WO2018073340A1 - Vaccin contre le virus de la grippe - Google Patents

Vaccin contre le virus de la grippe Download PDF

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Publication number
WO2018073340A1
WO2018073340A1 PCT/EP2017/076705 EP2017076705W WO2018073340A1 WO 2018073340 A1 WO2018073340 A1 WO 2018073340A1 EP 2017076705 W EP2017076705 W EP 2017076705W WO 2018073340 A1 WO2018073340 A1 WO 2018073340A1
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variant
influenza
subtype
amino acids
entire length
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PCT/EP2017/076705
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English (en)
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Edmond J. REMARQUE
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Redbiotec Ag
European Vaccine Initiative
Instituto De Biologia Experimental E Tecnológica (Ibet)
Etna Biotech S.R.L.
Biomedical Primate Research Centre
Stichting Wageningen Research (Wageningen Bioveterinary Research (Wbvr))
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Publication of WO2018073340A1 publication Critical patent/WO2018073340A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N2760/16123—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N2760/16223—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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 vaccine compositions comprising at least three variants of a subtype of an influenza virus antigen, wherein the at least three variants are selected on the basis of differences between their amino acid sequences.
  • the present invention further relates to said vaccine compositions for use in a method of generating an immune response against influenza viruses. Further provided is a method for producing said vaccine compositions.
  • Human influenza (human flu) is a highly contagious respiratory disease typically starting with an abrupt onset of fever, sore throat, blocked or running nose, headache, photophobia, dry cough and malaise. It gives rise to repeating and frequent epidemics and pandemics that occur suddenly, causing substantial morbidity and mortality.
  • the first recorded influenza pandemic dates back to 1580. Over the course of history there have been several influenza pandemics that have sickened and killed millions. Most cases of death have been found to be a result of an increased physiologic load in an already compromised host, or to be the outcome of the combined effects of the viral disease and a secondary bacterial infection.
  • the 1918 influenza virus, called the "Spanish flu” was particularly lethal, accounting for more than 40 million deaths worldwide and this was when rapid air travel was much less common. Albeit this strain caused pneumonia, also in this pandemic most deaths were associated with secondary bacterial pathogens.
  • Influenza viruses are RNA viruses that replicate their genome in the nucleus of the host cell. They belong to the family Orthomyxoviridae and are divided into three genera A, B and C, which can be distinguished by antigenic differences in two of the structural proteins of the virus, the matrix protein M2 and the nucleoprotein. Each of these types has many strains. These are enveloped viruses with a segmented genome containing seven or eight single- stranded segments of negative-sense RNA. Each of these RNA segments contains one or two genes. The genomes of influenza A and influenza B virus consist of eight RNA segments, which are coding for 12 viral proteins (Steinhauer, D.A. & Skehel, J.J., Ann. Rev. Genet.
  • the three largest gene segments of influenza A virus encode the subunits of the viral polymerase, PB2, PB1 , and PA.
  • the fourth segment encodes the hemagglutinin glycoprotein (HA), responsible for binding to cell-surface receptors and membrane fusion, and the fifth gene segment encodes the nucleoprotein (NP), which encapsidates cRNAs and vRNAs, which allows them to be recognized as templates for the viral polymerase.
  • HA hemagglutinin glycoprotein
  • NP nucleoprotein
  • Segment 6 encodes the neuraminidase (NA), which cleaves sialic acid from virus and host cell glycoconjugates to allow mature virus particles to be released.
  • the seventh segment generates two gene products, the matrix protein, M1 , and the M2 transmembrane protein, which has proton channel activity.
  • M1 matrix protein
  • M2 transmembrane protein
  • influenza B virus this segment encodes matrix protein M1 and BM2, thought to be a functional counterpart of M2.
  • the eighth gene segment encodes the protein NS1 , which inter alia sequesters ds RNA formed during virus replication, and the nuclear export protein (NEP).
  • NEP nuclear export protein
  • HA hemagglutinin surface proteins
  • NA neuraminidase
  • pandemic influenza A virus candidates exist. Once an influenza virus has become a seasonal virus, usually after a pandemic, it is going to drift or change over time.
  • Several seasonal viruses are presently in co-circulation: An influenza A virus of the subtype H3N2, and another of the subtype H1 N1 , and two influenza virus type B strains from the Yamagata and the Victoria lineages. After the recent swine flu pandemic, the new variant of the influenza A H1 N1 subtype (vH1 N1 or H1 N1 new) became the new seasonal H1 N1 strain.
  • Influenza B and C viruses can infect only humans, although there have been reports of influenza B virus isolation from seals and influenza C virus isolation from pigs. In contrast thereto, Influenza A viruses can infect both mammals and birds. The most devastating flu viruses of the 20th century, the Spanish flu pandemic in 1918 (H1 N1 ), the Asian flu pandemic in 1957 (H2N2) and the Hong Kong flu pandemic in 1968 (H3N2), were all of avian origin. Aquatic birds are natural reservoirs of influenza A viruses. These viruses are known to cross the species barrier and cause either transitory infections or establish permanent lineages in mammals including man. While influenza B viruses do not have pandemic potential, they cause significant disease and are the predominant circulating strain of influenza virus approximately one in every 3 years.
  • Influenza B virus is therefore an essential component of the influenza vaccine administered to susceptible groups such as the elderly and asthmatic.
  • approaches for pandemic influenza vaccines, as well as seasonal influenza vaccines warrant a combination of several influenza viruses.
  • pre-pandemic vaccines a combination of several strains into one vaccine candidate is indicated to either prime against several viruses simultaneously in a pre-pandemic setting or to limit a stockpile to a few vaccines (vaccine library), but each vaccine with a multivalent option, i.e. being protective against several strains to increase its potential.
  • seasonal vaccines should cover at least three strains, two A-strains and one B-strain.
