause increases in incidence of pneumonia and lower respiratory disease as witnessed by increased rates of hospitalisation or mortality.
  • the elderly or those with underlying chronic diseases are most likely to experience such complications, but young infants also may suffer severe disease.
  • novel influenza viruses emerge with a key surface antigen, the haemagglutinin, of a totally different subtype from strains circulating the season before.
  • the resulting antigens can vary from 20% to 50% from the corresponding protein of strains that were previously circulating in humans. This can result in virus escaping ‘herd immunity’ and establishing pandemics. This phenomenon is called ‘antigenic shift’. It is thought that at least in the past pandemics have occurred when an influenza virus from a different species, such as an avian or a porcine influenza virus, has crossed the species barrier. If such viruses have the potential to spread from person to person, they may spread worldwide within a few months to a year, resulting in a pandemic.
  • viruses of the H2N2 subtype replaced H1N1 viruses that had been circulating in the human population since at least 1918 when the virus was first isolated.
  • the H2 HA and N2 NA underwent antigenic drift between 1957 and 1968 until the HA was replaced in 1968 (Hong-Kong Flu pandemic) by the emergence of the H3N2 influenza subtype, after which the N2 NA continued to drift along with the H3 HA (Nakajima et al., 1991, Epidemiol. Infect. 106, 383-395).
  • an influenza virus strain that give it the potential to cause a pandemic outbreak are: it contains a new haemagglutinin compared to the haemagglutinin in the currently circulating strains, which may or not be accompanied by a change in neuraminidase subtype; it is capable of being transmitted horizontally in the human population; and it is pathogenic for humans.
  • a new haemagglutinin may be one which has not been evident in the human population for an extended period of time, probably a number of decades, such as H2. Or it may be a haemagglutinin that has not been circulating in the human population before, for example H5, H9, H7 or H6 which are found in birds. In either case the majority, or at least a large proportion of, or even the entire population has not previously encountered the antigen and is immunologically na ⁇ ve to it.
  • influenza vaccines especially in the case of influenza pandemics and for the elderly population.
  • a multivalent influenza immunogenic composition comprising an influenza virus or antigenic preparation thereof from at least two influenza virus strains, at least one strain being associated with a pandemic outbreak or having the potential to be associated with a pandemic outbreak, in combination with an oil-in-water emulsion adjuvant, wherein said oil-in-water emulsion adjuvant comprises a metabolisable oil, a sterol and an emulsifying agent.
  • said sterol is alpha-tocopherol.
  • Suitable pandemic strains are, but not limited to: H5N1, H9N2, H7N7, H2N2 and H1N1.
  • the invention provides a method for the production of an influenza immunogenic composition for a pandemic situation which method comprises admixing influenza virus antigen or antigenic preparation from at least two influenza virus strains, at least one of which is associated with a pandemic outbreak or has the potential to be associated with a pandemic outbreak, with an oil-in-water emulsion as herein above defined.
  • an immunogenic composition as herein defined for use in medicine.
  • an influenza virus antigen or antigenic preparation thereof and (b) an oil-in-water emulsion adjuvant in the manufacture of an immunogenic composition for inducing at least one of i) an improved CD4 T-cell immune response, ii) an improved B cell memory response, against said virus antigen or antigenic composition in a human, preferably in an immuno-compromised individual or population, such as a high risk adult or an elderly, which is preferred.
  • an immunogenic composition is as herein defined.
  • influenza virus or antigenic preparation thereof thereof and an oil-in-water emulsion adjuvant in the preparation of an immunogenic composition as herein defined for vaccination of human elderly against influenza.
  • the immunogenic composition is capable of inducing both an improved CD4 T-cell immune response and an improved B-memory cell response compared to that obtained with the un-adjuvanted antigen or antigenic composition.
  • an influenza virus or antigenic preparation thereof in the manufacture of an immunogenic composition for revaccination of humans previously vaccinated with a multivalent influenza immunogenic composition
  • a multivalent influenza immunogenic composition comprising an influenza antigen or antigenic preparation thereof from at least two influenza virus strains, at least one strain being associated with a pandemic outbreak or having the potential to be associated with a pandemic outbreak, in combination with an oil-in-water emulsion adjuvant as herein defined.
  • the composition used for the revaccination may be un-adjuvanted or may contain an adjuvant, in particular an oil-in-water emulsion adjuvant.
  • the immunogenic composition for revaccination contains an influenza virus or antigenic preparation thereof which shares common CD4 T-cell epitopes with the influenza virus or virus antigenic preparation thereof used for the first vaccination.
  • revaccination is made in subjects who have been vaccinated the previous season against influenza.
  • revaccination is made at least 6 months after the first vaccination, preferably 8 to 14 months after, more preferably at around 10 to 12 months after.
  • said oil-in-water emulsion comprises a metabolisable oil, alpha tocopherol and an emulsifying agent.
  • said oil-in-water emulsion adjuvant comprises at least one metabolisable oil in an amount of 0.5% to 20% of the total volume, and has oil droplets of which at least 70% by intensity have diameters of less than 1 ⁇ m.
  • an antigen or antigenic preparation from a first influenza strain in the manufacture of an immunogenic composition as herein defined for protection against influenza infections caused by a variant influenza strain.
  • influenza immunogenic composition comprising an influenza antigen or antigenic preparation thereof from at least two influenza virus strains, at least one strain being associated with a pandemic outbreak or having the potential to be associated with a pandemic outbreak, in combination with an oil-in-water emulsion adjuvant as herein defined.
  • the invention provides a method for revaccinating humans previously vaccinated with a multivalent influenza immunogenic composition comprising an influenza antigen or antigenic preparation thereof from at least two influenza virus strains, at least one strain being associated with a pandemic outbreak or having the potential to be associated with a pandemic outbreak, in combination with an oil-in-water emulsion adjuvant, said method comprising administering to said human an immunogenic composition comprising an influenza virus, either adjuvanted or un-adjuvanted.
  • a method for vaccinating a human population or individual against one influenza virus strain followed by revaccination of said human or population against a variant influenza virus strain comprising administering to said human (i) a first composition comprising an influenza virus or antigenic preparation thereof from a first influenza virus strain and an oil-in-water emulsion adjuvant, and (ii) a second immunogenic composition comprising a influenza virus strain variant of said first influenza virus strain.
  • said first strain is associated with a pandemic outbreak or has the potential to be associated with a pandemic outbreak.
  • said variant strain is associated with a pandemic outbreak or has the potential to be associated with a pandemic outbreak.
  • FIG. 1 Oil droplet particle size distribution in SB62 oil-in-water emulsion as measured by PCS.
  • FIG. 1B shows a schematic illustration of record 22 (upper part) and record 23 (lower part) by intensity.
  • FIG. 2 Schematic illustration of the preparation of MPL bulk.
  • FIG. 3 Schematic illustration of the preparation of AS03+MPL adjuvant.
  • FIG. 6 Explo Flu-001 clinical trial. Cross-reactive CD4 T-cell response to split influenza virus antigen after vaccination with Fluarix+AS03.
  • FIG. 7 Explo Flu-001 clinical trial. B cell memory response post vaccination.
  • FIG. 8 Explo Flu-002 clinical trial. CD4 T cell response against split influenza antigen following revaccination.
  • FIG. 9 Explo Flu-002 clinical trial. Anti-HI titers following revaccination.
  • FIG. 10 Ferret study 1. Temperature monitoring (priming and challenge). FIG. 10A is priming, FIG. 10B is challenge.
  • FIG. 11 Ferret study 1. Viral shedding.
  • FIG. 12 Ferret study II. Temperature monitoring (priming and challenge). FIG. 12A is priming, FIG. 12B is challenge.
  • FIG. 13 Ferret study II. Viral shedding.
  • FIG. 14 Ferret study II. HI titers to H3N2 A/Panama (vaccine strain) ( FIG. 14A ) and to H3N2 A/Wyoming (challenge strain) ( FIG. 14B ).
  • FIG. 15 Mice study. Frequencies of CD4 T cells in C57BI/6 primed mice using whole inactivated virus as re-stimulating antigen (day 7 post-immunisation).
  • FIG. 16 Mice study. Frequencies of CD8 T cells in C57BI/6 primed mice using whole inactivated virus as re-stimulating antigen (day 7 post-immunisation).
  • FIG. 17 Mice study. Frequencies of CD4 (upper part) and CD8 (lower part) T cells in C57BI/6 mice primed with heterologous strains, using whole inactivated virus as re-stimulating antigen (day 7 post-immunisation).
  • FIG. 18 Human clinical trial. B cell memory response post-vaccination of elderly with Fluarix, Fluarix+AS03, Fluarix+AS03+MPL (difference between pre- and post-).
  • FIG. 19 Ferret study III. Temperature monitoring before and after challenge.
  • FIG. 20 Ferret study III. Viral shedding before and after challenge.
  • FIG. 21 Ferret study III. HI titers to H3N2 A/Woming (vaccine strain).
  • FIG. 22 Ferret study III. HI titers to H3N2 A/Panama (challenge strain).
  • FIG. 23 Human clinical trial. HI titers (GMTs) at days 21, 90 and 180 post vaccination (persistence).
  • FIG. 24 Human clinical trial.
  • CD4 response all double test—Pool antigen at days 21, 90 and 180 post vaccination (persistence).
  • FIG. 25 Human clinical trial. HI titers in a revaccination clinical trial with AS03+MPL compared to Fluarix.
  • FIG. 26 Human clinical trial.
  • CMI for CD4 response all double test—Pool antigen at days 0 and 21.
  • FIG. 27 Human clinical trial with AS03+MPL at two concentrations. HI titers at days 0 and 21.
  • FIG. 28 Human clinical trial with AS03+MPL at two concentrations. Reactogenicity.
  • an influenza formulation comprising an influenza virus or antigenic preparation thereof together with an oil-in-water emulsion adjuvant comprising a metabolisable oil, a sterol such as alpha tocopherol and an emulsifying agent, was capable of improving the CD4 T-cell immune response and/or B cell memory response against said antigen or antigenic composition in a human compared to that obtained with the un-adjuvanted virus or antigenic preparation thereof.
  • the formulations adjuvanted with an oil-in-water emulsion adjuvant as herein defined will advantageously be used to induce anti-influenza CD4-T cell response capable of detection of influenza epitopes presented by MHC class II molecules.
  • the present Applicant has now found that it is effective to target the cell-mediated immune system in order to increase responsiveness against homologous and drift influenza strains (upon vaccination and infection).
  • compositions for use in the present invention have been able to provide better sero-protection against influenza following revaccination, as assessed by the number of human subjects meeting the influenza correlates of protections. Furthermore, the composition for use in the present invention have also been able to induce a trend for a higher B cell memory response following the first vaccination of a human subject, and a higher humoral response following revaccination, compared to the un-adjuvanted composition.
  • the Inventors have also been capable of demonstrating that the claimed adjuvanted composition was able to not only induce but also maintain protective levels of antibodies against all three strains present in the vaccine, in more individuals than those obtained with the un-advanted composition (see Table 43 for example).
  • the claimed composition is capable of ensuring a persistent immune response against influenza related disease.
  • persistence it is meant an HI antibody immune response which is capable of meeting regulatory criteria after at least three months, preferably after at least 6 months after the vaccination.
  • the claimed composition is able to induce protective levels of antibodies in >70% of individuals, suitably in >80% of individuals or suitably in >90% of individuals for at least one influenza strain, preferably for all strains present in the vaccine, after at least three months.
  • protective levels of antibodies of >90% are obtained at least 6 months post-vaccination against at least one, suitably two, or all strains present in the vaccine composition.
  • influenza virus or antigenic preparation thereof for use according to the present invention may be a split influenza virus or split virus antigenic preparation thereof.
  • the influenza preparation may contain another type of inactivated influenza antigen, such as inactivated whole virus or purified HA and NA (subunit vaccine), or an influenza virosome.
  • influenza virus may be a live attenuated influenza preparation.
  • split influenza virus or split virus antigenic preparation thereof for use according to the present invention is suitably an inactivated virus preparation where virus particles are disrupted with detergents or other reagents to solubilise the lipid envelope.
  • Split virus or split virus antigenic preparations thereof are suitably prepared by fragmentation of whole influenza virus, either infectious or inactivated, with solubilising concentrations of organic solvents or detergents and subsequent removal of all or the majority of the solubilising agent and some or most of the viral lipid material.
  • split virus antigenic preparation thereof is meant a split virus preparation which may have undergone some degree of purification compared to the split virus whilst retaining most of the antigenic properties of the split virus components.
  • split virus antigenic preparation may comprise split virus antigenic components of more than one viral strain.
  • Vaccines containing split virus called ‘influenza split vaccine’
  • split virus antigenic preparations generally contain residual matrix protein and nucleoprotein and sometimes lipid, as well as the membrane envelope proteins.
  • split virus vaccines will usually contain most or all of the virus structural proteins although not necessarily in the same proportions as they occur in the whole virus.
  • influenza virus may be in the form of a whole virus vaccine.
  • This may prove to be an advantage over a split virus vaccine for a pandemic situation as it avoids the uncertainty over whether a split virus vaccine can be successfully produced for a new strain of influenza virus.
  • the conventional detergents used for producing the split virus can damage the virus and render it unusable.
  • this would take time, which may not be available in a pandemic situation.
  • influenza virus preparation is in the form of a purified sub-unit influenza vaccine.
  • Sub-unit influenza vaccines generally contain the two major envelope proteins, HA and NA, and may have an additional advantage over whole virion vaccines as they are generally less reactogenic, particularly in young vaccinees.
  • Sub-unit vaccines can produced either recombinantly or purified from disrupted viral particles.
  • influenza virus preparation is in the form of a virosome.
  • Virosomes are spherical, unilamellar vesicles which retain the functional viral envelope glycoproteins HA and NA in authentic conformation, intercalated in the virosomes' phospholipids bilayer membrane.
  • Said influenza virus or antigenic preparation thereof may be egg-derived or tissue-culture derived.
  • influenza virus antigen or antigenic preparations thereof according to the invention may be derived from the conventional embryonated egg method, by growing influenza virus in eggs and purifying the harvested allantoic fluid. Eggs can be accumulated in large numbers at short notice. Alternatively, they may be derived from any of the new generation methods using tissue culture to grow the virus or express recombinant influenza virus surface antigens.
  • Suitable cell substrates for growing the virus include for example dog kidney cells such as MDCK or cells from a clone of MDCK, MDCK-like cells, monkey kidney cells such as AGMK cells including Vero cells, suitable pig cell lines, or any other mammalian cell type suitable for the production of influenza virus for vaccine purposes. Suitable cell substrates also include human cells e.g. MRC-5 cells. Suitable cell substrates are not limited to cell lines; for example primary cells such as chicken embryo fibroblasts and avian cell lines are also included.
  • the influenza virus antigen or antigenic preparation thereof may be produced by any of a number of commercially applicable processes, for example the split flu process described in patent no. DD 300 833 and DD 211 444, incorporated herein by reference.
  • split flu was produced using a solvent/detergent treatment, such as tri-n-butyl phosphate, or diethylether in combination with TweenTM (known as “Tween-ether” splitting) and this process is still used in some production facilities.
  • Other splitting agents now employed include detergents or proteolytic enzymes or bile salts, for example sodium deoxycholate as described in patent no. DD 155 875, incorporated herein by reference.
  • Detergents that can be used as splitting agents include cationic detergents e.g.
  • cetyl trimethyl ammonium bromide CAB
  • other ionic detergents e.g. laurylsulfate, taurodeoxycholate, or non-ionic detergents such as the ones described above including Triton X-100 (for example in a process described in Lina et al, 2000, Biologicals 28, 95-103) and Triton N-101, or combinations of any two or more detergents.
  • Triton X-100 for example in a process described in Lina et al, 2000, Biologicals 28, 95-103
  • Triton N-101 Triton N-101
  • the preparation process for a split vaccine may include a number of different filtration and/or other separation steps such as ultracentrifugation, ultrafiltration, zonal centrifugation and chromatography (e.g. ion exchange) steps in a variety of combinations, and optionally an inactivation step eg with heat, formaldehyde or ⁇ -propiolactone or U.V. which may be carried out before or after splitting.
  • the splitting process may be carried out as a batch, continuous or semi-continuous process.
  • a preferred splitting and purification process for a split immunogenic composition is described in WO 02/097072.
  • Preferred split flu vaccine antigen preparations according to the invention comprise a residual amount of Tween 80 and/or Triton X-100 remaining from the production process, although these may be added or their concentrations adjusted after preparation of the split antigen.
  • Tween 80 and Triton X-100 are present.
  • the preferred ranges for the final concentrations of these non-ionic surfactants in the vaccine dose are:
  • Tween 80 0.01 to 1%, more preferably about 0.1% (v/v)
  • Triton X-100 0.001 to 0.1 (% w/v), more preferably 0.005 to 0.02% (w/v).
  • the final concentration for Tween 80 ranges from 0.045%-0.09% w/v.
  • the antigen is provided as a 2 fold concentrated mixture, which has a Tween 80 concentration ranging from 0.045%-0.2% (w/v) and has to be diluted two times upon final formulation with the adjuvanted (or the buffer in the control formulation).
  • the final concentration for Triton X-100 ranges from 0.005%-0.017% w/v.
  • the antigen is provided as a 2 fold concentrated mixture, which has a Triton X-100 concentration ranging from 0.005%-0.034% (w/v) and has to be diluted two times upon final formulation with the adjuvanted (or the buffer in the control formulation).
  • influenza preparation is prepared in the presence of low level of thiomersal, or preferably in the absence of thiomersal.
  • the resulting influenza preparation is stable in the absence of organomercurial preservatives, in particular the preparation contains no residual thiomersal.
  • influenza virus preparation comprises a haemagglutinin antigen stabilised in the absence of thiomersal, or at low levels of thiomersal (generally 5 ⁇ g/ml or less).
  • the stabilization of B influenza strain is performed by a derivative of alpha tocopherol, such as alpha tocopherol succinate (also known as vitamin E succinate, i.e. VES).
  • alpha tocopherol succinate also known as vitamin E succinate, i.e. VES
  • a preferred composition contains three inactivated split virion antigens prepared from the WHO recommended strains of the appropriate influenza season.
  • influenza virus or antigenic preparation thereof and the oil-in-water emulsion adjuvant are contained in the same container. It is referred to as ‘one vial approach’.
  • the vial is a pre-filled syringe.
  • influenza virus or antigenic preparation thereof and the oil-in-water emulsion adjuvant are contained in separate containers or vials and admixed shortly before or upon administration into the subject. It is referred to as ‘two vials approach’.
  • the concentrated antigens for example the concentrated trivalent inactivated split virion antigens
  • the concentrated antigens are presented in one vial (335 ⁇ l) (antigen container) and a pre-filled syringe contains the adjuvant (360 ⁇ l) (adjuvant container).
  • the content of the vial containing the concentrated trivalent inactivated split virion antigens is removed from the vial by using the syringe containing the adjuvant followed by gentle mixing of the syringe.