  • pandemic vaccine In the case of a pandemic vaccine, generally the entire population should be administered with the vaccine, while in the case of a seasonal vaccine, primarily risk groups such as young children and the elderly should be administered with the vaccine.
  • priming in naive populations an induction of immunity as similar as possible to the wild type virus infection in regard to internal and external antigens is desired.
  • pre-existing immunity should not prohibit a sufficient booster response.
  • Priming can consist of one or several doses (a priming schedule) and boosting most often of only one vaccination.
  • the principal mechanism of action of current subunit or inactivated, detergent-disrupted influenza virus vaccines is to induce neutralizing antibodies (Doherty et al. 2008, The Journal of Clinical Investigation 1 18, 3273-3275).
  • influenza virus antigens such as HA or NA
  • HA or NA have variants and these variants are rather polymorph, i.e., they differ among each other in their amino acid sequence. All the more, these already polymorphic variants underlie the phenomenon of antigenic drift and antigenic shift. While influenza viruses are changing by antigenic drift all the time, antigenic shift happens only occasionally.
  • antigenic drift small changes in, e.g. the HA gene of influenza viruses happen continually over time as the virus replicates. These small genetic changes usually produce viruses that are rather closely related to one another. However, these small genetic changes can accumulate over time and result in viruses that are antigenically different (further away on the phylogenetic tree). When this happens, the immune system may no longer recognize those viruses. This is also why influenza vaccine composition must be reviewed each year, and updated as needed to keep up with evolving viruses. Antigenic shift is an abrupt, major change in the influenza viruses, resulting in new HA and/or NA proteins in influenza viruses.
  • Shift results in a new influenza subtype or a virus with a hemagglutinin or a hemagglutinin and neuraminidase combination that has emerged from an animal population that is so different from the same subtype in humans that most people do not have immunity to the new (e.g. novel) virus.
  • Such a "shift” occurred in the spring of 2009, when an H1 N1 virus with a new combination of genes emerged to infect people and quickly spread, causing a pandemic. When shift happens, most people have little or no protection against the new virus.
  • the technical problem underlying the present invention is to provide influenza vaccine compositions which are able to induce (cross)protection against influenza virus infections due to increased antibody breadth, yielding protection not only to the influenza variants of a subtype of an influenza virus antigen represented in the vaccine compositions, but also to variants of said subtype not included in the vaccine composition.
  • the present invention provides a solution to the technical problem of providing novel influenza vaccine compositions which are able to induce protection against influenza virus infections, wherein protection is not restricted to the specific variants of an influenza subtype represented in the vaccine compositions, but also to variants not included in the vaccine composition.
  • the present inventor found that immunization with a mixture of variants of a subtype of an influenza virus antigen yielded functional antibody levels to all variants comparable to levels induced by monovalent immunization and also to variants not included in the vaccine composition.
  • the mechanism behind the observed broadening was shown to be an increase in the fraction of cross-reactive antibodies, most likely because variant- specific epitopes are present at lower frequency relative to conserved epitopes. In other words, it is assumed that the broadening of the antibody response is due to the increased relative concentration of common epitopes diluting out variant specific epitopes.
  • the present inventor thereby developed the so-called epitope dilution phenomenon (EDiP) as a practical strategy for the induction of broad, cross-variant antibody responses against polymorphic antigens.
  • EDP epitope dilution phenomenon
  • the present inventor developed certain rules for the choice of variants of a subtype of an influenza virus antigen, such as HA or NA, with the aim of "educating" the immune system by a combinatorial immunization strategy to be able to recognize a broad range of variants of a subtype of an influenza virus antigen which also includes potential new subtypes.
  • the present invention demonstrates for influenza virus H1 , H3 and B strains that the immune response was broadened beyond the variants of the subtype included in a vaccine composition, i.e. strains isolated -10 years after the last strain included in the vaccine are neutralised by mouse sera.
  • H1 strains the following HA variants were used:
  • A/Brisbane/59/2007 (SEQ ID No: 5).
  • a broadening against A California/04/2009 (SEQ ID No: 6) or A/California/07/2009 (SEQ ID No: 7) or A/Mexico/INDRE4487/2009 (SEQ ID No: 8) can be expected. It may, however, not yet be measurable, since is assumed that the micro neutralization (MN) assay may lack sensitivity to detect A/California/4/2009 and/or A/Mexico/INDRE4487/2009 responses in mouse serum as has been previously observed with mini HA stem antigen constructs (Impagliazzo et al. (2015), Science 349(6254), 101-106).
  • MN micro neutralization
  • the inventor of the present application found that it is possible to broaden the scope of protection conferred to a subject upon vaccination compared to the scope of protection conferred by current influenza vaccines.
  • a specific combination of variants of a subtype of an influenza virus antigen is provided in an influenza vaccine composition, said specific combination is based on the rules for choosing variants as provided herein.
  • Immunity conferred by the vaccine composition of the present invention is advantageously not limited to variants which are actually represented in the vaccine composition. Rather, the vaccine composition of the present invention also confers immunity against other variants which are not included in the vaccine composition. These variants could have been circulating before or at the time point of vaccination of a subject.
  • the vaccine composition of the present invention may even confer protection against future variants which have not yet been circulating at the time of providing or administering the vaccine composition. Such future influenza virus strains might arise, e.g., from antigenic shift or antigenic drift.
  • the present invention provides a vaccine composition
  • a vaccine composition comprising
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza A virus antigen differ from each other by at least 10 amino acids over their entire length; and (iii) a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 35 amino acids over their entire length; and/or (b) at least three variants of a subtype of an influenza B virus antigen, wherein
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 20 amino acids over their entire length.
  • the first and second variant of a subtype of an influenza A virus antigen differ from each other by no more than 60, 55, 50, 45, 40, 35, or 30 amino acids over their entire length.
  • the first and third variant of a subtype of an influenza A virus antigen differ from each other by no more than 70, 65, 60, 55, or 50 amino acids over their entire length.
  • the first and second variant of a subtype of an influenza B virus antigen differ from each other by no more than 60, 55, 50, 45, 40, 35, or 30 amino acids over their entire length.
  • the first and third variant of a subtype of an influenza B virus antigen differ from each other by no more than 70, 65, 60, 55, 50, 45, 40 or 35 amino acids over their entire length.
  • the present invention also provides a vaccine composition as described herein for use in a method of generating an immune response against influenza virus A and/or B.
  • the present invention provides a vaccine composition for use in a method of effecting cross-neutralization against at least one variant of said subtype of said influenza virus A and/or B antigen which is different from said variants comprised in said vaccine.
  • the present invention provides the use of at least three variants of a subtype of an influenza A virus antigen, wherein
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza A virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 35 amino acids over their entire length
  • the present invention provides the use of at least three variants of a subtype of an influenza B virus antigen, wherein
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 20 amino acids over their entire length for the manufacture of a vaccine composition which effects cross-neutralization against at least one variant of said subtype of said influenza B virus antigen which is different from said variants comprised in said vaccine.
  • the present invention provides a method for producing a vaccine composition against influenza A virus, comprising
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 10 amino acids over their entire length
  • a second and third variant of a subtype of an influenza A virus antigen differ from each other by at least 10 amino acids over their entire length
  • a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 35 amino acids over their entire length
  • the present invention provides a method for producing a vaccine composition against influenza B virus, comprising
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 20 amino acids over their entire length
  • FIG. 7 Overview of the constructs for expression of VLPs. DETAILED DESCRIPTION OF THE INVENTION
  • such antigenic diversity can be manifested by simultaneous circulation of different strains of the pathogen and/or by frequent occurrence of minor and/or major changes in the structure of relevant antigens.
  • adaptive immunity resulting from vaccination is mostly limited to those influenza strains that have been represented in the vaccine by including in the vaccine viral antigens derived from these strains. Consequently, current influenza vaccines confer only surroundnarrow" immunity against influenza infections caused by a small number of influenza strains.
  • the present inventors found that the vaccine composition according to the present invention confers immunity against influenza infection which is bonnebroader" than protection conferred by current influenza vaccines.
  • the vaccine composition according to the present invention is useful in generating immunity both against influenza strains that are represented in the vaccine composition and against influenza strains that are not represented in the vaccine composition. This is achieved by specifically selecting variants of a subtype of an influenza virus antigen according to the explanations set out in the present application, and combining these variants in a vaccine composition.
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza A virus antigen differ from each other by at least 10 amino acids over their entire length; and (iii) a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 35 amino acids over their entire length;
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 20 amino acids over their entire length.
  • Influenza virus is a member of the orthomyxoviridae family. There are three subtypes of influenza viruses, designated influenza A, influenza B, and influenza C. The present invention focuses on influenza A and/or influenza B virus. Thus, when reference is made herein, e.g. to influenza virus, influenza virus vaccine or the like, preferably influenza A virus and/or influenza B virus is meant. The skilled person will understand from the context in which influenza A virus or influenza B virus, respectively, is used whether influenza A virus, influenza B virus, or both are meant.
  • the influenza virion contains a segmented negative- sense RNA genome, which encodes the following proteins: hemagglutinin (HA), neuraminidase (NA), matrix (Ml), proton ion- channel protein (M2), nucleoprotein (NP), polymerase basic protein 1 (PB 1 ), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), and nonstructural protein 2 (NS2).
  • HA hemagglutinin
  • NA neuraminidase
  • Ml matrix
  • NP proton ion- channel protein
  • NP nucleoprotein
  • PB 1 polymerase basic protein 1
  • PB2 polymerase basic protein 2
  • PA polymerase acidic protein
  • NS2 nonstructural protein 2
  • the HA, NA, Ml, and M2 are membrane associated, whereas NP, PB1 , PB2, PA, and NS2 are nucleocapsid associated proteins.
  • the Ml protein is the most abundant protein in
  • the HA and NA proteins are envelope glycoproteins, responsible for virus attachment and penetration of the viral particles into the cell, and the sources of the major immunodominant epitopes for virus neutralization and protective immunity. Both HA and NA proteins are considered the most important components for prophylactic influenza vaccines.
  • Influenza A viruses infect a wide variety of subjects including fowls and mammals, including, but not limited to humans, horses, marine mammals, pigs, ferrets, and chicken, ducks, birds, gooses, etc. In animals, most influenza A viruses cause mild localized infections of the respiratory and intestinal tract. However, highly pathogenic influenza A strains, such as H5N1 , cause systemic infections in poultry in which mortality may reach 100%. Animals infected with influenza A often act as a reservoir for the influenza viruses and certain subtypes have been shown to cross the species barrier to humans.
  • influenza virus antigen when used herein refers to a protein from influenza virus which elicits an immune response by a subject as mentioned herein.
  • the immune response may be a cellular immune response or a humoral immune response or both. In the context of the present invention, it may rather be a humoral immune response.
  • influenza virus comprises six additional internal genes, which give rise to eight different proteins, including polymerase genes PB1 , PB2 and PA, matrix proteins M1 and M2, nucleoprotein (NP), and non- structural proteins NS1 and NS2 (Horimoto et al., Clin Microbiol Rev. 14(1 ): 129-149, 2001 ).
  • HA, NA, PB1 , PB2, PA, M1 , M2, NP may be an influenza virus antigen.
  • an influenza virus antigen is a surface glycoprotein, which is preferably, hemagglutinin (HA) and/or neuraminidase (NA).
  • Influenza A viruses can be classified into subtypes based on allelic variations in antigenic regions of two genes that encode surface glycoproteins, namely, hemagglutinin (HA) and neuraminidase (NA) which are required for viral attachment and cellular release.