  • the used needle Prior to injection, the used needle is replaced by an intramuscular needle and the volume is corrected to 5301 ⁇ l.
  • One dose of the reconstituted adjuvanted influenza vaccine candidate corresponds to 530 ⁇ l.
  • At least one influenza strain in the multivalent immunogenic composition as herein defined is associated with a pandemic outbreak or have the potential to be associated with a pandemic outbreak.
  • Such strain may also be referred to as ‘pandemic strains’ in the text below.
  • the vaccine is a multivalent vaccine such as a bivalent, or a trivalent or a quadrivalent vaccine
  • at least one strain is associated with a pandemic outbreak or has the potential to be associated with a pandemic outbreak.
  • Suitable strains are, but not limited to: H5N1, H9N2, H7N7, H2N2 and H1N1.
  • Said influenza virus or antigenic preparation thereof is suitably multivalent such as bivalent or trivalent or quadrivalent.
  • the influenza virus or antigenic preparation thereof is trivalent or quadrivalent, having an antigen from three different influenza strains, at least one strain being associated with a pandemic outbreak or having the potential to be associated with a pandemic outbreak.
  • an influenza virus strain that give it the potential to cause a pandemic outbreak are: it contains a new haemagglutinin compared to the haemagglutinin in the currently circulating strains; it is capable of being transmitted horizontally in the human population; and it is pathogenic for humans.
  • a new haemagglutinin may be one which has not been evident in the human population for an extended period of time, probably a number of decades, such as H2. Or it may be a haemagglutinin that has not been circulating in the human population before, for example H5, H9, H7 or H6 which are found in birds. In either case the majority, or at least a large proportion of, or even the entire population has not previously encountered the antigen and is immunologically na ⁇ ve to it.
  • Certain parties are generally at an increased risk of becoming infected with influenza in a pandemic situation.
  • the elderly, the chronically ill and small children are particularly susceptible but many young and apparently healthy people are also at risk.
  • H2 influenza the part of the population born after 1968 is at an increased risk. It is important for these groups to be protected effectively as soon as possible and in a simple way.
  • Suitable strains are, but not limited to: H5N1, H9N2, H7N7, H2N2 and H1N1.
  • the composition may contain more than three valencies, for example two non pandemic strains plus a pandemic strain.
  • the composition may contain three pandemic strains.
  • the composition may contain three pandemic strains.
  • the adjuvant composition of the invention contains an oil-in-water emulsion adjuvant, preferably said emulsion comprises a metabolisable oil in an amount of 0.5% to 20% of the total volume, and having oil droplets of which at least 70% by intensity have diameters of less than 1 ⁇ m.
  • the oil phase of the emulsion system has to comprise a metabolisable oil.
  • metabolisable oil is well known in the art. Metabolisable can be defined as ‘being capable of being transformed by metabolism’ (Dorland's Illustrated Medical Dictionary, W.B. Sanders Company, 25th edition (1974)).
  • the oil may be any vegetable oil, fish oil, animal oil or synthetic oil, which is not toxic to the recipient and is capable of being transformed by metabolism. Nuts, seeds, and grains are common sources of vegetable oils. Synthetic oils are also part of this invention and can include commercially available oils such as NEOBEE® and others. A particularly suitable metabolisable oil is squalene.
  • Squalene (2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-tetracosahexaene) is an unsaturated oil which is found in large quantities in shark-liver oil, and in lower quantities in olive oil, wheat germ oil, rice bran oil, and yeast, and is a particularly preferred oil for use in this invention.
  • Squalene is a metabolisable oil by virtue of the fact that it is an intermediate in the biosynthesis of cholesterol (Merck index, 10th Edition, entry no. 8619).
  • Oil in water emulsions per se are well known in the art, and have been suggested to be useful as adjuvant compositions (EP 399843; WO 95/17210).
  • the metabolisable oil is present in an amount of 0.5% to 20% (final concentration) of the total volume of the immunogenic composition, preferably an amount of 1.0% to 10% of the total volume, preferably in an amount of 2.0% to 6.0% of the total volume.
  • the metabolisable oil is present in a final amount of about 0.5%, 1%, 3.5% or 5% of the total volume of the immunogenic composition. In another specific embodiment, the metabolisable oil is present in a final amount of 0.5%, 1%, 3.57% or 5% of the total volume of the immunogenic composition.
  • the oil-in-water emulsion systems of the present invention have a small oil droplet size in the sub-micron range.
  • the droplet sizes will be in the range 120 to 750 nm, more preferably sizes from 120 to 600 nm in diameter.
  • the oil-in water emulsion contains oil droplets of which at least 70% by intensity are less than 500 nm in diameter, more preferably at least 80% by intensity are less than 300 nm in diameter, more preferably at least 90% by intensity are in the range of 120 to 200 nm in diameter.
  • the oil droplet size i.e. diameter
  • the oil droplet size is given by intensity.
  • Intensity is measured by use of a sizing instrument, suitably by dynamic light scattering such as the Malvern Zetasizer 4000 or preferably the Malvern Zetasizer 3000HS.
  • a detailed procedure is given in Example II.2.
  • a first possibility is to determine the z average diameter ZAD by dynamic light scattering (PCS-Photon correlation spectroscopy); this method additionally give the polydispersity index (PDI), and both the ZAD and PDI are calculated with the cumulants algorithm. These values do not require the knowledge of the particle refractive index.
  • a second mean is to calculate the diameter of the oil droplet by determining the whole particle size distribution by another algorithm, either the Contin, or NNLS, or the automatic “Malvern” one (the default algorithm provided for by the sizing instrument). Most of the time, as the particle refractive index of a complex composition is unknown, only the intensity distribution is taken into consideration, and if necessary the intensity mean originating from this distribution.
  • the oil in water emulsion according to the invention comprises a sterol.
  • Sterols are well known in the art, for example cholesterol is well known and is, for example, disclosed in the Merck Index, 11th Edn., page 341, as a naturally occurring sterol found in animal fat.
  • Other suitable sterols include ⁇ -sitosterol, stigmasterol, ergosterol, alpha-tocopherol and ergocalciferol.
  • Said sterol is suitably present in an amount of 0.01% to 20% (w/v) of the total volume of the immunogenic composition, preferably at an amount of 0.1% to 5% (w/v).
  • the sterol when it is cholesterol, it is present in an amount of between 0.02% and 0.2% (w/v) of the total volume of the immunogenic composition, more preferably at an amount of 0.02% (w/v) in a 0.5 ml vaccine dose volume, or 0.07% (w/v) in 0.5 ml vaccine dose volume or 0.1% (w/v) in 0.7 ml vaccine dose volume.
  • the sterol is alpha-tocopherol or a derivative thereof such as alpha-tocopherol succinate.
  • alpha-tocopherol is present in an amount of between 0.2% and 5.0% (v/v) of the total volume of the immunogenic composition, more preferably at an amount of 2.5% (v/v) in a 0.5 ml vaccine dose volume, or 0.5% (v/v) in 0.5 ml vaccine dose volume or 1.7-1.9% (v/v), preferably 1.8% in 0.7 ml vaccine dose volume.
  • concentrations given in v/v can be converted into concentration in w/v by applying the following conversion factor: a 5% (v/v) alpha-tocopherol concentration is equivalent to a 4.8% (w/v) alpha-tocopherol concentration.
  • the oil in water emulsion may further comprise an emulsifying agent.
  • the emulsifying agent may be present at an amount of 0.01 to 5.0% by weight of the immunogenic composition (w/w), preferably present at an amount of 0.1 to 2.0% by weight (w/w). Preferred concentration are 0.5 to 1.5% by weight (w/w) of the total composition.
  • the emulsifying agent may suitably be polyoxyethylene sorbitan monooleate (Tween 80).
  • Tween 80 polyoxyethylene sorbitan monooleate
  • a 0.5 ml vaccine dose volume contains 1% (w/w) Tween 80
  • a 0.7 ml vaccine dose volume contains 0.7% (w/w) Tween 80.
  • the concentration of Tween 80 is 0.2% (w/w).
  • the oil in water emulsion adjuvant may be utilised with other adjuvants or immuno-stimulants and therefore an important embodiment of the invention is an oil in water formulation comprising squalene or another metabolisable oil, alpha tocopherol, and tween 80.
  • the oil in water emulsion may also contain span 85 and/or Lecithin.
  • the oil in water will comprise from 2 to 10% squalene of the total volume of the immunogenic composition, from 2 to 10% alpha tocopherol and from 0.3 to 3% Tween 80, and may be produced according to the procedure described in WO 95/17210.
  • the ratio of squalene:alpha tocopherol is equal or less than 1 as this provides a more stable emulsion.
  • Span 85 polyoxyethylene sorbitan trioleate
  • Span 85 polyoxyethylene sorbitan trioleate
  • the multivalent influenza composition is capable of inducing an improved CD4 T-cell immune response against at least one of the component antigen(s) or antigenic composition compared to the CD4 T-cell immune response obtained with the corresponding composition which in un-adjuvanted, i.e. does not contain any exogeneous adjuvant (herein also referred to as ‘plain composition’).
  • said improved CD4 T-cell immune response is against the pandemic influenza strain.
  • a higher CD4 T-cell immune response is meant that a higher CD4 response is obtained in a human patient after administration of the adjuvanted immunogenic composition than that obtained after administration of the same composition without adjuvant.
  • a higher CD4 T-cell response is obtained in a human patient upon administration of an immunogenic composition comprising an influenza virus or antigenic preparation thereof together with an oil-in-water emulsion adjuvant comprising a metabolisable oil, alpha tocopherol and an emulsifying agent, compared to the response induced after administration of an immunogenic composition comprising an influenza virus or antigenic preparation thereof which is un-adjuvanted.
  • Such formulation will advantageously be used to induce anti-influenza CD4-T cell response capable of detection of influenza epitopes presented by MHC class II molecules.
  • said immunological response induced by an adjuvanted split influenza composition for use in the present invention is higher than the immunological response induced by any other un-adjuvanted influenza conventional vaccine, such as sub-unit influenza vaccine or whole influenza virus vaccine.
  • said ‘improved CD4 T-cell immune response’ is obtained in an immunologically unprimed patient, i.e. a patient who is seronegative to said influenza virus or antigen.
  • This seronegativity may be the result of said patient having never faced such virus or antigen (so-called ‘naive’ patient) or, alternatively, having failed to respond to said antigen once encountered.
  • said improved CD4 T-cell immune response is obtained in an immunocompromised subject such as an elderly, typically at least 50 years of age, typically 65 years of age or above, or an adult below 65 years of age with a high risk medical condition (‘high risk’ adult), or a child under the age of two.
  • the improved CD4 T-cell immune response may be assessed by measuring the number of cells producing any of the following cytokines:
  • CD4 T-cell immune response when cells producing any of the above cytokines will be in a higher amount following administration of the adjuvanted composition compared to the administration of the un-adjuvanted composition. Typically at least one, preferably two of the five conditions mentioned herein above will be fulfilled. In a particular embodiment, the cells producing all four cytokines will be present at a higher amount in the adjuvanted group compared to the un-adjuvanted group.
  • the improved CD4 T-cell immune response conferred by the adjuvanted influenza composition of the present invention may be ideally obtained after one single administration.
  • the single dose approach will be extremely relevant for example in a rapidly evolving outbreak situation.
  • the second dose of said same composition (still considered as ‘composition for first vaccination’) may be administered during the on-going primary immune response and is adequately spaced.
  • the second dose of the composition is given a few weeks, or about one month, e.g. 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks after the first dose, to help prime the immune system in unresponsive or poorly responsive individuals.
  • the administration of said immunogenic composition alternatively or additionally induces an improved B-memory cell response in patients administered with the adjuvanted immunogenic composition compared to the B-memory cell response induced in individuals immunized with the un-adjuvanted composition.
  • An improved B-memory cell response is intended to mean an increased frequency of peripheral blood B lymphocytes capable of differentiation into antibody-secreting plasma cells upon antigen encounter as measured by stimulation of in-vitro differentiation (see Example sections, e.g. methods of Elispot B cells memory).
  • the vaccination with the composition for the first vaccination, adjuvanted has no measurable impact on the CD8 response.
  • compositions comprising an influenza virus or antigenic preparation thereof formulated with an oil-in-water emulsion adjuvant, in particular an oil-in-water emulsion adjuvant comprising a metabolisable oil, a sterol such as alpha tocopherol and an emulsifying agent, is effective in promoting T cell responses in an immuno-compromised human population.
  • an oil-in-water emulsion adjuvant comprising a metabolisable oil, a sterol such as alpha tocopherol and an emulsifying agent.
  • the claimed adjuvanted formulation has also been able to induce an improved CD4 T-cell immune response against influenza virus compared to that obtained with the un-adjuvanted formulation. This finding can be associated with an increased responsiveness upon vaccination or infection vis-à-vis influenza antigenic exposure.
  • this may also be associated with a cross-responsiveness, i.e. a higher ability to respond against variant influenza strains.
  • This improved response may be especially beneficial in an immuno-compromised human population such as the elderly population (65 years of age and above) and in particular the high risk elderly population. This may result in reducing the overall morbidity and mortality rate and preventing emergency admissions to hospital for pneumonia and other influenza-like illness. This may also be of benefit to the infant population (below 5 years, preferably below 2 years of age).
  • it allows inducing a CD4 T cell response which is more persistent in time, e.g. still present one year after the first vaccination, compared to the response induced with the un-adjuvanted formulation.
  • the CD4 T-cell immune response such as the improved CD4 T-cell immune response obtained in an unprimed subject, involves the induction of a cross-reactive CD4 T helper response.
  • the amount of cross-reactive CD4 T cells is increased.
  • cross-reactive CD4 response is meant CD4 T-cell targeting shared epitopes between influenza strains.
  • influenza vaccines are effective only against infecting strains of influenza virus that have haemagglutinin of similar antigenic characteristics.
  • the infecting (circulating) influenza virus has undergone minor changes (such as a point mutation or an accumulation of point mutations resulting in amino acid changes in the for example) in the surface glycoproteins in particular haemagglutinin (antigenic drift variant virus strain) the vaccine may still provide some protection, although it may only provide limited protection as the newly created variants may escape immunity induced by prior influenza infection or vaccination.
  • Antigenic drift is responsible for annual epidemics that occur during interpandemic periods (Wiley & Skehel, 1987, Ann. Rev. Biochem. 56, 365-394).
  • cross-reactive CD4 T cells provides an additional advantage to the composition of the invention, in that it may provide also cross-protection, in other words protection against heterologous infections, i.e. infections caused by a circulating influenza strain which is a variant (e.g. a drift) of the influenza strain contained in the immunogenic composition.
  • This may be advantageous when the circulating strain is difficult to propagate in eggs or to produce in tissue culture, rendering the use of a drifted strain a working alternative.
  • This may also be advantageous when the subject received a first and a second vaccination several months or a year apart, and the influenza strain in the immunogenic composition used for a second immunization is a drift variant strain of the strain used in the composition used for the first vaccination.
  • the adjuvanted influenza immunogenic composition as herein defined has therefore a higher ability to induce sero-protection and cross-reactive CD4 T cells in vaccinated elderly subjects. This characteristic may be associated with a higher ability to respond against a variant strain of the strain present in the immunogenic composition. This may prove to be an important advantage in a pandemic situation.
  • a multivalent influenza immunogenic composition comprising any or several of H5, a H2, a H9, H7 or H6 strain(s) may provide a higher ability to respond against a pandemic variant, i.e. a drift strain of said pandemic strain(s), either upon subsequent vaccination with or upon infection by said drift strain.
  • peripheral blood CD4 T-cells responding to antigenic strain preparation (whole virus or split antigen) that is homologous to the one present in the vaccine (H3N2: A/Panama/2007/99, H1N1: A/New Caledonia/20/99, B: B/Shangdong/7/97) (see Example 11).
  • H3N2 A/Panama/2007/99
  • H1N1 A/New Caledonia/20/99
  • B B/Shangdong/7/97
  • a comparable increase in frequency can be seen if peripheral blood CD4 T-cells are restimulated with influenza strains classified as drifted strains (H3N2: A/Sydney/5197, H1N1: A/Beijing/262/95, B: B/Yamanashi/166/98).
  • peripheral blood CD4 T-cells are restimulated with influenza strains classified as shift strains (H2N2: A/Singapore/1/57, H9N2: A/Hongkong/1073/99) by expert in the field, there is no observable increase following vaccination.
  • H2N2 A/Singapore/1/57
  • H9N2 A/Hongkong/1073/99
  • CD4 T-cells that are able to recognize both homologous and drifted Influenza strains have been named in the present document “cross-reactive”.
  • the adjuvanted influenza compositions as described herein have been capable to show heterosubtypic cross-reactivity since there is observable cross-reactivity against drifted Influenza strains.
  • the ability of a pandemic vaccine formulation to be effective against drift pandemic strains may prove to be an important characteristic in the case of pandemics.
  • CD4 T-cell epitopes shared by different Influenza strains have been identified in human (Gelder C et al. 1998, Int Immunol. 10(2):211-22; Gelder C M et al. 1996 J. Virol. 70(7):4787-90; Gelder C M et al. 1995 J. Virol. 1995 69(12):7497-506).
  • the adjuvanted composition may offer the additional benefit of providing better protection against circulating strains which have undergone a major change (such as gene recombination for example, between two different species) in the haemagglutinin (antigenic shift) against which currently available vaccines have no efficacy.
  • the composition may comprise an additional adjuvant, in particular a TRL-4 ligand adjuvant, suitably a non-toxic derivative of lipid A.
  • a TRL-4 ligand is 3 de-O-acylated monophosphoryl lipid A (3D-MPL).
  • Other suitable TLR-4 ligands are lipopolysaccharide (LPS) and derivatives, MDP (muramyl dipeptide) and F protein of RSV.
  • the composition may additionally include a Toll like receptor (TLR)-4 ligand, such as a non-toxic derivative of lipid A, particularly monophosphoryl lipid A or more particularly 3-Deacylated monophoshoryl lipid A (3D-MPL).
  • TLR Toll like receptor
  • 3D-MPL is sold under the trademark MPL® by Corixa corporation (herein MPL) and primarily promotes CD4+ T cell responses with an IFN- ⁇ (Th1) phenotype. It can be produced according to the methods disclosed in GB 2 220 211 A. Chemically it is a mixture of 3-deacylated monophosphoryl lipid A with 3, 4, 5 or 6 acylated chains.
  • small particle 3 D-MPL is used.
  • Small particle 3D-MPL has a particle size such that it may be sterile-filtered through a 0.22 ⁇ m filter. Such preparations are described in WO94/21292 and in Example II.
  • 3D-MPL can be used, for example, at an amount of 1 to 100 ⁇ g (w/v) per composition dose, preferably in an amount of 10 to 50 ⁇ g (w/v) per composition dose.
  • a suitable amount of 3D-MPL is for example any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 ⁇ g (w/v) per composition dose. More preferably, 3D-MPL amount ranges from 25 to 75 ⁇ g (w/v) per composition dose.