  • HA hemagglutinin
  • NA neuraminidase
  • subtype or “subtype of an influenza virus antigen” refers to allelic variations in antigenic regions of two genes that encode surface glycoproteins, namely, hemagglutinin (HA) and neuraminidase (NA).
  • HA hemagglutinin
  • NA neuraminidase
  • influenza virus preferably influenza A virus or influenza B virus, respectively.
  • influenza A virus preferably influenza A virus or influenza B virus, respectively.
  • influenza B virus preferably influenza B virus, respectively.
  • influenza B virus is usually not classified into subtypes, but lineages, the term "subtype” is also used for influenza B virus hemagglutinin (HA) and neuraminidase (NA).
  • HA hemagglutinin
  • NA neuraminidase
  • HA is a viral surface glycoprotein generally comprising on average approximately 560 amino acids and representing 25% of the total virus protein. It is responsible for adhesion of the viral particle to, and its penetration into, a host cell in the early stages of infection.
  • NA Neuraminidase
  • NA is a second membrane glycoprotein of the influenza viruses.
  • NA is 413 amino acid in length, and is encoded by a gene of 1413 nucleotides.
  • NA is involved in the destruction of the cellular receptor for the viral HA by cleaving terminal neuraminic acid (also called sialic acid) residues from carbohydrate moieties on the surfaces of infected cells.
  • NA also cleaves sialic acid residues from viral proteins, preventing aggregation of viruses.
  • influenza virus antigen any subtype of an influenza virus antigen can be used for providing a vaccine composition according to the present invention.
  • the influenza virus antigen is HA
  • the subtype can be any one of H1 , H2, H3, H4, H5, H6, H7, H8, H9, H10, H1 1 , H12, H13, H14, H15, H16, H17 or H18 according to WHO.
  • the HA subtype is H1 or H3 or B.
  • the subtype can be any one of NA1 , NA2, NA3, NA4, NA5, NA6, NA7, NA8, NA9, NA10, or NA1 1 according to WHO.
  • the NA subtype is NA1.
  • the term ..variant” or ..variant of a subtype of an influenza virus antigen refers to an amino acid sequence variant of a subtype of an influenza virus antigen.
  • variants differ from each other by at least one amino acid over their entire length.
  • Such variants may result from any known mechanism.
  • the variants may naturally occur, i.e. they may be associated with circulating influenza strains. It is also possible that the variants are newly generated, e.g., by antigenic drift, antigenic shift, antigenic diversity or by genetic manipulation. As such, the variants can be the result of targeted genetic manipulation or random genetic manipulation.
  • An "influenza virus strain " or, as also used herein in the context of influenza virus the term “strain” refers to an influenza virus that is characterized by its HA and NA subytoe, e.g. H1 N1 or H3N2.
  • the first variant is more ancient than the second and third variant and the second variant is more ancient than the third variant.
  • the first variant has been circulating before the second and third variant
  • the second variant has been circulating before the third variant.
  • variants designated by a higher number are preferably more modern (in terms of time) than variants designated by a lower number. Consequently, if the vaccine composition comprises more than three variants, any additional variants, e.g., a fourth, fifth, sixth, seventh, eighth, ninth or tenth variant, are preferably more modern than any variant designated by a lower number.
  • a vaccine composition may further include a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the carrier and composition can be sterile, and the formulation suits the mode of administration.
  • the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium.
  • first, second and third variant of a subtype of an influenza A virus antigen are chosen according to the following rule:
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 12 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 35 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 1 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 35 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 16 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza A virus antigen differ from each other by at least 16 amino acids over their entire length; and (iii) a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 35 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 18 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza A virus antigen differ from each other by at least 18 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 35 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 20 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza A virus antigen differ from each other by at least 20 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 35 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza A virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 40 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 12 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 40 amino acids over their entire length.
  • the at least three variants of a subtype of an influenza A virus antigen are chosen according to the following rule:
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 14 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 40 amino acids over their entire length.
  • the at least three variants of a subtype of an influenza A virus antigen are chosen according to the following rule:
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 16 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza A virus antigen differ from each other by at least 16 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 40 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 18 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza A virus antigen differ from each other by at least 18 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 40 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 20 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza A virus antigen differ from each other by at least 20 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 40 amino acids over their entire length.
  • the at least three variants of a subtype of an influenza A virus antigen are chosen according to the following rule: (i) a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza A virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 45 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 12 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 45 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 14 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 45 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 16 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza A virus antigen differ from each other by at least 16 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 45 amino acids over their entire length.
  • the at least three variants of a subtype of an influenza A virus antigen are chosen according to the following rule:
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 18 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza A virus antigen differ from each other by at least 18 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 45 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 20 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza A virus antigen differ from each other by at least 20 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 45 amino acids over their entire length.
  • the first and second variant of a subtype of an influenza A virus antigen differ from each other by no more than 60, 55, 50, 45, 40, 35, or 30 amino acids over their entire length.
  • the first and third variant of a subtype of an influenza A virus antigen differ from each other by no more than 70, 65, 60, 55, or 50 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 12 amino acids over their entire length and
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 14 amino acids over their entire length and (ii) differs from the first variant by at least 40 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 16 amino acids over their entire length and (ii) differs from the first variant by at least 40 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 18 amino acids over their entire length and (ii) differs from the first variant by at least 40 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 20 amino acids over their entire length and (ii) differs from the first variant by at least 40 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 10 amino acids over their entire length and (ii) differs from the first variant by at least 45 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 12 amino acids over their entire length and (ii) differs from the first variant by at least 45 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 14 amino acids over their entire length and (ii) differs from the first variant by at least 45 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 16 amino acids over their entire length and (ii) differs from the first variant by at least 45 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 18 amino acids over their entire length and (ii) differs from the first variant by at least 45 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 20 amino acids over their entire length and (ii) differs from the first variant by at least 45 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 10 amino acids over their entire length and (ii) differs from the first variant by at least 50 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 12 amino acids over their entire length and (ii) differs from the first variant by at least 50 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 14 amino acids over their entire length and (ii) differs from the first variant by at least 50 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 16 amino acids over their entire length and (ii) differs from the first variant by at least 50 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 18 amino acids over their entire length and (ii) differs from the first variant by at least 50 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 20 amino acids over their entire length and (ii) differs from the first variant by at least 50 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 10 amino acids over their entire length and (ii) differs from the first variant by at least 55 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 12 amino acids over their entire length and (ii) differs from the first variant by at least 55 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 14 amino acids over their entire length and (ii) differs from the first variant by at least 55 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 16 amino acids over their entire length and (ii) differs from the first variant by at least 55 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 18 amino acids over their entire length and (ii) differs from the first variant by at least 55 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza A virus antigen, wherein the fourth variant (i) differs from the third variant by at least 20 amino acids over their entire length and (ii) differs from the first variant by at least 55 amino acids over their entire length.
  • the third and fourth variant of a subtype of an influenza A virus antigen differ from each other by no more than 60, 55, 50, 45, 40, 35, or 30 amino acids over their entire length.
  • the first and fourth variant of a subtype of an influenza A virus antigen differ from each other by no more than 70, 65 or 60 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza A virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 1 1 amino acids over their entire length and (ii) differs from the first variant by at least 50 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza A virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 12 amino acids over their entire length and (ii) differs from the first variant by at least 50 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza A virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 15 amino acids over their entire length and (ii) differs from the first variant by at least 50 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza A virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 20 amino acids over their entire length and (ii) differs from the first variant by at least 50 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza A virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 10 amino acids over their entire length and (ii) differs from the first variant by at least 55 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza A virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 1 1 amino acids over their entire length and (ii) differs from the first variant by at least 55 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza A virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 12 amino acids over their entire length and (ii) differs from the first variant by at least 55 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza A virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 15 amino acids over their entire length and (ii) differs from the first variant by at least 55 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza A virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 20 amino acids over their entire length and (ii) differs from the first variant by at least 55 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza A virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 10 amino acids over their entire length and (ii) differs from the first variant by at least 60 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza A virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 1 1 amino acids over their entire length and (ii) differs from the first variant by at least 60 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza A virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 12 amino acids over their entire length and (ii) differs from the first variant by at least 60 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza A virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 15 amino acids over their entire length and (ii) differs from the first variant by at least 60 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza A virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 20 amino acids over their entire length and (ii) differs from the first variant by at least 60 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza A virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 10 amino acids over their entire length and (ii) differs from the first variant by at least 65 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza A virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 1 1 amino acids over their entire length and (ii) differs from the first variant by at least 65 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza A virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 12 amino acids over their entire length and (ii) differs from the first variant by at least 65 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza A virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 15 amino acids over their entire length and (ii) differs from the first variant by at least 65 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza A virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 20 amino acids over their entire length and (ii) differs from the first variant by at least 65 amino acids over their entire length.
  • the fourth and fifth variant of a subtype of an influenza A virus antigen differ from each other by no more than 60, 55, 50, 45, 40, 35 or 30 amino acids over their entire length.
  • the first and fifth variant of a subtype of an influenza A virus antigen differ from each other by no more than 90, 85, 80, 75 or 70 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a sixth, seventh, eighth, ninth or tenth variant of a subtype of an influenza A virus antigen or several of these variants. It is envisioned that the sixth variant of a subtype of an influenza A virus antigen differs from the fifth variant by at least 10 or 20 amino acids, but by no more than 50 or 40 amino acids, e.g. 10-40, 20-40, 10-50 or 20-50 amino acids difference.
  • the seventh variant of a subtype of an influenza A virus antigen differs from the sixth variant by at least 10 or 20 amino acids, but by no more than 50 or 40 amino acids, e.g. 10-40, 20-40, 10-50 or 20-50 amino acids difference.
  • the eighth variant of a subtype of an influenza A virus antigen differs from the seventh variant by at least 10 or 20 amino acids, but by no more than 50 or 40 amino acids, e.g. 10-40, 20-40, 10-50 or 20-50 amino acids difference.
  • the ninth variant of a subtype of an influenza A virus antigen differs from the eighth variant by at least 10 or 20 amino acids, but by no more than 50 or 40 amino acids, e.g. 10-40, 20-40, 10-50 or 20-50 amino acids difference.
  • the tenth variant of a subtype of an influenza A virus antigen differs from the ninth variant by at least 10 or 20 amino acids, but by no more than 50 or 40 amino acids, e.g. 10-40, 20-40, 10-50 or 20-50 amino acids difference.
  • the number of variants of the subtype of said influenza A virus antigen exceeds the number of variants of the subtype of said influenza virus antigens that is comprised in said vaccine.
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 12 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 12 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 20 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 14 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 14 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 20 amino acids over their entire length.
  • the at least three variants of a subtype of an influenza B virus antigen are chosen according to the following rule: (i) a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 16 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 16 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 20 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 18 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 18 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 20 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 20 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 20 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 20 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 25 amino acids over their entire length.
  • the at least three variants of a subtype of an influenza B virus antigen are chosen according to the following rule:
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 12 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 12 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 25 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 14 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 14 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 25 amino acids over their entire length.
  • the at least three variants of a subtype of an influenza B virus antigen are chosen according to the following rule:
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 16 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 16 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 25 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 18 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 18 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 25 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 20 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 20 amino acids over their entire length; and (iii) a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 25 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 30 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 12 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 12 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 30 amino acids over their entire length.
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 14 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 1 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 30 amino acids over their entire length.