  • composition dose will be ranging from about 0.5 ml to about 1 ml.
  • a typical vaccine dose are 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml or 1 ml.
  • a final concentration of 50 ⁇ g of 3D-MPL is contained per ml of vaccine composition, or 25 ⁇ g per 0.5 ml vaccine dose.
  • a final concentration of 35.7 ⁇ g or 71.4 ⁇ g of 3D-MPL is contained per ml of vaccine composition.
  • a 0.5 ml vaccine dose volume contains 25 ⁇ g or 50 ⁇ g of 3D-MPL per dose.
  • the dose of MPL is suitably able to enhance an immune response to an antigen in a human.
  • a suitable MPL amount is that which improves the immunological potential of the composition compared to the unadjuvanted composition, or compared to the composition adjuvanted with another MPL amount, whilst being acceptable from a reactogenicity profile.
  • Synthetic derivatives of lipid A are known, some being described as TLR-4 agonists, and include, but are not limited to:
  • OM174 (2-deoxy-6-o-[2-deoxy-2-[(R)-3-dodecanoyloxytetra-decanoylamino]-4-O— phosphono- ⁇ -D-glucopyranosyl]-2-[(R)-3-hydroxytetradecanoylamino]- ⁇ -D-glucopyranosyldihydrogenphosphate), (WO 95/14026) OM 294 DP (3S,9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9(R)-[(R)-3-hydroxytetradecanoylamino]decan-1,10-diol,1,10-bis(dihydrogenophosphate) (WO99/64301 and WO 00/0462) OM 197 MP-Ac DP (3S-,9R)-3-[(R)-dodecanoyloxytetradecanoylamin
  • TLR-4 ligands are, for example, lipopolysaccharide and its derivatives, muramyl dipeptide (MDP) or F protein of respiratory syncitial virus.
  • MDP muramyl dipeptide
  • Quil A is a saponin preparation isolated from the South American tree Quilaja Saponaria Molina and was first described by Dalsgaard et al. in 1974 (“Saponin adjuvants”, Archiv. für dienati Virusforschung, Vol. 44, Springer Verlag, Berlin, p 243-254) to have adjuvant activity. Purified fragments of Quil A have been isolated by HPLC which retain adjuvant activity without the toxicity associated with Quil A (EP 0 362 278), for example QS7 and QS21 (also known as QA7 and QA21).
  • QS-21 is a natural saponin derived from the bark of Quillaja saponaria Molina, which induces CD8+ cytotoxic T cells (CTLs), Th1 cells and a predominant IgG2a antibody response and is a preferred saponin in the context of the present invention.
  • CTLs cytotoxic T cells
  • Th1 cells Th1 cells
  • IgG2a antibody response is a preferred saponin in the context of the present invention.
  • compositions of QS21 have been described which are particularly preferred, these formulations further comprise a sterol (WO96/33739).
  • the saponins forming part of the present invention may be in the form of an oil in water emulsion (WO 95/17210).
  • An aspect of the present invention provides the use of an influenza antigen in the manufacture of an influenza immunogenic composition for revaccination of humans previously vaccinated with an multivalent influenza composition as claimed herein or with said multivalent influenza composition comprising a variant influenza strain, formulated with an oil-in-water emulsion adjuvant as herein defined.
  • revaccination is made at least 6 months after the first vaccination(s), preferably 8 to 14 months after, more preferably at around 10 to 12 months after.
  • the immunogenic composition for revaccination may contain any type of antigen preparation, either inactivated or live attenuated. It may contain the same type of antigen preparation i.e. split influenza virus or split influenza virus antigenic preparation thereof, a whole virion, a purified HA and NA (sub-unit) vaccine or a virosome, as the immunogenic composition used for the first vaccination.
  • the boosting composition may contain another type of influenza antigen, i.e. split influenza virus or split influenza virus antigenic preparation thereof, a whole virion, a purified HA and NA (sub-unit) vaccine or a virosome, than that used for the first vaccination.
  • a split virus or a whole virion vaccine is used.
  • the boosting composition may be adjuvanted or un-adjuvanted.
  • the un-adjuvanted boosting composition may be FluarixTM/ ⁇ -Rix®/Influsplit® given intramuscularly.
  • the formulation contains three inactivated split virion antigens prepared from the WHO recommended strains of the appropriate influenza season.
  • the invention provides for the use of an influenza virus or antigenic preparation thereof in the manufacture of an immunogenic composition for revaccination of humans previously vaccinated with an immunogenic composition as claimed herein.
  • the boosting composition may be adjuvanted or un-adjuvanted.
  • the boosting composition comprises an oil-in-water emulsion adjuvant, in particular an oil-in-water emulsion adjuvant comprising a metabolisable oil, a sterol such as alpha tocopherol and an emulsifying agent.
  • said oil-in-water emulsion adjuvant preferably comprises at least one metabolisable oil in an amount of 0.5% to 20% of the total volume, and has oil droplets of which at least 70% by intensity have diameters of less than 1 ⁇ m.
  • the first vaccination is made with an influenza composition, preferably a split influenza composition, containing at least one influenza strain that could potentially cause a pandemic outbreak and the re-vaccination is made with an influenza composition comprising at least one strain which is a circulating pandemic strain.
  • an influenza composition preferably a split influenza composition, containing at least one influenza strain that could potentially cause a pandemic outbreak and the re-vaccination is made with an influenza composition comprising at least one strain which is a circulating pandemic strain.
  • the immunogenic composition for revaccination (also called herein below the ‘boosting composition’) contains an influenza virus or antigenic preparation thereof which shares common CD4 T-cell epitopes with the influenza virus or antigenic preparation thereof used for the first vaccination.
  • a common CD4 T cell epitope is intended to mean peptides/sequences/epitopes from different antigens which can be recognised by the same CD4 cell (see examples of described epitopes in: Gelder C et al. 1998, Int Immunol. 10(2):211-22; Gelder C M et al. 1996 J Virol. 70(7):4787-90; Gelder C M et al. 1995 J Virol. 1995 69(12):7497-506).
  • the boosting composition is a monovalent influenza composition comprising an influenza strain which is associated with a pandemic outbreak or has the potential to be associated with a pandemic outbreak.
  • suitable strains are, but not limited to: H5N1, H9N2, H7N7, H2N2 and H1N1. Said strain may be the same as that, or one of those, present in the composition used for the first vaccination. In an alternative embodiment said strain may be a variant strain, i.e. a drift strain, of the strain present in the composition used for the first vaccination.
  • the boosting composition is a multivalent influenza vaccine.
  • the boosting composition is a multivalent vaccine such as a bivalent, trivalent or quadrivalent vaccine
  • at least one strain is associated with a pandemic outbreak or has the potential to be associated with a pandemic outbreak.
  • two or more strains in the boosting composition are pandemic strains.
  • the at least opne pandemic strain in the boosting composition is of the same type as that, or one of those, present in the composition used for the first vaccination.
  • the at least one strain may be a variant strain, i.e. a drift strain, of the at least one pandemic strain present in the composition used for the first vaccination.
  • an influenza virus or antigenic preparation thereof from a first pandemic influenza strain, in the manufacture of an immunogenic composition for protection against influenza infections caused by a influenza strain which is a variant of said first influenza strain
  • the boosting composition may be adjuvanted or not.
  • a boosting composition where used, is given at the next influenza season, e.g. approximately one year after the first immunogenic composition.
  • the boosting composition may also be given every subsequent year (third, fourth, fifth vaccination and so forth).
  • the boosting composition may be the same as the composition used for the first vaccination.
  • the boosting composition contains an influenza virus or antigenic preparation thereof which is a variant strain of the influenza virus used for the first vaccination.
  • influenza viral strains or antigenic preparation thereof are selected according to the reference material distributed by the World Health Organisation such that they are adapted to the influenza strain which is circulating on the year of the revaccination.
  • the influenza antigen or antigenic composition used in revaccination preferably comprises an adjuvant or an oil-in-water emulsion, suitably as described above.
  • the adjuvant may be an oil-in-water emulsion adjuvant as herein above described, which is preferred, optionally containing an additional adjuvant such as TLR-4 ligand such as 3D-MPL or a saponin, or may be another suitable adjuvant such as alum or alum alternatives such as polyphosphazene for example.
  • revaccination induces any, preferably two or all, of the following: (i) an improved CD4 response against the influenza virus or antigenic preparation thereof, or (ii) an improved B cell memory response or (iii) an improved humoral response, compared to the equivalent response induced after a first vaccination with the un-adjuvanted influenza virus or antigenic preparation thereof.
  • the immunological responses induced after revaccination with the adjuvanted influenza virus or antigenic preparation thereof as herein defined are higher than the corresponding response induced after the revaccination with the un-adjuvanted composition.
  • the immunological responses induced after revaccination with an un-adjuvanted, preferably split, influenza virus are higher in the population first vaccinated with the adjuvanted, preferably split, influenza composition than the corresponding response in the population first vaccinated with the un-adjuvanted, preferably split, influenza composition.
  • the revaccination of the subjects with a boosting composition comprising an influenza virus and an oil-in-water emulsion adjuvant comprising a metabolisable oil, a sterol such as alpha tocopherol and an emulsifying agent, as defined herein above, shows higher antibody titers than the corresponding values in the group of people first vaccinated with the un-adjuvanted composition and boosted with the un-adjuvanted composition.
  • the effect of the adjuvant in enhancing the antibody response to revaccination is especially of importance in the elderly population which is known to have a low response to vaccination or infection by influenza virus.
  • the adjuvanted composition-associated benefit was also marked in terms of improving the CD4 T-cell response following revaccination.
  • the adjuvanted composition of the invention is capable of inducing a better cross-responsiveness against drifted strain (the influenza strain from the next influenza season) compared to the protection conferred by the control vaccine. Said cross-responsiveness has shown a higher persistence compared to that obtained with the un-adjuvanted formulation.
  • the effect of the adjuvant in enhancing the cross-responsiveness against drifted strain is of important in a pandemic situation.
  • the invention relates to a vaccination regime in which the first vaccination is made with an influenza composition, preferably a split influenza composition, containing at least one influenza strain that could potentially cause a pandemic outbreak and the revaccination is made with a circulating strain, either a pandemic strain or a classical strain.
  • influenza composition preferably a split influenza composition
  • a circulating strain either a pandemic strain or a classical strain.
  • This antigenic drift mainly resides in epitope regions of the viral surface proteins haemagglutinin (HA) and neuraminidase (NA). It is known that any difference in CD4 and B cell epitopes between different influenza strains, being used by the virus to evade the adaptive response of the host immune system, will play a major role in influenza vaccination and is.
  • HA haemagglutinin
  • NA neuraminidase
  • CD4 T-cell epitopes shared by different Influenza strains have been identified in human (see for example: Gelder C et al. 1998, Int Immunol. 10(2):211-22; Gelder C M et al. 1996 J. Virol. 70(7):4787-90; and Gelder C M et al. 1995 J. Virol. 1995 69(12):7497-506).
  • the revaccination is made by using a boosting composition which contains an influenza virus or antigenic preparation thereof which shares common CD4 T-cell epitopes with the influenza virus antigen or antigenic preparation thereof used for the first vaccination.
  • the invention thus relates to the use of the immunogenic composition comprising a pandemic influenza virus or antigenic preparation thereof and an oil-in-water emulsion adjuvant, in particular an oil-in-water emulsion adjuvant comprising a metabolisable oil, a sterol such as alpha tocopherol and an emulsifying agent, in the manufacture of a first vaccination-component of a multi-dose vaccine, the multi-dose vaccine further comprising, as a boosting dose, an influenza virus or antigenic preparation thereof which shares common CD4 T-cell epitopes with the pandemic influenza virus antigen or virus antigenic preparation thereof of the dose given at the first vaccination.
  • composition of the invention may be administered by any suitable delivery route, such as intradermal, mucosal e.g. intranasal, oral, intramuscular or subcutaneous.
  • suitable delivery route such as intradermal, mucosal e.g. intranasal, oral, intramuscular or subcutaneous.
  • Other delivery routes are well known in the art.
  • the intramuscular delivery route is preferred for the adjuvanted influenza composition.
  • Intradermal delivery is another suitable route.
  • Any suitable device may be used for intradermal delivery, for example short needle devices such as those described in U.S. Pat. No. 4,886,499, U.S. Pat. No. 5,190,521, U.S. Pat. No. 5,328,483, U.S. Pat. No. 5,527,288, U.S. Pat. No. 4,270,537, U.S. Pat. No. 5,015,235, U.S. Pat. No. 5,141,496, U.S. Pat. No. 5,417,662.
  • Intradermal vaccines may also be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in WO99/34850 and EP1092444, incorporated herein by reference, and functional equivalents thereof.
  • jet injection devices which deliver liquid vaccines to the dermis via a liquid jet injector or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis. Jet injection devices are described for example in U.S. Pat. No. 5,480,381, U.S. Pat. No. 5,599,302, U.S. Pat. No. 5,334,144, U.S. Pat. No. 5,993,412, U.S. Pat. No. 5,649,912, U.S. Pat. No.
  • Another suitable administration route is the subcutaneous route.
  • Any suitable device may be used for subcutaneous delivery, for example classical needle.
  • a needle-free jet injector service is used, such as that published in WO 01/05453, WO 01/05452, WO 01/05451, WO 01/32243, WO 01/41840, WO 01/41839, WO 01/47585, WO 01/56637, WO 01/58512, WO 01/64269, WO 01/78810, WO 01/91835, WO 01/97884, WO 02/09796, WO 02/34317. More preferably said device is pre-filled with the liquid vaccine formulation.
  • the vaccine is administered intranasally.
  • the vaccine is administered locally to the nasopharyngeal area, preferably without being inhaled into the lungs. It is desirable to use an intranasal delivery device which delivers the vaccine formulation to the nasopharyngeal area, without or substantially without it entering the lungs.
  • Preferred devices for intranasal administration of the vaccines according to the invention are spray devices.
  • Suitable commercially available nasal spray devices include AccusprayTM (Becton Dickinson).
  • Nebulisers produce a very fine spray which can be easily inhaled into the lungs and therefore does not efficiently reach the nasal mucosa. Nebulisers are therefore not preferred.
  • Preferred spray devices for intranasal use are devices for which the performance of the device is not dependent upon the pressure applied by the user. These devices are known as pressure threshold devices. Liquid is released from the nozzle only when a threshold pressure is applied. These devices make it easier to achieve a spray with a regular droplet size.
  • Pressure threshold devices suitable for use with the present invention are known in the art and are described for example in WO 91/13281 and EP 311 863 B and EP 516 636, incorporated herein by reference. Such devices are commercially available from Pfeiffer GmbH and are also described in Bommer, R. Pharmaceutical Technology Europe, September 1999.
  • Preferred intranasal devices produce droplets (measured using water as the liquid) in the range 1 to 200 ⁇ m, preferably 10 to 120 ⁇ m. Below 10 ⁇ m there is a risk of inhalation, therefore it is desirable to have no more than about 5% of droplets below 10 ⁇ m. Droplets above 120 ⁇ m do not spread as well as smaller droplets, so it is desirable to have no more than about 5% of droplets exceeding 120 ⁇ m.
  • Bi-dose delivery is a further preferred feature of an intranasal delivery system for use with the vaccines according to the invention.
  • Bi-dose devices contain two sub-doses of a single vaccine dose, one sub-dose for administration to each nostril. Generally, the two sub-doses are present in a single chamber and the construction of the device allows the efficient delivery of a single sub-dose at a time. Alternatively, a monodose device may be used for administering the vaccines according to the invention.
  • epidermal or transdermal vaccination route is also contemplated in the present invention.
  • the adjuvanted immunogenic composition for the first administration may be given intramuscularly, and the boosting composition, either adjuvanted or not, may be administered through a different route, for example intradermal, subcutaneous or intranasal.
  • the composition for the first administration may contain a standard HA content of 15 ⁇ g per influenza strain, and the boosting composition may contain a low dose of HA, i.e. below 15 ⁇ g, and depending on the administration route, may be given in a smaller volume.
  • the target population to vaccinate may be immuno-compromised human.
  • Immuno-compromised humans generally are less well able to respond to an antigen, in particular to an influenza antigen, in comparison to healthy adults.
  • the target population is a population which is unprimed against influenza, either being na ⁇ ve (such as vis à vis a pandemic strain), or having failed to respond previously to influenza infection or vaccination.
  • the target population is elderly persons suitably aged at least 50, typically at least 55, or at least 60, or 65 years and over, younger high-risk adults (i.e. between 18 and 64 years of age) such as people working in health institutions, or those young adults with a risk factor such as cardiovascular and pulmonary disease, or diabetes.
  • Another target population is all children 6 months of age and over, especially children 6-23 months of age who experience a relatively high influenza-related hospitalization rate.
  • the target population is elderly above 65 years of age.
  • the immunogenic compositions according to the present invention are a standard 0.5 ml injectable dose in most cases, and contains 15 ⁇ g of haemagglutinin antigen component from the or each influenza strain, as measured by single radial immunodiffusion (SRD) (J. M. Wood et al.: J. Biol. Stand. 5 (1977) 237-247; J. M. Wood et al., J. Biol. Stand. 9 (1981) 317-330).
  • the vaccine dose volume will be between 0.5 ml and 1 ml, in particular a standard 0.5 ml, or 0.7 ml vaccine dose volume. Slight adaptation of the dose volume will be made routinely depending on the HA concentration in the original bulk sample.
  • said immunogenic composition contains a low dose of HA antigen—e.g any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ⁇ g of HA per influenza strain.
  • a suitable low dose of HA is between 1 to 7.5 ⁇ g of HA per influenza strain, suitably between 3.5 to 5 ⁇ g such as 3.75 ⁇ g of HA per influenza strain, typically about 5 ⁇ g of HA per influenza strain.
  • a vaccine dose according to the invention in particular a low dose vaccine, may be provided in a smaller volume than the conventional injected split flu vaccines, which are generally around 0.5, 0.7 or 1 ml per dose.
  • the low volume doses according to the invention are preferably below 500 gi, more preferably below 300 ⁇ l and most preferably not more than about 200 ⁇ l or less per dose.
  • a preferred low volume vaccine dose is a dose with a low antigen dose in a low volume, e.g. about 15 ⁇ g or about 7.5 ⁇ g HA or about 3.0 ⁇ g HA (per strain) in a volume of about 200 ⁇ l.
  • the influenza medicament of the invention preferably meets certain international criteria for vaccines.
  • At least two or all three of the criteria will need to be met for all strains, particularly for a new vaccine such as a new vaccine for delivery via a different route. Under some circumstances two criteria may be sufficient. For example, it may be acceptable for two of the three criteria to be met by all strains while the third criterion is met by some but not all strains (e.g. two out of three strains). The requirements are different for adult populations (18-60 years) and elderly populations (>60 years).
  • the invention provides a method of designing a vaccine for diseases known to be cured or treated through a CD4+ T cell activation, comprising
  • Example I describes immunological read-out methods used in mice, ferret and human studies.
  • Example II describes the preparation and characterization of the oil in water emulsion and adjuvant formulations used in the studies exemplified.