  • the at least three variants of a subtype of an influenza B virus antigen are chosen according to the following rule:
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 16 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 16 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 30 amino acids over their entire length.
  • the at least three variants of a subtype of an influenza B virus antigen are chosen according to the following rule:
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 18 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 18 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 30 amino acids over their entire length.
  • the at least three variants of a subtype of an influenza B virus antigen are chosen according to the following rule:
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 20 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 20 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 30 amino acids over their entire length.
  • the first and second variant of a subtype of an influenza B virus antigen differ from each other by no more than 60, 55, 50, 45, 40, 35, or 30 amino acids over their entire length.
  • the first and third variant of a subtype of an influenza B virus antigen differ from each other by no more than 70, 65, 60, 55, 50, 45, 40 or 35 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 12 amino acids over their entire length and (ii) differs from the first variant by at least 20 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 14 amino acids over their entire length and (ii) differs from the first variant by at least 20 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 16 amino acids over their entire length and (ii) differs from the first variant by at least 20 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 18 amino acids over their entire length and (ii) differs from the first variant by at least 20 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 20 amino acids over their entire length and (ii) differs from the first variant by at least 20 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 10 amino acids over their entire length and (ii) differs from the first variant by at least 25 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 12 amino acids over their entire length and (ii) differs from the first variant by at least 25 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 1 amino acids over their entire length and (ii) differs from the first variant by at least 25 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 16 amino acids over their entire length and (ii) differs from the first variant by at least 25 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 18 amino acids over their entire length and (ii) differs from the first variant by at least 25 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 20 amino acids over their entire length and (ii) differs from the first variant by at least 25 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 10 amino acids over their entire length and (ii) differs from the first variant by at least 30 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 12 amino acids over their entire length and (ii) differs from the first variant by at least 30 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 14 amino acids over their entire length and (ii) differs from the first variant by at least 30 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 16 amino acids over their entire length and (ii) differs from the first variant by at least 30 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 18 amino acids over their entire length and (ii) differs from the first variant by at least 30 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 20 amino acids over their entire length and (ii) differs from the first variant by at least 30 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 10 amino acids over their entire length and (ii) differs from the first variant by at least 35 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 12 amino acids over their entire length and (ii) differs from the first variant by at least 35 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 14 amino acids over their entire length and (ii) differs from the first variant by at least 35 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 16 amino acids over their entire length and (ii) differs from the first variant by at least 35 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 18 amino acids over their entire length and (ii) differs from the first variant by at least 35 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fourth variant of a subtype of an influenza B virus antigen, wherein the fourth variant (i) differs from the third variant by at least 20 amino acids over their entire length and (ii) differs from the first variant by at least 35 amino acids over their entire length.
  • the third and fourth variant of a subtype of an influenza B virus antigen differ from each other by no more than 60, 55, 50, 45, 40, 35, or 30 amino acids over their entire length.
  • the first and fourth variant of a subtype of an influenza B virus antigen differ from each other by no more than 70, 65 or 60 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza B virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 12 amino acids over their entire length and (ii) differs from the first variant by at least 30 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza B virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 15 amino acids over their entire length and (ii) differs from the first variant by at least 30 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza B virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 20 amino acids over their entire length and (ii) differs from the first variant by at least 30 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza B virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 10 amino acids over their entire length and (ii) differs from the first variant by at least 35 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza B virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 12 amino acids over their entire length and (ii) differs from the first variant by at least 35 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza B virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 15 amino acids over their entire length and (ii) differs from the first variant by at least 35 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza B virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 20 amino acids over their entire length and (ii) differs from the first variant by at least 35 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza B virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 10 amino acids over their entire length and (ii) differs from the first variant by at least 40 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza B virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 12 amino acids over their entire length and (ii) differs from the first variant by at least 40 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza B virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 15 amino acids over their entire length and (ii) differs from the first variant by at least 40 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a fifth variant of a subtype of an influenza B virus antigen, wherein the fifth variant (i) differs from the fourth variant by at least 20 amino acids over their entire length and (ii) differs from the first variant by at least 40 amino acids over their entire length.
  • the fourth and fifth variant of a subtype of an influenza B virus antigen differ from each other by no more than 60, 55, 50, 45, 40, 35 or 30 amino acids over their entire length.
  • the first and fifth variant of a subtype of an influenza B virus antigen differ from each other by no more than 100, 90, 85, 80, 75, 70, 65, 55 or 50 amino acids over their entire length.
  • the vaccine composition according to the present invention comprises a sixth, seventh, eighth, ninth or tenth variant of a subtype of an influenza B virus antigen or several of these variants.
  • the sixth variant of a subtype of an influenza B virus antigen differs from the fifth variant by at least 10 or 20 amino acids, but by no more than 50 or 40 amino acids, e.g. 10-40, 20-40, 10-50 or 20-50 amino acids difference.
  • the seventh variant of a subtype of an influenza B virus antigen differs from the sixth variant by at least 10 or 20 amino acids, but by no more than 50 or 40 amino acids, e.g. 10-40, 20-40, 10-50 or 20-50 amino acids difference.
  • the eighth variant of a subtype of an influenza B virus antigen differs from the seventh variant by at least 10 or 20 amino acids, but by no more than 50 or 40 amino acids, e.g. 10-40, 20-40, 10-50 or 20-50 amino acids difference. It is envisioned that the ninth variant of a subtype of an influenza B virus antigen differs from the eighth variant by at least 10 or 20 amino acids, but by no more than 50 or 40 amino acids, e.g. 10-40, 20-40, 10-50 or 20-50 amino acids difference.
  • the tenth variant of a subtype of an influenza B virus antigen differs from the ninth variant by at least 10 or 20 amino acids, but by no more than 50 or 40 amino acids, e.g. 10-40, 20-40, 10-50 or 20-50 amino acids difference.
  • the number of variants of the subtype of said influenza B virus antigen exceeds the number of variants of the subtype of said influenza virus antigens that is comprised in said vaccine.
  • the subtype is H1 and the vaccine composition comprises the following five variants of H1 HA:
  • the subtype is H3 and the vaccine composition comprises the following five variants of H3 HA:
  • the subtype is B and the vaccine composition comprises the following five variants of B HA:
  • the subtype is NA and the vaccine composition comprises the following three variants of N1 NA:
  • the vaccine composition may comprise one or more of these N1 variants.
  • the subtype is H4 and the vaccine composition comprises the following five variants of HA:
  • H7 A/cinnamon teal/Bolivia/4537/2001 (SEQ ID No: 35),
  • H14 A/herring gull/Astrakhan/267/1982 (SEQ ID No: 37), and/or
  • H15 A/Australian shelduck/Western Australia/1762/1979 (SEQ ID No: 38). It is also envisioned that the vaccine composition may comprise one or more of these H4, H7, H10, H14, H15 variants.
  • Sequence alignments Differences between amino acid sequences of variants as referred to herein may be translated into a certain degree of “identity”.
  • % identity is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues by the total number of residues and gaps and multiplying the product by 100. Preferably, identity is determined over the entire length of the sequences being compared. "Gaps" are spaces in an alignment that are the result of additions or deletions of amino acids. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved, and have deletions, additions, or replacements, may have a lower degree of identity.
  • the preferred computer program for determining sequence identity is MUSCLE (multiple sequence alignment with high accuracy and high throughput); Edgar RC (2004) Nucleic Acids Res. 32: 1792-1797.
  • the MUSCLE program is, e.g. available at www.fludb.org.
  • the term "over their entire length” means that amino acid sequences of at least two variants of a subtype of influenza virus antigens, except for a signal sequence or pro-sequence that may be contained in the amino acid sequence of a variant, are aligned and inspected in order to determine the degree of identity as mentioned above.
  • H1 contains a 17 amino acid pro-sequence
  • H3 a 16 amino acid pro-sequence
  • HB a 15 amino acid pro-sequence.
  • pro- sequences from HA is readily in a position to determine the same in an HA polypeptide (see, e.g., Kovacova et al. (2002) Virus Genes 24:1 , 57-63)
  • the vaccine composition according to the present invention effects cross-neutralization against at least one variant of said subtype of said influenza virus A and/or B antigen that is different from said variants comprised in said vaccine.
  • Cross-neutralization means that serum obtained from a subject after immunization with a vaccine composition of the present invention is capable of neutralizing at least 25% infection, more preferably at least 50% infection of MDCK cells, as determined in an influenza microneutralization assay, caused by an influenza virus having a variant of a subtype of an influenza virus antigen which is different from a variants comprised in a vaccine composition of the present invention.
  • a micro neutralization assay is preferably performed as follows:
  • virus stocks are titrated.
  • a series of log 10 dilutions are made and 0.1 ml/well (10 wells per dilution) are added into flat-bottomed 96-well plates containing a monolayer of confluent MDCK cells. Plates are incubated at room temperature for 30 minutes before replacing inoculum with infection medium (DMEM containing 2mM glutamine, sodium bicarbonate , penicillin-streptomycin 1/100, amphotericin B and 0.0025 ⁇ g/ml TPCK trypsin). Plates are further incubated for 72 hours at 35°C. 50 ⁇ per well supernatants are harvested and run in HA assays using 0.7% turkey red blood cells. 50 % Tissue culture infectious doses (TCID 50 ) are calculated using the Spearman-Karber formula.
  • Sera samples are heat treated at 56°C for 50 minutes then added in duplicate into flat- bottomed 96-well plates using a starting dilution of 1/20, followed by a further seven doubling dilutions.
  • 10 2 TCID 50 (100 ⁇ ) viruses is then added into each well. Plates are incubated at room temperature for 1 hour before adding the mixtures to flat-bottomed 96-well plates containing a monolayer of confluent MDCK cells. After 30 minutes incubation at room temperature the serum-virus mixture is replaced with 100 ⁇ of infection media and incubated for 72 hours at 35°C. Supernatants are screened using an HA assay, as above. Serum neutralisation titres are expressed as the reciprocal of the highest dilution whereby 50% infection was prevented. Titres are the average of duplicate samples. Each assay run includes a back-titration of the viruses used and validation criteria of 10 2 +/- 10° 5 /100 ⁇ .
  • the variant against which cross-neutralization in an influenza microneutralization assay is effected differs from each variant comprised in the vaccine composition by at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or 70 or more amino acids over their entire length.
  • the variant of a subtype of an influenza A virus antigen against which cross- neutralization in an influenza microneutralization assay is effected differs from each variant of a subtype of an influenza A virus antigen comprised in the vaccine composition by at least 20 amino acids over their entire length.
  • Said variant of a subtype of an influenza A virus antigen against which cross-neutralization in an influenza microneutralization assay is effected may also differ from each variant of a subtype of an influenza A virus antigen comprised in the vaccine composition by at least 25, 30, 40, 50, 60, 70, 80, 90 or even 100 amino acids. However, it may not differ by more than 120 or preferably 1 10 amino acids.
  • the variant of a subtype of an influenza B virus antigen against which cross- neutralization in an influenza microneutralization assay is effected differs from each variant of a subtype of an influenza B virus antigen comprised in the vaccine composition by at least 15 amino acids over their entire length.
  • Said variant of a subtype of an influenza B virus antigen against which cross-neutralization in an influenza microneutralization assay is effected may also differ from each variant of a subtype of an influenza A virus antigen comprised in the vaccine composition by at least 16, 18, 20, 25, 30, 35, 40 or even 50 amino acids. However, it may not differ by more than 70 or preferably 60 amino acids.
  • the vaccine composition according to the present invention can provide vaccinated subjects with broad protection against influenza strains both circulating at the time of vaccination and predicted to arise in the future.