  • Example III describes a clinical trial in an elderly population aged over 65 years with a vaccine containing a split influenza antigen preparation and AS03 adjuvant
  • Example IV describes a second clinical trial—revaccination trial—in an elderly population aged over 65 years with a vaccine containing a split influenza antigen preparation and AS03 adjuvant.
  • Example V shows a pre-clinical evaluation of adjuvanted and un-adjuvanted influenza vaccines in ferrets (study I and study II). The temperature monitoring, viral shedding and CD4 T-cell response were measured.
  • Example VI shows a pre-clinical evaluation of adjuvanted and un-adjuvanted influenza vaccines in C57BI/6 na ⁇ ve and primed mice.
  • Example VII shows a pre-clinical evaluation of adjuvanted and un-adjuvanted split and sub-unit influenza vaccines in C57BI/6 mice primed with heterologous strains.
  • Example VIII describes a clinical trial in an elderly population aged over 65 years with a vaccine containing a split influenza antigen preparation containing AS03 adjuvant, AS03+MPL adjuvant, or no exogeneous adjuvant.
  • Example IX shows a pre-clinical evaluation of adjuvanted and un-adjuvanted influenza vaccines in ferrets (study III).
  • Example X shows a clinical trial in an elderly population aged over 65 years with a vaccine containing a split influenza antigen preparation containing AS03 with or without MPL adjuvant: immunogenicity persistence data at day 90 and day 180.
  • Example XI shows a clinical trial in an elderly population aged over 65 years with a vaccine containing a split influenza antigen preparation containing AS03 with MPL adjuvant.
  • Example XII shows a clinical trial in an elderly population aged over 65 years with a vaccine containing a split influenza antigen preparation containing AS03 with MPL adjuvant at two concentrations.
  • Anti-Hemagglutinin antibody titers to the three influenza virus strains were determined using the hemagglutination inhibition test (HI).
  • the principle of the HI test is based on the ability of specific anti-Influenza antibodies to inhibit hemagglutination of chicken red blood cells (RBC) by influenza virus hemagglutinin (HA). Heat inactivated sera were previously treated by Kaolin and chicken RBC to remove non-specific inhibitors. After pretreatment, two-fold dilutions of sera were incubated with 4 hemagglutination units of each influenza strain. Chicken red blood cells were then added and the inhibition of agglutination was scored.
  • the titers were expressed as the reciprocal of the highest dilution of serum that completely inhibited hemagglutination. As the first dilution of sera was 1:20, an undetectable level was scored as a titer equal to 10.
  • effector T cells and/or effector-memory T cells produce IFN- ⁇ and/or central memory T cells produce IL-2.
  • PBMCs are harvested at day 7 post-immunization.
  • Lymphoid cells are re-stimulated in vitro in the presence of secretion inhibitor (Brefeldine).
  • results are expressed as a frequency of cytokine positive cell within CD4/CD8 T cells.
  • Intracellular staining of cytokines of T cells was performed on PBMC 7 days after the second immunization. Blood was collected from mice and pooled in heparinated medium RPMI+Add. For blood, RPMI+Add-diluted PBL suspensions were layered onto a Lympholyte-Mammal gradient according to the recommended protocol (centrifuge 20 min at 2500 rpm and R.T.). The mononuclear cells at the interface were removed, washed 2 ⁇ in RPMI+Add and PBMCs suspensions were adjusted to 2 ⁇ 10 6 cells/ml in RPMI 5% fetal calf serum.
  • PBMC are incubated overnight at 37° C. in presence of Brefeldin (1 ⁇ g/ml) at 37° C. to inhibit cytokine secretion.
  • IFN- ⁇ /IL-2/CD4/CD8 staining was performed as follows: Cell suspensions were washed, resuspended in 50 ⁇ l of PBS 1% FCS containing 2% Fc blocking reagent ( 1/50; 2.4G2). After 10 min incubation at 4° C., 50 ⁇ l of a mixture of anti-CD4-PE ( 2/50) and anti-CD8 perCp ( 3/50) was added and incubated 30 min at 4° C. After a washing in PBS 1% FCS, cells were permeabilized by resuspending in 200 ⁇ l of Cytofix-Cytoperm (Kit BD) and incubated 20 min at 4° C.
  • Cytofix-Cytoperm Kerat BD
  • Anti-Hemagglutinin antibody titers to the three influenza virus strains were determined using the hemagglutination inhibition test (HI).
  • the principle of the HI test is based on the ability of specific anti-influenza antibodies to inhibit hemagglutination of chicken red blood cells (RBC) by influenza virus hemagglutinin (HA).
  • Sera were first treated with a 25% neuraminidase solution (RDE) and were heat-inactivated to remove non-specific inhibitors. After pre-treatment, two-fold dilutions of sera were incubated with 4 hemagglutination units of each influenza strain. Chicken red blood cells were then added and the inhibition of agglutination was scored. The titers were expressed as the reciprocal of the highest dilution of serum that completely inhibited hemagglutination. As the first dilution of sera was 1:10, an undetectable level was scored as a titer equal to 5.
  • the nasal washes were performed by administration of 5 ml of PBS in both nostrils in awoke animals.
  • the inoculum was collected in a Petri dish and placed into sample containers on dry ice.
  • the culture medium is gently removed and 100 ⁇ l of a 1/20 WST-1 containing medium is added and incubated for another 18 hrs.
  • the intensity of the yellow formazan dye produced upon reduction of WST-1 by viable cells is proportional to the number of viable cells present in the well at the end of the viral titration assay and is quantified by measuring the absorbance of each well at the appropriate wavelength (450 nanometers).
  • the cut-off is defined as the OD average of uninfected control cells—0.3 OD (0.3 OD correspond to +/ ⁇ 3 StDev of OD of uninfected control cells).
  • a positive score is defined when OD is ⁇ cut-off and in contrast a negative score is defined when OD is >cut-off.
  • Viral shedding titers were determined by “Reed and Muench” and expressed as Log TCID50/ml.
  • the immune response was determined by measuring HI antibodies using the method described by the WHO Collaborating Centre for influenza, Centres for Disease Control, Atlanta, USA (1991).
  • Antibody titre measurements were conducted on thawed frozen serum samples with a standardised and comprehensively validated micromethod using 4 hemagglutination-inhibiting units (4 HIU) of the appropriate antigens and a 0.5% fowl erythrocyte suspension. Non-specific serum inhibitors were removed by heat treatment and receptor-destroying enzyme.
  • the sera obtained were evaluated for HI antibody levels.
  • a dilution series (by a factor of 2) was prepared up to an end dilution of 1:20480.
  • the titration end-point was taken as the highest dilution step that showed complete inhibition (100%) of hemagglutination. All assays were performed in duplicate.
  • the assay was performed in fetuin-coated microtitre plates.
  • a 2-fold dilution series of the antiserum was prepared and mixed with a standardised amount of influenza A H3N2, H1N1 or influenza B virus.
  • the test was based on the biological activity of the neuraminidase which enzymatically releases neuraminic acid from fetuin. After cleavage of the terminal neuraminic acid ⁇ -D-glactose-N-acetyl-galactosamin was unmasked.
  • HRP horseradish peroxidase
  • Virus neutralisation by antibodies contained in the serum was determined in a microneutralization assay. The sera were used without further treatment in the assay. Each serum was tested in triplicate. A standardised amount of virus was mixed with serial dilutions of serum and incubated to allow binding of the antibodies to the virus. A cell suspension, containing a defined amount of MDCK cells was then added to the mixture of virus and antiserum and incubated at 33° C. After the incubation period, virus replication was visualised by hemagglutination of chicken red blood cells. The 50% neutralisation titre of a serum was calculated by the method of Reed and Muench.
  • Peripheral blood antigen-specific CD4 and CD8 T cells can be restimulated in vitro to produce IL-2, CD40L, TNF-alpha and IFN if incubated with their corresponding antigen. Consequently, antigen-specific CD4 and CD8 T cells can be enumerated by flow cytometry following conventional immunofluorescence labelling of cellular phenotype as well as intracellular cytokines production.
  • Influenza vaccine antigen as well as peptides derived from specific influenza protein were used as antigen to restimulate Influenza-specific T cells. Results were expressed as a frequency of cytokine(s)-positive CD4 or CD8 T cell within the CD4 or CD8 T cell sub-population.
  • CMI Cell Mediated Immune
  • the immunogenicity analysis was based on the total vaccinated cohort. For each treatment group, the following parameters (with 95% confidence intervals) were calculated:
  • the oil/water emulsion used in the subsequent examples is composed an organic phase made of 2 oils (alpha-tocopherol and squalene), and an aqueous phase of PBS containing Tween 80 as emulsifying agent.
  • the oil in water emulsion adjuvant formulations used in the subsequent examples were made comprising the following oil in water emulsion component (final concentrations given): 2.5% squalene (v/v), 2.5% alpha-tocopherol (v/v), 0.9% polyoxyethylene sorbitan monooleate (v/v) (Tween 80), see WO 95/17210.
  • Tween 80 is dissolved in phosphate buffered saline (PBS) to give a 2% solution in the PBS.
  • PBS phosphate buffered saline
  • To provide 100 ml two-fold concentrate emulsion 5 g of DL alpha tocopherol and 5 ml of squalene are vortexed to mix thoroughly. 90 ml of PBS/Tween solution is added and mixed thoroughly. The resulting emulsion is then passed through a syringe and finally microfluidised by using an M110S microfluidics machine.
  • the resulting oil droplets have a size of approximately 120-180 nm (expressed as Z average measured by PCS).
  • the other adjuvants/antigen components are added to the emulsion in simple admixture.
  • the preparation of the SB62 emulsion is made by mixing under strong agitation of an oil phase composed of hydrophobic components ( ⁇ -tocopherol and squalene) and an aqueous phase containing the water soluble components (Tween 80 and PBS mod (modified), pH 6.8). While stirring, the oil phase ( 1/10 total volume) is transferred to the aqueous phase ( 9/10 total volume), and the mixture is stirred for 15 minutes at room temperature. The resulting mixture then subjected to shear, impact and cavitation forces in the interaction chamber of a microfluidizer (15000 PSI-8 cycles) to produce submicron droplets (distribution between 100 and 200 nm). The resulting pH is between 6.8 ⁇ 0.1.
  • the SB62 emulsion is then sterilised by filtration through a 0.22 ⁇ m membrane and the sterile bulk emulsion is stored refrigerated in Cupac containers at 2 to 8° C.
  • Sterile inert gas nitrogen or argon
  • the final composition of the SB62 emulsion is as follows:
  • Tween 80 1.8% (v/v) 19.4 mg/ml; Squalene: 5% (v/v) 42.8 mg/ml; ⁇ -tocopherol: 5% (v/v) 47.5 mg/ml; PBS-mod: NaCl 121 mM, KCl 2.38 mM, Na2HPO4 7.14 mM, KH2PO4 1.3 mM; pH 6.8 ⁇ 0.1.
  • the size of the diameter of the oil droplets is determined according to the following procedure and under the following experimental conditions.
  • the droplet size measure is given as an intensity measure and expressed as z average measured by PCS.
  • PL-Nanocal Particle size standards 100 nm (cat n o 6011-1015) was diluted in 10 mM NaCl.
  • the Automatic Malvern algorithm uses a combination of cumulants, Contin and non negative least squares (NNLS) algorithms.
  • the intensity distribution may be converted into volume distribution thanks to the Mie theory.
  • the z-average diameter (ZAD) size is weighed by the amount of light scattered by each size of particles in the sample. This value is related to a monomodal analysis of the sample and is mainly used for reproducibility purposes.
  • the count rate (CR) is a measure of scattered light: it corresponds to thousands of photons per second.
  • the polydispersity (Poly) index is the width of the distribution. This is a dimensionless measure of the distribution broadness.
  • FIG. 1 A schematic representation of these results is shown in FIG. 1 for formulation 1023.
  • the great majority of the particles e.g. at least 80%
  • SB62 formulation was measured at different dilutions with the Malvern Zetasizer 3000HS and two optical models.
  • the particle size ZAD i.e. intensity mean by cumulant analysis
  • MPL (as used throughout the document it is an abbreviation for 3D-MPL, i.e. 3-O-deacylated monophosphoryl lipid A) liquid bulk is prepared from MPL® lyophilized powder.
  • MPL liquid bulk is a stable concentrated (around 1 mg/ml) aqueous dispersion of the raw material, which is ready-to-use for vaccine or adjuvant formulation.
  • FIG. 2 A schematic representation of the preparation process is given in FIG. 2 .
  • MPL liquid bulk preparation is carried over in sterile glass containers.
  • the dispersion of MPL consists of the following steps:
  • MPL powder is lyophilized by microfluidisation resulting in a stable colloidal aqueous dispersion (MPL particle size smaller than 200 nm).
  • the MPL lyophilized powder is dispersed in water for injection in order to obtain a coarse 10 mg/ml suspension.
  • the suspension then undergoes a thermal treatment under stirring. After cooling to room temperature, the microfluidization process is started in order to decrease the particle size.
  • Microfluidization is conducted using Microfluidics apparatus M110EH, by continuously circulating the dispersion through a microfluidization interaction chamber, at a defined pressure for a minimum amount of passages (number of cycles: n min ).
  • the microfluidization duration representing the number of cycles, is calculated on basis of the measured flow rate and the dispersion volume.
  • the resulting flow rate may vary from one interaction chamber to another, and throughout the lifecycle of a particular interaction chamber.
  • the interaction chamber used is of the type F20Y Microfluidics.
  • the processing time may vary from one batch to another.
  • the time required for 1 cycle is calculated on basis of the flow rate.
  • the flow rate to be considered is the flow rate measured with water for injection just before introduction of MPL into the apparatus.
  • One cycle is defined as the time (in minutes) needed for the total volume of MPL to pass once through the apparatus.
  • the time needed to obtain n cycles is calculated as follows:
  • the number of cycles is thus adapted accordingly.
  • Minimum amount of cycles to perform (n min ) are described for the preferred equipment and interaction chambers used.
  • the total amount of cycles to run is determined by the result of a particle size measurement performed after n min cycles.
  • a particle size limit (d lim ) is defined, based on historical data. The measurement is realized by photon correlation spectroscopy (PCS) technique, and d lim is expressed as an unimodal result (Z average ). Under this limit, the microfluidization can be stopped after n min cycles. Above this limit, microfluidization is continued until satisfactory size reduction is obtained, for maximum another 50 cycles.
  • the dispersed MPL is stored at +2 to +8° C. awaiting transfer to the filtration area.
  • the dispersion is diluted with water for injection, and sterile filtered through a 0.22 ⁇ m filter under laminal flow.
  • the final MPL concentration is 1 mg/ml (0.80-1.20 mg/ml).
  • MPL is added at a final concentration of between 10 and 50 ⁇ g per vaccine dose.
  • PBS10 fold concentrated (pH 7.4 when one fold concentrated) as well as a SB62 mixture containing Tween, Triton X-100 and VES (vitamin E succinate) is added to water for injection.
  • the quantities take into account the detergent present in the influenza strains so as to reach a target final concentration of 750 ⁇ g/ml Tween 80, 110 ⁇ g/ml Triton X-100 and 100 ⁇ g/ml VES.
  • 15 ⁇ g of each influenza strain of interest for example strain H1N1, H3N2 and B in a classical tri-valent vaccine
  • 250 ⁇ l of SB62 emulsion is added and then 25 ⁇ g or 50 ⁇ g of MPL.
  • the same formulation can be prepared from a 2 vials approach by mixing 2 fold concentrated antigen or antigenic preparation with the AS03 (SB62 250 ⁇ l) or the AS03+MPL (SB62 250 ⁇ l+25 ⁇ g or 50 ⁇ g MPL) adjuvant. In this instance it is proceeded as follows.
  • the manufacturing of the AS25-adjuvanted influenza vaccine consists of three main steps:
  • the volumes of the three monovalent bulks are based on the HA content measured in each monovalent bulk prior to the formulation and on a target volume of 1100 ml.
  • Concentrated phosphate buffered saline and a pre-mixture of Tween 80, Triton X-100 and ⁇ -tocopheryl hydrogen succinate are diluted in water for injection.
  • the three concentrated monobulks (A/New Caledonia, A/New York, B/Jiangsu) are then successively diluted in the resulting phosphate buffered saline/Tween 80-Triton X-100- ⁇ -tocopheryl hydrogen succinate solution (pH 7.4, 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.47 mM KH2PO4, 990 ⁇ g/ml Tween 80, 150 ⁇ g/ml Triton X-100 and 130 ⁇ g/ml ⁇ -tocopheryl hydrogen succinate) in order to have a final concentration of 39.47 ⁇ g HA of A strains (H1N1, H3N2) per ml of trivalent final bulk (15 ⁇ g HA/A strain/380 ⁇ l trivalent final bulk) and 46 ⁇ g HA of B strain (17.5 ⁇ g HA/B strain/380 ⁇ l trivalent final bulk). Between addition of each mono
  • the trivalent final bulk of antigens is aseptically filled into 3-ml sterile Type I (Ph. Eur.) glass vials. Each vial contains a volume of 470 ⁇ l (380 ⁇ l+90 ⁇ l overfill).
  • the adjuvant AS03/MPL is prepared by mixing of two components: SB62 emulsion (method in section II.1.2) and MPL (method in section II.3.1).
  • One-fold concentrated PBS mod prepared by diluting 10 ⁇ concentrated PBS mod in water for injection
  • SB62 bulk and MPL liquid bulk at 1 mg/ml.
  • MPL concentration will be determined so as to reach a final content of between 10 to 50 ⁇ g, suitably around 25 ⁇ g per final human vaccine dose.
  • the mixture is stirred for 5-30 minutes at room temperature, and the pH is adjusted to 6.8 ⁇ 0.1 with NAOH (0.05 or 0.5 M)/HCl (0.03 M or 0.3 M).
  • the mixture is sterilised by filtration through a 0.22 ⁇ m membrane. Sterile inert gas (nitrogen) flushing is performed to produce inert head space in the filled containers during minimum 1 minute.
  • the sterile AS03+MPL adjuvant is stored at +2-8° C. until aseptical filling into 1.25-ml sterile Type I (Ph. Eur.) glass syringes. Each syringe contains a volume overage of 80 ⁇ l (320 ⁇ l+80 ⁇ l overfill).
  • the content of the prefilled syringe containing the adjuvant is injected into the vial that contains the concentrated trivalent inactivated split virion antigens. After mixing the content is withdrawn into the syringe and the needle is replaced by an intramuscular needle.
  • One dose of the reconstituted the AS25-adjuvanted influenza candidate vaccine corresponds to 0.7 mL.
  • a phase I, open, randomised study was conducted in an elderly population aged over 65 years in 2003 in order to evaluate the reactogenicity and the immunogenicity of GlaxoSmithKline Biologicals influenza candidate vaccine containing the adjuvant AS03.
  • the humoral immune response i.e. anti-hemagglutinin, neutralising and anti-neuraminidase antibody titres
  • cell mediated immune response CD4 and/or CD8 T cell responses
  • Vaccination schedule one injection of influenza vaccine at day 0, blood sample collection, read-out analysis at day 21 (HI antibody determination, NI antibody determination, determination of neutralising antibodies, and CMI analysis) and study conclusion.
  • the standard trivalent split influenza vaccine—FluarixTM used in this study, is a commercial vaccine from the year 2003 developed and manufactured by GlaxoSmithKline Biologicals.
  • the AS03-adjuvanted influenza vaccine candidate is a 2 components vaccine consisting of a concentrated trivalent inactivated split virion antigens presented in a type I glass vial (335 ⁇ l) (antigen container) and of a pre-filled type I glass syringe containing the SB62 emulsion (335 ⁇ l) (adjuvant container).
  • the content of the antigen container is removed from the with the help of the SB62 emulsion pre-filled syringe, followed by gently mixing of the syringe. Mixing of the SB62 emulsion with the vaccine antigens reconstitute the AS03 adjuvant.
  • the used needle Prior to injection, the used needle is replaced by an intramuscular needle and the volume is corrected to 500 ⁇ l.
  • One dose of the reconstituted AS03-adjuvanted influenza vaccine corresponds to 0.5 ml, contains 15 ⁇ g HA of each influenza virus strain as in the registered FluarixTM/ ⁇ -Rix® vaccine and contains 10.68 mg squalene, 11.86 mg DL-alpha tocopherol, and 4.85 mg polysorbate 80 (Tween 80).
  • the manufacturing of the AS03-adjuvanted influenza vaccine consists of three main steps:
  • the volumes of the three monovalent bulks are based on the HA content measured in each monovalent bulk prior to the formulation and on a target volume of 800 ml.
  • Concentrated phosphate buffered saline and a pre-mixture of Tween 80, Triton X-100 and ⁇ -tocopheryl hydrogen succinate are diluted in water for injection.
  • the three concentrated monobulks (strain A/New Caledonia-, strain A/Panama- and strain B/Shangdong-) are then successively diluted in the resulting phosphate buffered saline/Tween 80-Triton X-100- ⁇ -tocopheryl hydrogen succinate solution (pH 7.4, 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2 HPO 4 , 1.47 mM KH 2 PO 4 , 1500 ⁇ g/ml Tween 80, 220 ⁇ g/ml Triton X-100 and 200 ⁇ g/ml ⁇ -tocopheryl hydrogen succinate) in order to have a final concentration of 60 ⁇ g HA of A strains per ml of trivalent final bulk
  • the trivalent final bulk of antigens is aseptically filled into 3-ml sterile Type I glass vials. Each vial contains a 34% volume overage (335 ⁇ l total volume).
  • the final composition of the SB62 emulsion is as follows:
  • the sterile SB62 bulk emulsion is then aseptically filled into 1.25-ml sterile Type I glass syringes. Each syringe contains a 34% volume overage (335 ⁇ l total volume).
  • the content of the vial containing the concentrated trivalent inactivated split virion antigens is removed from the vial with the help the syringe containing the SB62 emulsion followed by gently mixing of the syringe. Mixing of the SB62 emulsion with the vaccine antigens reconstitutes the AS03 adjuvant.
  • the vaccines were administered intramuscularly in the deltoid region of the non-dominant arm.
  • the vaccinees were observed closely for at least 30 minutes, with appropriate medical treatment readily available in case of a rare anaphylactic reaction following the administration of vaccine.
  • the mean age of the total vaccinated cohort at the time of vaccination was 71.8 years with a standard deviation of 6.0 years.
  • influenza vaccine adjuvanted with AS03 was safe and clinically well tolerated in the study population, i.e. elderly people aged over 65 years.
  • the GMTs for HI antibodies with 95% Cl are shown in Table 7 (GMT for anti-HI antibody).
  • Pre-vaccination GMTs of antibodies for all vaccine strains were within the same range in the three groups. After vaccination, anti-haemagglutinin antibody levels increased significantly. Post vaccination, there was a trend for higher GMTs of HI antibody for all three vaccine strains in the FluAS03 and Fluarix groups although there was some overlap of 95% Cl between the Fluarix group and the FluWVV group.
  • the conversion factors represent the fold increase in serum HI GMTs for each vaccine strain on day 21 compared to day 0.
  • the conversion factor varies from 6.1 to 13.6 according to the virus strain and the vaccine. This conversion factor is largely superior to the 2.0 fold increase in GMT required by the European authorities.
  • the seroprotection rates represent the proportion of subjects with a serum HI titre ⁇ 40 on day 21.
  • half of the subjects (range 34.0%-69.4%) in all groups had protective levels of antibodies for all strains
  • the seroprotection rates in the three groups ranged from 88.0% to 100% for the different virus strains. In terms of protection, this means that more than 88% of the subjects had a serum HI titre ⁇ 40 after vaccination and were deemed to be protected against the three strains. This rate is largely superior to the seroprotection rate of 60% required in the ⁇ 60 years old population, by the European authorities.
  • the GMTs and seroconversion rates for NI antibodies with 95% Cl are shown in Table 11 (anti-NA antibody GMT) and Table 12 (Seroconversion rates of NA at post-vaccination (day 21) (4-fold-increase)).
  • Peripheral blood antigen-specific CD4 and CD8 T cells can be restimulated in vitro to produce IL-2, CD40L, TNF-alpha and IFN ⁇ if incubated with their corresponding antigen. Consequently, antigen-specific CD4 and CD8 T cells can be enumerated by flow cytometry following conventional immunofluorescence labelling of cellular phenotype as well as intracellular cytokines production.
  • Influenza vaccine antigen as well as peptides derived from specific influenza protein were used as antigen to restimulate Influenza-specific T cells. Results are presented for the CD4 and CD8 T-cell response in Tables 13 to 18.
  • Results were also expressed as a frequency of cytokine(s)-positive CD4 or CD8 T cell within the CD4 or CD8 T cell sub-population and presented in FIG. 4 and FIG. 5 .
  • the cross-reactive CD4 T-cells response was evaluated using influenza antigen from drifted strains (A/H1N1/Beijing/262/95 (H1N1d), A/H3N2/Sydney/5/97 (H3N2d), B/Yamanashi/166/98 (Bd)) or shift strains (A/Singapore/1/57 (H2N2), A/Hongkong/1073/99 (H9N2)). Results expressed as a frequency of cytokine(s)-positive CD4 T cells are presented in FIG. 6 .
  • Vaccination with Fluarix only induces low levels of cross-reactive CD4 T-cell response ( FIG. 6 ).
  • Vaccination with FluAS03 induces a strong CD4 T-cell response against drifted influenza strains and this is statistically significant ( FIG. 6 ). A little response was detected against shift strains.
  • the B-cells Elispot memory response induced to differentiate into plasma cells in vitro using influenza vaccine strains or anti-human immunoglobulin was evaluate in order to enumerate anti-influenza or IgG secreting plasma. The results are described in Table 19 and Table 20 and in FIG. 7 .
  • a subset of 22 first subjects having received one dose of FluAS03 vaccine and 21 first subjects having received one dose of Fluarix vaccine was selected to evaluate the impact of vaccination on influenza-specific memory B-cells using the B-cell memory Elispot technology. The following endpoints were determined
  • Descriptive statistics for each vaccination group at days 0 and day 21 expressed as a frequency of Influenza specific-antibody forming cells per million (10 6 ) of antibody forming cells. Descriptive statistics in individual difference between day 21 and day 0 (Post-Pre) as a frequency of Influenza specific-antibody forming cells per million (10 6 ) of antibody forming cells.
  • B-cells Memory descriptive statistics on pre (Day 0) and post (Day 21) and inferential statistics of post (Day 21) frequency of antigen- plasma within a 10 6 of IgG-producing plasma cells (subset of subjects) Time- STRAIN Group point N Mean SD Min A/NEW 1 Day 0 22 9751.58 6630.335 0.00 CALEDONIA 1 Day 21 22 22001.65 11308.261 3981.90 2 Day 0 21 9193.61 4339.421 1300.81 2 Day 21 21 12263.08 7285.698 789.47 A/PANAMA 1 Day 0 22 4329.17 2923.497 0.00 1 Day 21 22 18066.69 14604.842 714.29 2 Day 0 21 4860.41 3392.373 0.00 2 Day 21 21 13872.95 12052.163 0.00 B/SHANDONG 1 Day 0 22 3722.80 2347.315 0.00 1 Day 21 22 15949.60 12385.965 0.00 2 Day 0
  • the three vaccines exceeded the requirements of the European authorities for annual registration of split virion influenza vaccines (“Note for Guidance on Harmonisation of Requirements for influenza Vaccines” for the immuno-logical assessment of the annual strain changes—CPMP/BWP/214/96).
  • the three influenza vaccines tested in this study were immunogenic in the healthy elderly, who developed a good antibody response to influenza hemagglutinin and neutralising antigens (Table 21).
  • CMI cell-mediated immunity
  • a phase I/II, open, controlled study has been conducted in order to evaluate the reactogenicity and the immunogenicity of the GlaxoSmithKline Biologicals influenza candidate vaccine containing the adjuvant AS03, in an elderly population aged over 65 years and previously vaccinated in 2003 with the candidate vaccine in the Explo-Flu-001 clinical trial.
  • FluarixTM vaccine (known as ⁇ -rixTM in Belgium) has been used as reference.
  • the humoral immune response i.e. anti-hemagglutinin antibody titres
  • cell mediated immune response CD4 and/or CD8 T cell responses
  • B memory cell response were measured 21 days after intramuscular administration of one dose of an AS03 adjuvanted vaccine. FluarixTM was used as reference.
  • the objectives were:
  • the vaccine composition is similar to that used for the study Explo-Flu-001 except for the influenza strains included in the vaccine (year 2004 vaccine).
  • the strains are as follows:
  • cells producing at least two different cytokines CD40L, IL-2, IFN ⁇ , TNF ⁇
  • cells producing at least CD40L and another cytokine IL-2, TNF ⁇ , IFN ⁇
  • cells producing at least IL-2 and another cytokine CD40L, TNF ⁇ , IFN ⁇
  • cells producing at least IFN ⁇ and another cytokine IL-2, TNF ⁇ , CD40L
  • cells producing at least TNF ⁇ and another cytokine IL-2, CD40L, IFN ⁇
  • Results were expressed as a frequency of cytokine(s)-positive CD4 or CD8 T cell within the CD4 or CD8 T cell sub-population.
  • the frequency of antigen-specific CD4 T-lymphocytes secreting in response was summarised by descriptive statistics for each antigen, for each cytokine, for each vaccine group and at each timepoint (pre- and post-vaccination).
  • Vaccine-induced CD4 T-cells are shown to be able to persist at least for one year since there is an observable difference in prevaccination levels of CD4 T-cell responses between individuals vaccinated with Fluarix has compared to those vaccinated with Fluarix/AS03 the year before.
  • the results are also shown in FIG. 8 , showing the CD4 T-cell response to split Flu antigen before and after revaccination. D0 corresponds to 12 months after first year vaccination and thus indicates persistence.
  • the frequency of antigen-specific CD8 T-lymphocytes secreting in response was summarised by descriptive statistics for each antigen, for each cytokine, for each vaccine group and at each timepoint (pre- and post-vaccination), similarly to the procedure followed in respect of CD4 T cell response.
  • the frequency of antigen-specific CD4 T-lymphocytes secreting in response at prevaccination was summarised by descriptive statistics for each cytokine and for each vaccine group and for each of the two studies in Table 25, for each of the two studies study and for each vaccine group in Table 27. Inferential statistics are given in Table 26 and Table 28.
  • the frequency of antigen-specific CD4 T-lymphocytes secreting in response at (post-pre) timepoint was summarised by descriptive statistics for each cytokine and for each vaccine group and for each study in Table 29, for each study and for each vaccine group in Table 31. Inferential statistics are given in Table 30 and Table 32.
  • the adjuvanted vaccine Flu-AS03 is superior to the equivalent unadjuvated vaccine Fluarix in terms of frequency of influenza specific CD4 T cells, and also in terms of persistence of the immune response elicited by the first Flu-AS03 vaccination (primo-vaccination in Explo Flu 001) until D0 of the revaccination study (Explo Flu 002 i.e. +/ ⁇ 1 year later). Furthermore this response is capable to recognise drifted influenza strains present in the new vaccine and to recognise the strains of the 2004 influenza vaccine.
  • Influenza infection in the ferret model closely mimics human influenza, with regards both to the sensitivity to infection and the clinical response.
  • the ferret is extremely sensitive to infection with both influenza A and B viruses without prior adaptation of viral strains. Therefore, it provides an excellent model system for studies of protection conferred by administered influenza vaccines.
  • the objective of this experiment was to demonstrate the efficacy of an adjuvanted influenza vaccine compared to the plain (un-adjuvanted) vaccine.
  • ferrets Mustela putorius furo (6 ferrets/group) aged 14-20 weeks were obtained from MISAY Consultancy (Hampshire, UK). Ferrets were primed on day 0 with heterosubtypic strain H1N1 A/Stockholm/24/90 (4 Log TCID 50 /ml). On day 21, ferrets were injected intramuscularly with a full human dose (500 ⁇ g vaccine dose, 15 ⁇ g HA/strain) of a combination of H1N1 A/New Caledonia/20/99, H3N2 A/Panama/2007/99 and B/Shangdong/7/97. Ferrets were then challenged on day 41 by intranasal route with an homotypic strain H3N2 A/Panama/2007/99 (4.51 Log TCID 50 /ml).
  • Trivalent Full HD 15 ⁇ g IM; Day 21 Priming H1N1 Plain HA/strain (A/Stockolm/24/ 90) Day 0 2 Trivalent Full HD: 15 ⁇ g IM; Day 21 Priming H1N1 AS03 HA/strain (A/Stockolm/24/90) Day 0 3 Trivalent Full HD: 15 ⁇ g IM; Day 21 Priming H1N1 AS03+MPL HA/strain (A/Stockolm/24/ 90) Day 0 4 PBS IM; Day 21 Priming H1N1 (A/Stockolm/24/ 90) Day 0
  • Formulation 1 Trivalent Plain (Un-Adjuvanted) Formulation (500 ⁇ l):
  • PBS10 fold concentrated (pH 7.4 when one fold concentrated) as well as a mixture containing Tween 80, Triton X-100 and VES (quantities taking into account the detergents present in the strains) are added to water for injection.
  • the detergents quantities reached are the following: 750 ⁇ g Tween 80, 110 ⁇ g Triton X-100 and 100 ⁇ g VES per 1 ml.
  • 15 ⁇ g of each strain H1N1, H3N2 and 17.5 ⁇ g of B strain are added in sequence with 10 min stirring between each addition. The formulation is stirred for 15 minutes at room temperature and stored at 4° C. if not administered directly.
  • PBS10 fold concentrated (pH 7.4 when one fold concentrated) as well as a mixture containing Tween 80, Triton X-100 and VES (quantities taking into account the detergents present in the strains) is added to water for injection.
  • the detergents quantities reached are the following: 750 ⁇ g Tween 80, 110 ⁇ g Triton X-100 and 100 ⁇ g VES per 1 ml.
  • 15 ⁇ g of each strain H1N1, H3N2 and 17.5 ⁇ g of B strain are added with 10 min stirring between each addition.
  • 250 ⁇ l of SB62 emulsion (prepared as in taught in Example II.1) is added. The formulation is stirred for 15 minutes at room temperature and stored at 4° C. if not administered directly.
  • PBS 10 fold concentrated (pH 7.4 when one fold concentrated) as well as a mixture containing Tween 80, Triton X-100 and VES (quantities taking into account the detergents present in the strains) is added to water for injection.
  • the detergents quantities reached are the following: 750 ⁇ g Tween 80, 110 ⁇ g Triton X-100 and 100 ⁇ g VES per 1 ml.
  • 15 ⁇ g of each strain H1N 1 , H3N2 and 17.5 ⁇ g of B strain are added with 10 min stirring between each addition.
  • 250 ⁇ l of SB62 emulsion (prepared as in taught in Example II.1) is added.
  • the mixture is stirred again for 15 min just prior addition of 25 ⁇ g of MPL from a suspension prepared as detailed in Example II.3.1.
  • the formulation is stirred for 15 minutes at room temperature and stored at 4° C. if not administered directly.
  • FIG. 10 A schematic representation of the results is given in FIG. 10 and FIG. 11 .
  • Viral titration of nasal washes was performed on 6 animals per group.
  • the nasal washes were performed by administration of 5 ml of PBS in both nostrils in awake animals.
  • the inoculation was collected in a Petri dish and placed into sample containers at ⁇ 80° C. (dry ice).
  • the intensity of the yellow formazan dye produced upon reduction of WST-1 by viable cells is proportional to the number of viable cells present in the well at the end of the viral titration assay and is quantified by measuring the absorbance of each well at the appropriate wavelength (450 nanometers).
  • the cut-off is defined as the OD average of uninfected control cells—0.3 OD (0.3 OD correspond to +/ ⁇ 3 StDev of OD of uninfected control cells).
  • a positive score is defined when OD is ⁇ cut-off and in contrast a negative score is defined when OD is >cut-off.
  • Viral shedding titers were determined by “Reed and Muench” and expressed as Log TCID50/ml.
  • Trivalent Split adjuvanted with AS03 or AS03+MPL were observed with Trivalent Split adjuvanted with AS03 or AS03+MPL compared to the Trivalent Split Plain for all 3 strains (at least 2-fold for 2 out of 3 strains, i.e. H3N2 and B strains).
  • AS03 and AS03+MPL formulations showed added benefit in terms of protective efficacy in ferrets (lower viral shedding and temperature) ( FIGS. 10 and 11 ).
  • Ferrets were primed on day 0 with heterosubtypic strain H1N1 A/Stockholm/24/90 (4 Log TCID 50 /ml).
  • ferrets were injected intramuscularly with a full human dose (500 ⁇ g vaccine dose, 15 ⁇ g HA/strain) of a combination of H1N1 A/New Caledonia/20/99, H3N2 A/Panama/2007/99 and B/Shangdong/7/97 (17.5 ⁇ g HA).
  • Ferrets were then challenged on day 43 by intranasal route with an heterosubtypic strain H3N2 A/Wyoming/3/2003 (4.51 Log TCID 50 /ml).
  • FIG. 12 A schematic representation of the results is given in FIG. 12 and in FIG. 13 .
  • the nasal washes were performed by administration of 5 ml of PBS in both nostrils in awake animals.
  • the inoculation was collected in a Petri dish and placed into sample containers at ⁇ 80° C. (dry ice).
  • the intensity of the yellow formazan dye produced upon reduction of WST-1 by viable cells is proportional to the number of viable cells present in the well at the end of the viral titration assay and is quantified by measuring the absorbance of each well at the appropriate wavelength (450 nanometers).
  • the cut-off is defined as the OD average of uninfected control cells—0.3 OD (0.3 OD corresponds to +/ ⁇ 3 St Dev of OD of uninfected control cells).
  • a positive score is defined when OD is ⁇ cut-off and in contrast a negative score is defined when OD is >cut-off.
  • Viral shedding titers were determined by “Reed and Muench” and expressed as Log TCID50/ml.
  • Viral shedding was measured for 12 ferrets from Day 1 Pre-priming- to Day 7 Post-priming. Results are expressed in pool.
  • Viral shedding was measured for 6 ferrets/group from Day 1 Pre-challenge to Day 7 Post-challenge.
  • Anti-Hemagglutinin antibody titers to the H3N2 influenza virus were determined using the hemagglutination inhibition test (HI).
  • the principle of the HI test is based on the ability of specific anti-Influenza antibodies to inhibit hemagglutination of chicken red blood cells (RBC) by influenza virus hemagglutinin (HA).
  • Sera were first treated with a 25% neuraminidase solution (RDE) and were heat-inactivated to remove non-specific inhibitors. After pre-treatment, two-fold dilutions of sera were incubated with 4 hemagglutination units of each influenza strain. Chicken red blood cells were then added and the inhibition of agglutination was scored. The titers were expressed as the reciprocal of the highest dilution of serum that completely inhibited hemagglutination. As the first dilution of sera was 1:10, an undetectable level was scored as a titer equal to 5.
  • results are shown in FIGS. 14A and 14B .
  • H3N2 A/Panama After immunization with H3N2 A/Panama, higher humoral responses (HI titers) were observed in ferrets immunized with the trivalent split vaccine adjuvanted with AS03 or AS03+MPL, as compared to the humoral response observed after immunization of ferrets with the un-adjuvanted (plain) trivalent split vaccine (FluarixTM).
  • a boost of A/Wyoming-specific HI titers was observed in ferrets immunized with the heterologous strain A/Panama H3N2 and challenged with A/Wyoming H3N2.
  • the heterologous challenge resulted in an increase of A/Panama-specific HI titers in ferrets immunized with A/Panama H3N2 adjuvanted with AS03 and AS03+MPL.
  • the purpose was to select readouts to induce in mice similar CMI responses than observed in humans. Particularly, the purpose was to show higher CMI responses in mice by using Split AS03 or split AS03+MPL compared to Split plain.
  • mice/group mice Female C57BI/6 mice (15 mice/group) aged 6-8 weeks were obtained from Harlan Horst, Netherland. The groups tested were:
  • mice were primed on day 0 with heterosubtypic strains (5 ⁇ g HA whole inactivated H1N1 A/Johnannesburg/82/96, H3N2 A/Sydney/5197, B/Harbin/7/94). On day 28, mice were injected intramuscularly with 1.5 ⁇ g HA Trivalent split (A/New Caledonia/20/99, A/Panama/2007/99, B/Shangdong/7/97) plain or adjuvanted (see groups below).
  • PBS 10 fold concentrated is added to reach isotonicity and is 1 fold concentrated in the final volume.
  • H 2 O volume is calculated to reach the targeted volume.
  • Formulation 1 (for 500 ⁇ l): PBS 10 fold concentrated (pH 7.4 when one fold concentrated) as well as a mixture containing Tween 80, Triton X-100 and VES (quantities taking into account the detergents present in the strains) are added to water for injection.
  • the detergents quantities reached are the following: 750 ⁇ g Tween 80, 110 ⁇ g Triton X-100 and 100 ⁇ g VES per 1 ml
  • 15 ⁇ g of each strain H1N1, H3N2 and B are added with 10 min stirring between each addition.
  • the formulation is stirred for 15 minutes at room temperature and stored at 4° C. if not administered directly.
  • PBS 10 fold concentrated (pH 7.4 when one fold concentrated) as well as a mixture containing Tween 80, Triton X-100 and VES (quantities taking into account the detergents present in the strains) is added to water for injection.
  • the detergents quantities reached are the following: 750 ⁇ g Tween 80, 110 ⁇ g Triton X-100 and 100 ⁇ g VES per 1 ml.
  • 15 ⁇ g of each strain H1N1, H3N2 and B are added with 10 min stirring between each addition.
  • 250 ⁇ l of SB62 emulsion (prepared as taught in Example II.1) is added. The formulation is stirred for 15 minutes at room temperature and stored at 4° C. if not administered directly.
  • PBS 10 fold concentrated (pH 7.4 when one fold concentrated) as well as a mixture containing Tween 80, Triton X-100 and VES (quantities taking into account the detergents present in the strains) is added to water for injection.
  • the detergents quantities reached are the following: 750 ⁇ g Tween 80, 110 ⁇ g Triton X-100 and 100 ⁇ g VES per 1 ml
  • 15 ⁇ g of each strain H1N1, H3N2 and B are added with 10 min stirring between each addition.
  • 250 ⁇ l of SB62 emulsion (prepared as taught in Example II.1) is added. The mixture is stirred again for 15 min just prior addition of 25 ⁇ g of MPL.
  • the formulation is stirred for 15 minutes at room temperature and stored at 4° C. if not administered directly.
  • PBMCs from primed mice were harvested 7 days post-immunization. They were tested in pools/group.
  • the purpose was to compare the CMI response induced by a GlaxoSmithKline commercially available split vaccine (FluarixTM) versus a subunit vaccine (Chiron's vaccine FluadTM) as well as the CMI response obtained with these vaccines adjuvanted with AS03, or AS03+MPL or another oil-in-water emulsion adjuvant (OW).
  • mice Female C57BI/6 mice (24 mice/group) aged 6-8 weeks were obtained from Harlan Horst, Netherland. Mice were primed intranasally on day 0 with heterosubtypic strains (5 ⁇ g HA whole formaldehyde inactivated H1N1 A/Johnannesburg/82/96, H3N2 A/Sydney/5/97, B/Harbin/7/94). On day 29, mice were injected intramuscularly with 1.5 ⁇ g HA Trivalent split (A/New Caledonia/20/99, A/Wyoming/3/2003, B/Jiangsu/10/2003) plain or adjuvanted (see groups in Table 39 below).
  • An oil-in-water emulsion called OW is prepared following the recipe published in the instruction booklet contained in Chiron Behring FluAd vaccine.
  • Equal volume of PBS and FluAdTM/GripguardTM (commercial vaccine) vaccine are mixed.
  • the formulation is stirred for 15 minutes and stored at 4° C. if not administered directly.
  • PBS mod pH 7.4 (to reach a final volume of 1 ml) is added to a 500 ⁇ l dose of AggripalTM (commercial vaccine). After 15 min stirring, 250 ⁇ l of SB62 is added (prepared according to the methodoly detailed for the scaled-up production). 25 ⁇ g of MPL are then added. The formulation is stirred for 15 minutes and stored at 4° C. if not administered directly.
  • 250 ⁇ l of PBS mod pH 7.4 are added to a 500 ⁇ l dose of Aggripal. After 15 min stirring, 250 ⁇ l of OW as prepared for group 2 is added and the formulation is stirred 15 min and stored at 4° C. if not administered directly.
  • Equal volume of PBS mod pH 7.4 and Aggripal are mixed. The formulation is stirred for 15 minutes and stored at 4° C. if not administered directly.
  • IHA/neutralization assay 21 Days Post-immunization.
  • mice/group PBMCs from 24 mice/group were harvested 7 days post-immunization and tested in pools/group.
  • Haemagglutination inhibition activity against the 3 vaccine strains was detected in sera from 24 animals per group at Day 14 after intranasal heterologous priming and at Day 16 Post-immunization.
  • mice from 24 mice per group were harvested at Day 7 Post-immunization and tested in one pool/group. Inactivated trivalent whole viruses (1 ⁇ g/ml) were used as re-stimulating antigen. Results are shown in FIG. 17 upper part.
  • PBMCs from 24 mice per group were harvested at Day 7 Post-immunization and tested in one pool/group. Inactivated trivalent whole viruses (1 ⁇ g/ml) were used as re-stimulating antigen.
  • the strains used in the three vaccines were the ones that had been recommended by the WHO for the 2004-2005 Northern Hemisphere season, i.e. A/New Caledonia/20/99 (H1N1), A/New California/3/2003 (H3N2) and B/Jiangsu/10/2003.
  • the commercially available vaccine used as a comparator the adjuvanted vaccines (AS03, or AS03+MPL) contain 15 ⁇ g haemagglutinin (HA) of each influenza virus strain per dose.
  • the adjuvanted influenza candidate vaccines are 2 component vaccines consisting of a concentrated trivalent inactivated split virion antigens presented in a type I glass vial and of a pre-filled type I glass syringe containing the adjuvant (AS03 or AS03+MPL). They have been prepared as detailed in Example II.
  • the three inactivated split virion antigens (monovalent bulks) used in formulation of the adjuvanted influenza candidate vaccines, are exactly the same as the active ingredients used in formulation of the commercial FluarixTM/ ⁇ -Rix.
  • the AS03-adjuvanted influenza candidate vaccine is a 2 components vaccine consisting of a concentrated trivalent inactivated split virion antigens presented in a type I glass vial (335 ⁇ l) (antigen container) and of a pre-filled type I glass syringe containing the SB62 emulsion (335 ⁇ l) (adjuvant container). Description and composition of the AS03 candidate vaccine is explained in Example III.
  • the AS03+MPL-adjuvanted influenza candidate vaccine is a 2 components vaccine consisting of a concentrated trivalent inactivated split virion antigens presented in a type I glass vial (335 ⁇ l) (antigen container) and of a pre-filled type I glass syringe containing the AS03+MPL adjuvant (360 ⁇ l) (adjuvant container).
  • the content of the antigen container is removed from the vial by using the syringe containing the AS03+MPL adjuvant, followed by gently mixing of the syringe.
  • the used needle Prior to injection, the used needle is replaced by an intramuscular needle and the volume is corrected to 530 ⁇ l.
  • One dose of the reconstituted the AS03+MPL-adjuvanted influenza candidate vaccine corresponds to 530 ⁇ l.
  • the inactivated split virion antigen are concentrated two-fold in the antigen container (i.e. 60 ⁇ g HA/ml) as compared to FluarixTM (i.e. 30 ⁇ g HA/ml).
  • composition of one dose of the reconstituted adjuvanted influenza vaccine is identical to that reported in Table 45 (see Example XI) except for the influenza strains. Both vaccines were given intramuscularly.
  • the CMI objectives were to determine which immunogenic composition between the formulation adjuvanted with AS03, or AS03+MPL versus the composition without any adjuvant has the strongest immunostimulating activity on CD4- and CD8-mediated immunity of individuals vaccinated with influenza antigens.
  • the CMI analysis was based on the Total vaccinated cohort.
  • Results were expressed as a frequency of cytokine(s)-positive CD4 or CD8 T cell within the CD4 or CD8 T cell sub-population.
  • the objective of the study was to investigate whether the frequency of memory B cell specific to Flu Antigen are significantly induced upon one intramuscular vaccination with the Flu candidate vaccine containing the Adjuvant AS03+MPL or AS03, as compared to Fluarix in elderly population.
  • the frequency of memory B cell has been assessed by B cell Elispot assay.
  • the end points are:
  • the objective of this experiment was to evaluate the ability of these vaccines to reduce disease symptoms (body temperature and viral shedding) in nasal secretions of ferrets challenged with heterologous strains.
  • ferrets Mustela putorius furo
  • H1N1 A/Stockholm/24/90 4 Log TCID 50 /ml
  • ferrets were injected intramuscularly with a full human dose (1 ml vaccine dose, 15 ⁇ g HA/strain) of a combination of H1N1 A/New Caledonia/20/99, H3N2 A/Wyoming/3/2003 and B/Jiangsu/10/2003.
  • PBS 10 fold concentrated (pH 7.4 when one fold concentrated) as well as a mixture containing Tween 80, Triton X-100 and VES (quantities taking into account the detergents present in the strains) are added to water for injection.
  • the detergents quantities reached are the following: 375 ⁇ g Tween 80, 55 ⁇ g Triton X-100 and 50 ⁇ g VES per 1 ml.
  • 15 ⁇ g of each strain H1N1, H3N2 and 17.5 ⁇ g of B strain are added with 10 min stirring between each addition.
  • the formulation is stirred for 15 minutes at room temperature and stored at 4° C. if not administered directly.
  • PBS 10 fold concentrated (pH 7.4 when one fold concentrated) as well as a mixture containing Tween 80, Triton X-100 and VES (quantities taking into account the detergents present in the strains) is added to water for injection.
  • the detergents quantities reached are the following: 375 ⁇ g Tween 80, 55 ⁇ g Triton X-100 and 50 ⁇ g VES per 1 ml.
  • 15 ⁇ g of each strain H1N1, H3N2 and B are added with 10 min stirring between each addition.
  • 250 ⁇ l of SB62 emulsion (prepared as detailed in Example II.1) is added. The mixture is stirred again for 15 minutes just prior addition of 25 ⁇ g of MPL.
  • the formulation is stirred for 15 minutes at room temperature and stored at 4° C. if not administered directly.
  • Viral titration of nasal washes was performed on 6 animals per group.
  • the nasal washes were performed by the administration of 5 ml of PBS in both nostrils in awake animals.
  • the inoculation was collected in a Petri dish and placed into sample containers on dry ice ( ⁇ 80° C.).
  • the intensity of the yellow formazan dye produced upon reduction of WST-1 by viable cells is proportional to the number of viable cells present in the well at the end of the viral titration assay and is quantified by measuring the absorbance of each well at the appropriate wavelength (450 nanometers).
  • the cut-off is defined as the OD average of uninfected control cells—0.3 OD (0.3 OD corresponds to +/ ⁇ 3 St Dev of OD of uninfected control cells).
  • a positive score is defined when OD is ⁇ cut-off and in contrast a negative score is defined when OD is >cut-off.
  • Viral shedding titers were determined by “Reed and Muench” and expressed as Log TCID50/ml.
  • Results are shown in FIG. 20 .
  • Lower viral shedding was observed post-challenge with the trivalent split vaccine adjuvanted with AS03+MPL, or with the AgrippalTM sub-unit vaccine adjuvanted with AS03, as compared to the very low viral shedding reduction observed after immunization of ferrets with the un-adjuvanted (plain) trivalent split vaccine (FluarixTM) or with FluadTM sub-unit vaccine.
  • Anti-Hemagglutinin antibody titers to the H3N2 influenza virus strains were determined using the hemagglutination inhibition test (HI).
  • the principle of the HI test is based on the ability of specific anti-Influenza antibodies to inhibit hemagglutination of chicken red blood cells (RBC) by influenza virus hemagglutinin (HA).
  • Sera were first treated with a 25% neuraminidase solution (RDE) and were heat-inactivated to remove non-specific inhibitors. After pre-treatment, two-fold dilutions of sera were incubated with 4 hemagglutination units of each influenza strain. Chicken red blood cells were then added and the inhibition of agglutination was scored. The titers were expressed as the reciprocal of the highest dilution of serum that completely inhibited hemagglutination. As the first dilution of sera was 1:10, an undetectable level was scored as a titer equal to 5.
  • the GMTs for HI antibodies with 95% Cl are shown in FIG. 23 .
  • Pre-vaccination GMTs of antibodies for all 3 vaccine-strains were within the same range in the 3 groups. After vaccinations, anti-haemagglutinin antibody levels increased significantly.
  • Post-vaccination GMTs of antibodies for the 3 vaccine strains remained however within the same ranges for all vaccines.
  • influenza vaccines fulfilled the requirements of the European authorities for annual registration of influenza inactivated vaccines [“Note for Guidance on Harmonisation of Requirements for Influenza Vaccines for the immunological assessment of the annual strain changes” (CPMP/BWP/214/96)] in subjects aged over 60 years.
  • One objective of this study was to evaluate the humoral immune response (anti-haemagglutinin and anti-MPL titres) of the revaccination with the adjuvanted influenza vaccine Flu AS03+MPL administered about one year after administration of the first dose.
  • subjects who had already received FluarixTM in the previous trial received a dose of commercial vaccine and formed the control group of this trial.
  • the strains used in the three vaccines were the ones that had been recommended by the WHO for the 2005-2006 Northern Hemisphere season, i.e. A/New Caledonia/20/99 (H1N1), A/New California/7/2004 (H3N2) and B/Jiangsu/10/2003.
  • the commercially available vaccine used as a comparator the (AS03+MPL—adjuvanted vaccine, hereinafter in short “the adjuvanted vaccine”) contains 15 ⁇ g haemagglutinin (HA) of each influenza virus strain per dose.
  • the adjuvanted influenza candidate vaccine is a 2 component vaccine consisting of a concentrated trivalent inactivated split virion antigens presented in a type I glass vial and of a pre-filled type I glass syringe containing the AS03+MPL adjuvant. It has been prepared according the method detailed in Example II.
  • the content of the prefilled syringe containing the adjuvant is injected into the vial that contains the concentrated trivalent inactivated split virion antigens. After mixing the content is withdrawn into the syringe and the needle is replaced by an intramuscular needle.
  • One dose of the reconstituted the adjuvanted influenza candidate vaccine corresponds to 0.7 mL.
  • the adjuvanted influenza candidate vaccine is a preservative-free vaccine.
  • composition of one dose of the reconstituted adjuvanted influenza vaccine is given in Table 45. Both vaccines were given intramuscularly.
  • influenza vaccines fulfilled the requirements of the European authorities for annual registration of influenza inactivated vaccines [“Note for Guidance on Harmonisation of Requirements for Influenza Vaccines for the immunological assessment of the annual strain changes” (CPMP/BWP/214/96)] in subjects aged over 60 years (Table 46).
  • the primary objective is to demonstrate the non inferiority 21 days post-vaccination of the influenza adjuvanted vaccines administered in elderly subjects (aged 65 years and older) as compared to FluarixTM administered in adults (aged 18-40 years) in terms of frequency of influenza-specific CD4 T-lymphocytes producing at least two different cytokines (CD40L, IL-2, TNF- ⁇ , IFN- ⁇ ).
  • the secondary objectives are:
  • the tertiary objective is to evaluate the cell mediated immune response (production of IFN- ⁇ , IL-2, CD40L, and TNF- ⁇ and memory B-cell response) 21, 90 and 180 days after vaccination with adjuvanted influenza-vaccines. FluarixTM is used as reference.
  • influenza vaccine adjuvanted with AS03+MPL (25 ⁇ g per dose) system is also used in study illustrated in Example XI.
  • the influenza vaccine adjuvanted with AS03+MPL (50 ⁇ g per dose) system is of identical composition except that the concentration of MPL is doubled.
  • the process is the same as the one described in Example VIII for the influenza vaccine adjuvanted with AS03+MPL, with as only difference that the concentration of MPL is doubled.
  • the GM ratio in term of influenza-specific CD4 frequency between groups vaccinated with adjuvanted vaccines and Flu YNG is obtained using an ANCOVA model on the logarithm-transformed titres.
  • the ANCOVA model includes the vaccine group as fixed effect and the pre-vaccination log-transformed titre as regressor.
  • the GM ratio and their 98.75% Cl are derived as exponential-transformation of the corresponding group contrast in the model.
  • the 98.75% Cl for the adjusted GM is obtained by exponential-transformation of the 98.75% Cl for the group least square mean of the above ANCOVA model.
  • the adjusted GM and GM ratios (with their 98.75% Cl) of influenza-specific CD4 T-lymphocyte producing at least two cytokines (IL-2, IFN- ⁇ , TNF- ⁇ and CD40L) at day 21, after in vitro restimulation with “pooled antigens II”, are presented in Table 47.
  • the upper limit of two-sided 98.75% Cl of GM ratio is far below the clinical limit of 2.0. This shows the non-inferiority of both adjuvanted influenza vaccines administered to elderly subjects compared to the FluarixTM vaccine administered in adults aged between 18 and 40 years in term of post-vaccination frequency of influenza-specific CD4.
  • Adjusted GM ratio of influenza-specific CD4 producing at least two cytokines Day 21 (ATP cohort for immunogenicity) Adjusted GM ratio (Flu YNG/ AS03+MPL (conc. 1) Flu YNG AS03+MPL (conc. 1) 98.8% CI N Adjusted GM N Adjusted GM Value LL UL 70 1995.3 72 2430.0 0.82 0.65 1.04 Adjusted GM ratio (Flu YNG/ AS03+MPL (conc. 2) Flu YNG AS03+MPL (conc.
  • the frequency of influenza-specific CD4/CD8 T-lymphocytes and memory B-cells were measured at days 0, 21, 90 and 180.
  • the frequency of influenza-specific cytokine-positive CD4/CD8 T-lymphocytes was summarised (descriptive statistics) for each vaccination group at days 0 and 21, for each antigen.
  • Non-parametric test (Wilcoxon test) was used to compare the location of difference between the two groups (influenza adjuvanted vaccine versus FluarixTM) and the statistical p-value is calculated for each antigen at each different test.
  • Descriptive statistics in individual difference between day 21/day 0 (Post-/Pre-vaccination) responses is calculated for each vaccination group and each antigen at each different test.
  • Non-parametric test (Wilcoxon test) is used to compare the individual difference Post-/Pre-vaccination) and the statistical p-value will be calculated for each antigen at each different test.
  • HI serum haemagglutination-inhibition
  • the cut-off value for HI antibody against all vaccine antigens was defined by the laboratory before the analysis (and equals 1:10).
  • a seronegative subject is a subject whose antibody titre is below the cut-off value.
  • a seropositive subject is a subject whose antibody titre is greater than or equal to the cut-off value.
  • Antibody titre below the cut-off of the assay is given an arbitrary value of half the cut-off.
  • the 95% Cl for GM is obtained within each group separately.
  • the 95% Cl for the mean of log-transformed titre is first obtained assuming that log-transformed titres are normally distributed with unknown variance.
  • the 95% Cl for the GM is then obtained by exponential-transformation of the 95% Cl for the mean of log-transformed titre.
  • Missing serological result for a particular antibody measurement is not replaced. Therefore a subject without serological result at a given time point do not contribute to the analysis of the assay for that time point.
  • Pre-vaccination GMTs of HI antibodies for all 3 vaccine strains were within the same range in the 4 treatment groups. After vaccination, there is clear impact of the 2 adjuvants which increase the humoral response in elderly, compared to standard Fluarix in the same population.
  • the adjuvanted influenza vaccines exceeded the requirements of the European authorities for annual registration of split virion influenza vaccines [“Note for Guidance on Harmonization of Requirements for Influenza Vaccines for the immunological assessment of the annual strain changes” (CPMP/BWP/214/96)] in subjects aged over 60 years.
  • the seroprotection rates for the 2 influenza adjuvanted vaccine groups are in the same range compared to Fluarix (18-40 years) group.
  • the seroconversion rates for the 2 influenza adjuvanted vaccine groups are in the same range compared to Fluarix (18-40 years) group excepted for New Caledonia strain.
  • the maximum intensity of local injection site redness/swelling is scored as follows:
  • the maximum intensity of fever is scored as follows:
  • the investigator makes an assessment of intensity for all other AEs, i.e. unsolicited symptoms, including SAEs reported during the study. The assessment is based on the investigator's clinical judgement. The intensity of each AE recorded is assigned to one of the following categories:
  • Grade 3 symptoms showed a trend to be higher in the group who received the vaccine adjuvanted with the highest MPL concentration compared to the group who received the adjuvanted vaccine wherein the MPL is at a lower concentration.
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080181911A1 (en) * 2005-03-23 2008-07-31 Emmanuel Jules Hanon Use Of An Influenza Virus, An Oil-In-Water Emulsion Adjuvant, To Induce Cd4 T-Cell And/Or Improved B-Memory Cell Response
US20090136543A1 (en) * 2007-04-20 2009-05-28 William Ripley Ballou Vaccine
US8221761B1 (en) * 1999-02-26 2012-07-17 Novartis Ag Enhancement of bactericidal activity of neisseria antigens with oligonucleotides containing CG motifs
US20130095130A1 (en) * 2010-01-06 2013-04-18 Vaxinnate Corporation Methods and Compositions for Providing Protective Immunity in the Elderly
US9056901B2 (en) 2006-03-07 2015-06-16 Vaxinnate Corporation Methods of making hemagglutinin proteins
US9205138B2 (en) 2008-04-18 2015-12-08 Vaxinnate Corporation Deletion mutants of flagellin and methods of use
US9278127B2 (en) 2006-07-17 2016-03-08 Glaxosmithkline Biologicals, Sa Influenza vaccine

Families Citing this family (77)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9923176D0 (en) * 1999-09-30 1999-12-01 Smithkline Beecham Biolog Novel composition
US20100221284A1 (en) * 2001-05-30 2010-09-02 Saech-Sisches Serumwerk Dresden Novel vaccine composition
DE10144906B4 (de) 2001-09-12 2013-11-28 Novartis Vaccines And Diagnostics Gmbh Verfahren zur großtechnischen Herstellung von Impfstoffen
US11707520B2 (en) 2005-11-03 2023-07-25 Seqirus UK Limited Adjuvanted vaccines with non-virion antigens prepared from influenza viruses grown in cell culture
WO2007052059A2 (en) * 2005-11-04 2007-05-10 Novartis Vaccines And Diagnostics Srl Changing th1/th2 balance in split influenza vaccines with adjuvants
JP2009514839A (ja) * 2005-11-04 2009-04-09 ノバルティス ヴァクシンズ アンド ダイアグノスティクス エスアールエル サイトカイン誘導剤を含むアジュバントインフルエンザワクチン
KR20080069232A (ko) * 2005-11-04 2008-07-25 노바티스 백신즈 앤드 다이아그노스틱스 에스.알.엘. 스플리트 인플루엔자 백신에 대한 보조제로서 유리 수성상계면활성제를 갖는 에멀젼
ES2420829T3 (es) * 2005-11-04 2013-08-27 Novartis Vaccines And Diagnostics S.R.L. Vacunas adyuvantadas con antígeno de no virión preparadas a partir de virus de la gripe cultivados en cultivo celular
FR2896162B1 (fr) * 2006-01-13 2008-02-15 Sanofi Pasteur Sa Emulsion huile dans eau thermoreversible
EP2004226A1 (en) * 2006-03-24 2008-12-24 Novartis Vaccines and Diagnostics GmbH & Co. KG Storage of influenza vaccines without refrigeration
ES2401484T3 (es) 2006-06-15 2013-04-22 Novartis Ag Régimen de vacunación contra la gripe multidosis ahorrador de adyuvantes
CA2583555C (en) * 2006-07-17 2020-01-07 Glaxosmithkline Biologicals S.A. Influenza vaccine
CN105727276A (zh) * 2006-07-17 2016-07-06 葛兰素史密丝克莱恩生物有限公司 流感疫苗
EA015817B1 (ru) * 2006-10-12 2011-12-30 Глаксосмитклайн Байолоджикалс С.А. Иммуногенная композиция, содержащая адъювант в виде эмульсии "масло в воде"
EP2086582B1 (en) * 2006-10-12 2012-11-14 GlaxoSmithKline Biologicals s.a. Vaccine comprising an oil in water emulsion adjuvant
GB0622282D0 (en) * 2006-11-08 2006-12-20 Novartis Ag Quality control methods
EP1923070A1 (en) * 2006-11-15 2008-05-21 Intervet International BV Vaccine for vaccinating feline against influenza virus
US20110045022A1 (en) * 2006-12-06 2011-02-24 Theodore Tsai Vaccines including antigen from four strains of influenza virus
EP1938835A1 (en) * 2006-12-29 2008-07-02 Pevion Biotech AG Non-specific immunostimulating agents
NZ603245A (en) * 2007-02-23 2014-04-30 Baylor Res Inst Therapeutic applications of activation of human antigen-presenting cells through dectin-1
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DK2200642T3 (da) * 2007-10-19 2012-07-16 Novartis Ag Meningococvaccinepræparater
GB0810305D0 (en) † 2008-06-05 2008-07-09 Novartis Ag Influenza vaccination
US8668904B2 (en) * 2007-12-06 2014-03-11 Glaxosmithkline Biologicals S.A. Influenza composition
US8466259B2 (en) * 2007-12-07 2013-06-18 National Health Research Institutes Adjuvants
US8426163B2 (en) * 2007-12-07 2013-04-23 National Health Research Institutes Production of lipidated proteins in E. coli
EA201071086A1 (ru) * 2008-03-18 2011-04-29 Новартис Аг Усовершенствованный способ получения вакцинных антигенов вируса гриппа
GB0905570D0 (en) * 2009-03-31 2009-05-13 Novartis Ag Combined vaccines
WO2009156852A1 (en) * 2008-06-25 2009-12-30 Novartis Ag Rapid responses to delayed booster immunisations
AU2015203072B2 (en) * 2009-02-10 2017-05-25 Seqirus UK Limited Influenza vaccine regimens for pandemic-associated strains
CN102307590A (zh) 2009-02-10 2012-01-04 诺华有限公司 具有减少量的角鲨烯的流感疫苗
EA201171032A1 (ru) 2009-02-10 2012-02-28 Новартис Аг Схемы лечения с помощью вакцины против гриппа, связанного с пандемическими штаммами
US8287880B2 (en) * 2009-06-02 2012-10-16 National Health Research Institutes Lipidated vaccine against dengue virus infection
CN102482327B (zh) * 2009-06-22 2014-10-29 财团法人卫生研究院 脂质化肿瘤相关抗原及其免疫治疗的组成物及方法
EP2289575B1 (de) * 2009-08-06 2017-07-05 Biotronik VI Patent AG Medizinisches Implantat enthaltend eine antioxidative Substanz
GB2485327A (en) * 2009-08-12 2012-05-09 Sigmoid Pharma Ltd Immunomodulatory compositions comprising a polymer matrix and an oil phase
EP2308506A1 (en) * 2009-10-02 2011-04-13 Mucosis B.V. Adjuvanted intranasal vaccine formulations
GB0918830D0 (en) 2009-10-27 2009-12-09 Glaxosmithkline Biolog Niederl Process
GB0919117D0 (en) 2009-10-30 2009-12-16 Glaxosmithkline Biolog Sa Process
SI2506834T1 (sl) 2009-12-03 2016-04-29 Novartis Ag Cirkulacija sestavin med homogenizacijo in mikrofluidizacijo emulzij
CA2780425C (en) * 2009-12-03 2014-02-04 Novartis Ag Hydrophilic filtration during manufacture of vaccine adjuvants
DE102009056883B4 (de) 2009-12-03 2012-08-16 Novartis Ag Impfstoff-Adjuvantien und verbesserte Verfahren zur Herstellung derselben
DE102009056871A1 (de) * 2009-12-03 2011-06-22 Novartis AG, 4056 Impfstoff-Adjuvantien und verbesserte Verfahren zur Herstellung derselben
CL2012001399A1 (es) 2009-12-03 2013-03-08 Novartis Ag Metodo para fabricar adyuvante para vacuna (emulsion aceite/agua con escualeno, polisorbato 80 y trioleato de sorbitan), que comprende (i) formar primera emulsion en homogenizador desde un contendor a otro para formar segunda emulsion, (ii) y microfluidizar primera emulsion para formar segunda emulsion.
AU2013202690B2 (en) * 2009-12-03 2016-02-11 Novartis Ag Hydrophilic filtration during manufacture of vaccine adjuvants
US20130189311A1 (en) * 2009-12-03 2013-07-25 Novartis Ag Arranging interaction and back pressure chambers for microfluidization
DE102009056884B4 (de) 2009-12-03 2021-03-18 Novartis Ag Impfstoff-Adjuvantien und verbesserte Verfahren zur Herstellung derselben
CA2783955A1 (en) * 2009-12-22 2011-06-30 Sanofi Pasteur Limited Immunogenic compositions
WO2011088451A1 (en) * 2010-01-15 2011-07-21 Novavax, Inc. Uses of influenza virus-like particles (vlps) for characterization of neuraminidase and hemagglutinin activity
RU2527688C2 (ru) * 2010-02-27 2014-09-10 Общество С Ограниченной Ответственностью "Научно-Производственная Фирма "Материа Медика Холдинг" Способ лечения инфекционных заболеваний, вызванных вирусом гриппа с типом поверхностого антигена н1n1 и лекарственное средство для лечения инфекционных заболеваний, вызванных вирусом гриппа с типом поверхностного антигена н1n1
GB201009676D0 (en) 2010-06-10 2010-07-21 Glaxosmithkline Biolog Sa Novel process
GB201009671D0 (en) 2010-06-10 2010-07-21 Glaxosmithkline Biolog Sa Novel process
GB201009673D0 (en) 2010-06-10 2010-07-21 Glaxosmithkline Biolog Sa Novel process
US9821051B1 (en) 2010-10-28 2017-11-21 Seqirus UK Limited Reducing hospitalization in elderly influenza vaccine recipients
TWI507413B (zh) * 2010-11-15 2015-11-11 Nat Health Research Institutes 脂質化多抗原表位疫苗
TW201221642A (en) 2010-11-15 2012-06-01 Nat Health Research Institutes Method of producing lipidated polypeptides
WO2012116714A1 (en) 2011-03-02 2012-09-07 Curevac Gmbh Vaccination in elderly patients
MX371154B (es) * 2012-12-28 2020-01-20 Japan As Represented By Director General Of Nat Institute Of Infectious Diseases Composicion nasal de vacuna para la influenza.
CN105828835A (zh) * 2013-05-10 2016-08-03 诺华股份有限公司 避免流感疫苗中的发作性嗜睡病风险
US10080792B2 (en) * 2013-09-23 2018-09-25 Engen Bio, Inc. Influenza vaccine and therapy
CN104436157A (zh) * 2013-09-23 2015-03-25 恩金生物有限公司 流感疫苗和治疗
EP3197487B1 (en) * 2014-09-26 2022-08-03 Seqirus UK Limited Vaccination of immunocompromised subjects
WO2019108928A1 (en) * 2017-11-30 2019-06-06 Ohio State Innovation Foundation Mucoadhesive nanoparticle entrapped influenza virus vaccine delivery system
US20220080043A1 (en) * 2019-01-30 2022-03-17 Glaxosmithkline Biologicals Sa Oil/surfactant mixtures for self-emulsification
KR20220118399A (ko) * 2019-12-20 2022-08-25 프레제니우스 카비 오스트리아 게엠베하 수중유 에멀젼의 제조 방법
RU2741003C1 (ru) * 2020-03-27 2021-01-22 Евгений Дмитриевич Некрасов Способ производства четырехвалентной субъединичной вакцины против гриппа без адъювантов
US20230256090A1 (en) 2020-06-29 2023-08-17 Glaxosmithkline Biologicals Sa Adjuvants
KR20230054719A (ko) 2020-08-24 2023-04-25 사노피 파스퇴르 인크 Sars-cov-2 감염에 대한 백신
CA3190375A1 (en) 2020-08-24 2022-03-03 Natalie ANOSOVA Covid-19 vaccines with tocopherol-containing squalene emulsion adjuvants
KR102270165B1 (ko) 2020-10-22 2021-06-28 한국화학연구원 세정제 조성물
WO2022200582A1 (en) 2021-03-26 2022-09-29 Glaxosmithkline Biologicals Sa Immunogenic compositions
CA3222568A1 (en) 2021-06-28 2023-01-05 Glaxosmithkline Biologicals Sa Novel influenza antigens
RU2766292C1 (ru) * 2021-08-05 2022-03-14 Федеральное государственное унитарное предприятие "Санкт-Петербургский научно-исследовательский институт вакцин и сывороток и предприятие по производству бактерийных препаратов" Федерального медико-биологического агентства (ФГУП СПбНИИВС ФМБА России) Состав вакцины против covid-19
WO2023020993A1 (en) 2021-08-16 2023-02-23 Glaxosmithkline Biologicals Sa Novel methods
WO2023020994A1 (en) 2021-08-16 2023-02-23 Glaxosmithkline Biologicals Sa Novel methods
WO2023020992A1 (en) 2021-08-16 2023-02-23 Glaxosmithkline Biologicals Sa Novel methods
WO2023061993A1 (en) 2021-10-13 2023-04-20 Glaxosmithkline Biologicals Sa Polypeptides

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3095974A (en) * 1961-10-27 1963-07-02 Anthony R Perini Collapsible book rack
US4047869A (en) * 1973-02-23 1977-09-13 Mulvany Jr R F Apparatus for forming plastic articles
US4071734A (en) * 1975-12-03 1978-01-31 Zarechensky Anatoly Vasilievic Powder electrode strip for surfacing with wear-resistant alloy
US5149531A (en) * 1990-06-27 1992-09-22 University Of Pittsburgh Of The Commonwealth System Of Higher Education Method of using cold-adapted live influenza virus vaccine as an antiviral agent against influenza
US5376369A (en) * 1987-11-03 1994-12-27 Syntex (U.S.A.) Inc. Vaccine adjuvant
US5858368A (en) * 1993-09-13 1999-01-12 Protein Sciences Corporation Vaccine comprising a baculovirus produced influenza hemagglutinin protein fused to a second protein
US5969109A (en) * 1990-02-28 1999-10-19 Bona; Constantin Chimeric antibodies comprising antigen binding sites and B and T cell epitopes
US6372227B1 (en) * 1997-09-05 2002-04-16 Smithkline Beecham Biologicals, S.A. Vaccines
US20040071734A1 (en) * 2001-02-23 2004-04-15 Nathalie Garcon Novel vaccine

Family Cites Families (107)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4270537A (en) 1979-11-19 1981-06-02 Romaine Richard A Automatic hypodermic syringe
DD155875A1 (de) 1980-12-31 1982-07-14 Willy Nordheim Verfahren zur herstellung eines ballaststoffarmen inaktivierten influenzaimpfstoffes
US4436727A (en) * 1982-05-26 1984-03-13 Ribi Immunochem Research, Inc. Refined detoxified endotoxin product
US4866034A (en) * 1982-05-26 1989-09-12 Ribi Immunochem Research Inc. Refined detoxified endotoxin
DD211444A3 (de) 1982-08-19 1984-07-11 Saechsisches Serumwerk Verfahren zur herstellung von influenza-impfstoffen
GB8300467D0 (en) 1983-01-08 1983-02-09 Wellcome Found Equine influenza
US4596556A (en) 1985-03-25 1986-06-24 Bioject, Inc. Hypodermic injection apparatus
DD300833A7 (de) 1985-10-28 1992-08-13 Saechsische Landesgewerbefoerd Verfahren zur herstellung von inaktivierten influenza-vollvirusimpfstoffen
CA1283827C (en) 1986-12-18 1991-05-07 Giorgio Cirelli Appliance for injection of liquid formulations
GB8704027D0 (en) 1987-02-20 1987-03-25 Owen Mumford Ltd Syringe needle combination
AU614755B2 (en) 1987-06-05 1991-09-12 United States of America, as represented by the Secretary, U.S. Department of Commerce, The Autocrine motility factors in cancer diagnosis and management
US4941880A (en) 1987-06-19 1990-07-17 Bioject, Inc. Pre-filled ampule and non-invasive hypodermic injection device assembly
US4940460A (en) 1987-06-19 1990-07-10 Bioject, Inc. Patient-fillable and non-invasive hypodermic injection device assembly
US4790824A (en) 1987-06-19 1988-12-13 Bioject, Inc. Non-invasive hypodermic injection device
DE3734306A1 (de) 1987-10-10 1989-04-27 Pfeiffer Erich Gmbh & Co Kg Austragvorrichtung fuer fliessfaehige medien
US5339163A (en) 1988-03-16 1994-08-16 Canon Kabushiki Kaisha Automatic exposure control device using plural image plane detection areas
US4912094B1 (en) 1988-06-29 1994-02-15 Ribi Immunochem Research Inc. Modified lipopolysaccharides and process of preparation
GB8821049D0 (en) 1988-09-08 1988-10-05 Health Lab Service Board Method & composition for treatment & prevention of viral infections
FR2638359A1 (fr) 1988-11-03 1990-05-04 Tino Dalto Guide de seringue avec reglage de la profondeur de penetration de l'aiguille dans la peau
EP0382271B1 (en) * 1989-02-04 1994-12-21 Akzo Nobel N.V. Tocols as adjuvant in vaccine
US5092235A (en) 1989-05-24 1992-03-03 Tektronix, Inc. Pressure fixing and developing apparatus
WO1990014837A1 (en) * 1989-05-25 1990-12-13 Chiron Corporation Adjuvant formulation comprising a submicron oil droplet emulsion
US5312335A (en) 1989-11-09 1994-05-17 Bioject Inc. Needleless hypodermic injection device
US5064413A (en) 1989-11-09 1991-11-12 Bioject, Inc. Needleless hypodermic injection device
DE4005528C2 (de) 1990-02-22 1998-01-15 Pfeiffer Erich Gmbh & Co Kg Austragvorrichtung für Medien
WO1991013261A1 (en) 1990-03-02 1991-09-05 Experimentalny Nauchno-Issledovatelsky Institut Metallorezhuschikh Stankov Digital servo drive
US5190521A (en) 1990-08-22 1993-03-02 Tecnol Medical Products, Inc. Apparatus and method for raising a skin wheal and anesthetizing skin
US5527288A (en) 1990-12-13 1996-06-18 Elan Medical Technologies Limited Intradermal drug delivery device and method for intradermal delivery of drugs
GB9105992D0 (en) 1991-03-21 1991-05-08 Smithkline Beecham Biolog Vaccine
GB9118204D0 (en) 1991-08-23 1991-10-09 Weston Terence E Needle-less injector
SE9102652D0 (sv) 1991-09-13 1991-09-13 Kabi Pharmacia Ab Injection needle arrangement
US5328483A (en) 1992-02-27 1994-07-12 Jacoby Richard M Intradermal injection device with medication and needle guard
MY111880A (en) 1992-03-27 2001-02-28 Smithkline Beecham Biologicals S A Hepatitis vaccines containing 3-0 deacylated monophosphoryl lipid a
US5383851A (en) 1992-07-24 1995-01-24 Bioject Inc. Needleless hypodermic injection device
US5569189A (en) 1992-09-28 1996-10-29 Equidyne Systems, Inc. hypodermic jet injector
US5334144A (en) 1992-10-30 1994-08-02 Becton, Dickinson And Company Single use disposable needleless injector
JPH08506592A (ja) * 1993-02-19 1996-07-16 スミスクライン・ビーチャム・コーポレイション ３−ｏ−脱アシル化モノホスホリル脂質Ａ含有のインフルエンザワクチン組成物
EP0684838A1 (en) 1993-02-19 1995-12-06 Smithkline Beecham Corporation Influenza vaccine compositions containing 3-o-deacylated monophosphoryl lipid a
DK0812593T4 (da) 1993-03-23 2008-05-13 Smithkline Beecham Biolog Vaccinepræparater indeholdende 3-O-deacyleret monophosphoryllipid-A
WO1995011700A1 (en) 1993-10-29 1995-05-04 Pharmos Corp. Submicron emulsions as vaccine adjuvants
SK281944B6 (sk) 1993-11-17 2001-09-11 Laboratoires Om S. A. Beta(1->6)glukozamínové disacharidy, spôsob ich prípravy, farmaceutický prostriedok, ktorý ich obsahuje, a ich použitie
GB9326253D0 (en) * 1993-12-23 1994-02-23 Smithkline Beecham Biolog Vaccines
DE69533693T2 (de) 1994-02-24 2005-12-22 Novavax Inc.(n.d.Ges.d.Staates Delaware) Impfstoffe, die paucilamelläre Lipidvesikel als Adjuvans enthalten
WO1995024176A1 (en) 1994-03-07 1995-09-14 Bioject, Inc. Ampule filling device
US5466220A (en) 1994-03-08 1995-11-14 Bioject, Inc. Drug vial mixing and transfer device
WO1995026204A1 (en) 1994-03-25 1995-10-05 Isis Pharmaceuticals, Inc. Immune stimulation by phosphorothioate oligonucleotide analogs
US5599302A (en) 1995-01-09 1997-02-04 Medi-Ject Corporation Medical injection system and method, gas spring thereof and launching device using gas spring
UA56132C2 (uk) 1995-04-25 2003-05-15 Смітклайн Бічем Байолоджікалс С.А. Композиція вакцини (варіанти), спосіб стабілізації qs21 відносно гідролізу (варіанти), спосіб приготування композиції вакцини
US5730723A (en) 1995-10-10 1998-03-24 Visionary Medical Products Corporation, Inc. Gas pressured needle-less injection device and method
US5893397A (en) 1996-01-12 1999-04-13 Bioject Inc. Medication vial/syringe liquid-transfer apparatus
GB9607549D0 (en) 1996-04-11 1996-06-12 Weston Medical Ltd Spring-powered dispensing device
US5916879A (en) 1996-11-12 1999-06-29 St. Jude Children's Research Hospital DNA transcription unit vaccines that protect against avian influenza viruses and methods of use thereof
TW570803B (en) 1997-04-09 2004-01-11 Duphar Int Res Influenza vaccine
US5993412A (en) 1997-05-19 1999-11-30 Bioject, Inc. Injection apparatus
GB9711990D0 (en) 1997-06-11 1997-08-06 Smithkline Beecham Biolog Vaccine
WO1999011241A1 (en) * 1997-09-05 1999-03-11 Smithkline Beecham Biologicals S.A. Oil in water emulsions containing saponins
IT1298087B1 (it) 1998-01-08 1999-12-20 Fiderm S R L Dispositivo per il controllo della profondita' di penetrazione di un ago, in particolare applicabile ad una siringa per iniezioni
ES2273482T3 (es) * 1998-03-09 2007-05-01 Glaxosmithkline Biologicals S.A. Composiciones de vacunas combinadas.
US6838269B1 (en) * 1998-04-15 2005-01-04 Genencor International, Inc. Proteins producing an altered immunogenic response and methods of making and using the same
NZ508013A (en) 1998-05-07 2003-08-29 Corixa Corp Adjuvant composition for use with an antigen in a vaccine composition
AU7924598A (en) 1998-06-08 1999-12-30 Sca Emballage France Fast flattening packaging
DK1091928T3 (da) 1998-06-30 2007-07-23 Om Pharma Nye acylerede pseudodipeptider, fremgangsmåde til disses fremstilling og sammensætninger, som indeholder dem
AT408615B (de) 1998-09-15 2002-01-25 Immuno Ag Neue influenzavirus-impfstoffzusammensetzung
US20040006242A1 (en) 1999-02-01 2004-01-08 Hawkins Lynn D. Immunomodulatory compounds and method of use thereof
US6551600B2 (en) 1999-02-01 2003-04-22 Eisai Co., Ltd. Immunological adjuvant compounds compositions and methods of use thereof
AT407958B (de) 1999-02-11 2001-07-25 Immuno Ag Inaktivierte influenza-virus-vakzine zur nasalen oder oralen applikation
CA2689696C (en) 1999-02-26 2013-08-06 Novartis Vaccines And Diagnostics, Inc. Microemulsions with adsorbed macromolecules and microparticles
FR2796290B1 (fr) 1999-07-16 2001-09-14 Cross Site Technologies Seringue sans aiguille fonctionnant avec un generateur d'onde de choc a travers une paroi
FR2796289B1 (fr) 1999-07-16 2001-08-10 Cross Site Technologies Seringue sans aiguille avec injecteur a elements superposes
FR2796291B1 (fr) 1999-07-16 2001-09-21 Cross Site Technologies Seringue sans aiguille munie d'un systeme de declenchement piezo-electrique
GB9921147D0 (en) * 1999-09-07 1999-11-10 Smithkline Beecham Biolog Novel composition
GB9921146D0 (en) * 1999-09-07 1999-11-10 Smithkline Beecham Biolog Novel composition
GB9923176D0 (en) * 1999-09-30 1999-12-01 Smithkline Beecham Biolog Novel composition
US6494865B1 (en) 1999-10-14 2002-12-17 Becton Dickinson And Company Intradermal delivery device including a needle assembly
FR2800619B1 (fr) 1999-11-05 2002-02-08 Cross Site Technologies Seringue sans aiguille avec un moyen de poussee temporairement retenu
FR2802102B1 (fr) 1999-12-08 2002-07-12 Poudres & Explosifs Ste Nale Seringue sans aiguille munie d'un tube d'ejection a section constante
FR2802103B1 (fr) 1999-12-08 2003-10-03 Poudres & Explosifs Ste Nale Seringue sans aiguille fonctionnant avec entrainement du principe actif par effet tube a choc
AU1581400A (en) 1999-12-22 2001-07-03 Om Pharma Acyl pseudopeptides bearing a functionalised auxiliary spacer
FR2802820B1 (fr) 1999-12-27 2002-10-18 Poudres & Explosifs Ste Nale Seringue sans aiguille fonctionnant par effet tube a choc, avec maintien prealable du principe actif sur le cote
CN1326873C (zh) 2000-01-31 2007-07-18 史密丝克莱恩比彻姆生物有限公司 用于hiv预防或治疗性免疫的疫苗
FR2804329B1 (fr) 2000-02-02 2002-12-13 Poudres & Explosifs Ste Nale Seringue sans aiguille munie d'un opercule contenant le principe actif
FR2804869B1 (fr) 2000-02-11 2002-05-17 Poudres & Explosifs Ste Nale Seringue sans aiguille pour l'injection d'un liquide contenu dans une ampoule pre-remplie
FR2805749B1 (fr) 2000-03-01 2002-05-17 Poudres & Explosifs Ste Nale Seringue sans aiguille a deux niveaux de vitesse d'injection
FR2807946B1 (fr) 2000-04-19 2002-06-07 Poudres & Explosifs Ste Nale Seringue sans aiguille fonctionnant avec un chargement pyrotechnique bicomposition
FR2809626B1 (fr) 2000-05-30 2003-03-07 Poudres & Explosifs Ste Nale Seringue sans aiguille avec membrane d'isolation d'un ejecteur multiconduit
FR2810554B1 (fr) 2000-06-22 2003-05-16 Poudres & Explosifs Ste Nale Seringue sans aiguille munie d'un reservoir modulable
FR2812202B1 (fr) 2000-07-28 2002-09-13 Poudres & Explosifs Ste Nale Seringue sans aiguille fonctionnant par mise en compression du reservoir contenant le principe actif liquide
GB0025577D0 (en) 2000-10-18 2000-12-06 Smithkline Beecham Biolog Vaccine
FR2815544B1 (fr) 2000-10-23 2003-02-14 Poudres & Explosifs Ste Nale Seringue sans aiguille securisee a architecture compacte
EP1201250A1 (en) 2000-10-25 2002-05-02 SMITHKLINE BEECHAM BIOLOGICALS s.a. Immunogenic compositions comprising liver stage malarial antigens
MY134424A (en) 2001-05-30 2007-12-31 Saechsisches Serumwerk Stable influenza virus preparations with low or no amount of thiomersal
US7361352B2 (en) * 2001-08-15 2008-04-22 Acambis, Inc. Influenza immunogen and vaccine
US6861410B1 (en) 2002-03-21 2005-03-01 Chiron Corporation Immunological adjuvant compositions
MXPA04009554A (es) * 2002-04-01 2005-01-25 Euro Celtique Sa Construcciones epitopes que comrpenden antigeno presentando mecanismos de marcacion celular.
DE602004020189D1 (de) 2003-01-30 2009-05-07 Novartis Vaccines & Diagnostic Adjuvante influenza-vakzine
DE202005022108U1 (de) * 2004-03-09 2013-11-12 Novartis Vaccines And Diagnostics, Inc. Influenza-Virus-Impfstoffe
US20090028903A1 (en) * 2005-03-23 2009-01-29 Glaxosmithkline Biologicals, S.A. Novel use
PT1861120T (pt) * 2005-03-23 2016-08-18 Glaxosmithkline Biologicals Sa Utilização de um vírus influenza e de um adjuvante de emulsão óleo-em-água para induzir células t cd4 e/ou melhorar a resposta de células b de memória
AR054822A1 (es) 2005-07-07 2007-07-18 Sanofi Pasteur Emulsion inmuno adyuvante
NZ594482A (en) 2005-11-04 2012-11-30 Novartis Vaccines & Diagnostic Influenza vaccines with reduced amount of oil-in-water emulsion as adjuvant
FR2896162B1 (fr) 2006-01-13 2008-02-15 Sanofi Pasteur Sa Emulsion huile dans eau thermoreversible
WO2007130330A2 (en) 2006-05-01 2007-11-15 Technovax, Inc. Polyvalent influenza virus-like particle (vlp) compositions
MX2009000660A (es) * 2006-07-17 2009-04-08 Glaxosmithkline Biolog Sa Vacuna de influenza.
CN105727276A (zh) * 2006-07-17 2016-07-06 葛兰素史密丝克莱恩生物有限公司 流感疫苗
EA015817B1 (ru) 2006-10-12 2011-12-30 Глаксосмитклайн Байолоджикалс С.А. Иммуногенная композиция, содержащая адъювант в виде эмульсии "масло в воде"
PE20090146A1 (es) * 2007-04-20 2009-03-23 Glaxosmithkline Biolog Sa Composicion inmunogenica contra el virus influenza
US8668904B2 (en) * 2007-12-06 2014-03-11 Glaxosmithkline Biologicals S.A. Influenza composition

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3095974A (en) * 1961-10-27 1963-07-02 Anthony R Perini Collapsible book rack
US4047869A (en) * 1973-02-23 1977-09-13 Mulvany Jr R F Apparatus for forming plastic articles
US4071734A (en) * 1975-12-03 1978-01-31 Zarechensky Anatoly Vasilievic Powder electrode strip for surfacing with wear-resistant alloy
US5376369A (en) * 1987-11-03 1994-12-27 Syntex (U.S.A.) Inc. Vaccine adjuvant
US5969109A (en) * 1990-02-28 1999-10-19 Bona; Constantin Chimeric antibodies comprising antigen binding sites and B and T cell epitopes
US5149531A (en) * 1990-06-27 1992-09-22 University Of Pittsburgh Of The Commonwealth System Of Higher Education Method of using cold-adapted live influenza virus vaccine as an antiviral agent against influenza
US5858368A (en) * 1993-09-13 1999-01-12 Protein Sciences Corporation Vaccine comprising a baculovirus produced influenza hemagglutinin protein fused to a second protein
US6372227B1 (en) * 1997-09-05 2002-04-16 Smithkline Beecham Biologicals, S.A. Vaccines
US20040071734A1 (en) * 2001-02-23 2004-04-15 Nathalie Garcon Novel vaccine

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8221761B1 (en) * 1999-02-26 2012-07-17 Novartis Ag Enhancement of bactericidal activity of neisseria antigens with oligonucleotides containing CG motifs
US9730999B2 (en) 2005-03-23 2017-08-15 Glaxosmithkline Biologicals Sa Adjuvanted influenza virus compositions
US20080181911A1 (en) * 2005-03-23 2008-07-31 Emmanuel Jules Hanon Use Of An Influenza Virus, An Oil-In-Water Emulsion Adjuvant, To Induce Cd4 T-Cell And/Or Improved B-Memory Cell Response
US9200042B2 (en) 2006-03-07 2015-12-01 Vaxinnate Corporation Flagellin fusion proteins
US9056901B2 (en) 2006-03-07 2015-06-16 Vaxinnate Corporation Methods of making hemagglutinin proteins
US11564984B2 (en) 2006-07-17 2023-01-31 Glaxosmithkline Biologicals Sa Influenza vaccine
US9278127B2 (en) 2006-07-17 2016-03-08 Glaxosmithkline Biologicals, Sa Influenza vaccine
US9943588B2 (en) 2006-07-17 2018-04-17 Glaxosmithkline Biologicals S.A. Influenza vaccine
US10016495B2 (en) 2007-04-20 2018-07-10 Glaxosmithkline Biologicals S.A. Oil-in-water emulsion influenza vaccine
US10548969B2 (en) 2007-04-20 2020-02-04 Glaxosmithkline Biologicals Sa Oil-in-water emulsion influenza vaccine
US20100189741A1 (en) * 2007-04-20 2010-07-29 William Ripley Ballou Oil-in-water emulsion influenza vaccine
US9452209B2 (en) * 2007-04-20 2016-09-27 Glaxosmithkline Biologicals Sa Influenza vaccine
US9597389B2 (en) 2007-04-20 2017-03-21 Glaxosmithkline Biologicals Sa Oil-in-water emulsion influenza vaccine
US20090136543A1 (en) * 2007-04-20 2009-05-28 William Ripley Ballou Vaccine
US9211320B2 (en) 2008-04-18 2015-12-15 Vaxinnate Corporation Deletion mutants of flagellin and methods of use
US9205138B2 (en) 2008-04-18 2015-12-08 Vaxinnate Corporation Deletion mutants of flagellin and methods of use
US20130095130A1 (en) * 2010-01-06 2013-04-18 Vaxinnate Corporation Methods and Compositions for Providing Protective Immunity in the Elderly

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