  • the vaccine of the present invention is envisaged to be useful in providing a "universal vaccine" both for seasonal and pandemic flu.
  • the vaccine composition according to the present invention provides a significant improvement of public health.
  • the vaccine composition according to the present invention is highly advantageous. Annual reformulation of influenza vaccines as required for control of seasonal flu is a highly cost-intensive process. Also the cost of annual distribution and administration to subjects is significant. Due to the fact that the vaccine composition according to the present invention provides vaccinated subjects with broad protection as described hereinbefore, it is no longer necessary to annually reformulate and re-administer the vaccine while still maintaining or even improving public health.
  • influenza virus antigen can be comprised in the vaccine composition in any form such as in the form of one or more proteins or in the form of one or more nucleic acids or combinations of both. It is also possible that the proteins or nucleic acids or combinations of both are present in the form of aggregates. If nucleic acids are used, the rules regarding the necessary amino acid differences between the corresponding variants can be applied in an analogous manner, in consideration of basic genetic principles.
  • the variants comprised in the vaccine composition are proteins. These proteins can be produced or isolated by any method know in the art. These proteins can be natural proteins or they can be artificial proteins such as genetically engineered proteins. In a specific embodiment, the variants are totally or partially comprised in a fusion protein. To create a fusion protein, the nucleic acid sequences must be in the same reading frame. For example, a fusion protein includes an influenza HA variant or NA variant fused to a heterologous protein.
  • the variants comprised in the vaccine composition are totally or partially immobilized on the surface of a virus-like particle (VLP).
  • VLP virus-like particle
  • a single VLP can comprise identical or different variants of a subtype of an influenza virus antigen.
  • each variant comprised in the vaccine composition can be present on a distinct population of VLPs (monovalent VLP), i.e. there are several VLP populations, each representing an individual variant.
  • several variants such as two, three, four, five, six, seven, eight, nine, ten or more variants can be present on the same VLP (polyvalent VLP), i.e. there is less than one VLP population per variant.
  • a specific VLP population can carry two, three, four, five, six, seven, eight, nine, ten or more variants on its surface, with five (pentavalent) being preferred.
  • a vaccine composition may comprise monovalent and polyvalent VLPs that have variants as described herein totally or partially immobilized on their surface.
  • VLPs Virus-like particles
  • They are well known. They are especially useful in the field of vaccination since they can be tailored to various applications. Furthermore, they do preferably not contain any viral genetic material, preventing their replication in host cells and rendering them non-infectious, which is preferred from a medical point of view in order to reduce the risk associated with vaccinations.
  • the main components of VLPs are viral structural proteins.
  • VLPs can often be produced by heterologous expression and can be easily purified. Most VLPs comprise at least a viral core protein that drives budding and release of particles from a host cell.
  • a viral core protein is influenza M1 .
  • Influenza VLPs can be produced by transfection of host cells with plasmids encoding the HA and NA proteins, and optionally the M1 protein. After incubation of the transfected cells for an appropriate time to allow for protein expression (such as for approximately 72 hours), VLPs can be isolated from cell culture supernatants.
  • the variants comprised in the vaccine composition are comprised in a scaffold.
  • An especially preferred scaffold is a virosome.
  • a virosome is a particle in which a membrane such as a phospholipid membrane encloses the virus derived proteins.
  • the membrane may comprise viral surface antigens such as HA and/or NA.
  • Virosomes are generally useful for protein delivery to target cells by membrane fusion.
  • the variants are comprised in a vector.
  • the influenza virus antigens may be expressed by said vector upon delivery to host cells.
  • the vector may be DNA-based or RNA-based.
  • the vector is a viral vector.
  • the concept of including adjuvants in vaccine compositions is well known in the field of vaccination.
  • the presence of adjuvants in a vaccine composition may allow to minimize the amount of antigenic material to be administered while maintaining stimulation of an immune response sufficient for inducing immunity against the antigenic material, e.g., by inducing generation of specific antibodies.
  • the vaccine composition may further comprise one or more adjuvants.
  • An "adjuvant" shall mean any agent suitable for enhancing the immunogenicity of an antigen and boosting an immune response in a subject.
  • adjuvants including particulate adjuvants, suitable for use with both protein- and nucleic acid-based vaccines, and methods of combining adjuvants with antigens, are well known to those skilled in the art.
  • Adjuvants suitable for use with protein immunization include, but are not limited to, alum, Freund's incomplete adjuvant (FIA), saponin, Quil A, and QS-21 .
  • Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants.
  • Adjuvants also include biological molecules, such as costimulatory molecules.
  • Exemplary biological adjuvants include IL-2, ANTES, GM-CSF, TNF-a, IFN- ⁇ , G-CSF, LFA-3, CD72, B7-1 , B7-2, OX-40L and 4-1 BBL.
  • the vaccine compositions according to the present invention may be administered by any known route of administration.
  • Exemplary routes of administration of a vaccine composition of the invention include oral, transdermal, and parenteral delivery. Suitable routes of administration may, for example, include depot, oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.
  • the vaccine compositions according to the present invention are useful in prevention and/or treatment of influenza infections.
  • the vaccine composition according to the present invention can be used in a method of generating an immune response against influenza virus in a subject.
  • the vaccine composition can be used in a method of effecting cross- neutralization against at least one variant of said subtype of said influenza A and/or B virus antigen which is different from said variants comprised in said vaccine.
  • the variant of a subtype of an influenza A virus antigen against which cross- neutralization in an influenza microneutralization assay is effected differs from each variant of a subtype of an influenza A virus antigen comprised in the vaccine composition by at least 20 amino acids over their entire length.
  • Said variant of a subtype of an influenza A virus antigen against which cross-neutralization in an influenza microneutralization assay is effected may also differ from each variant of a subtype of an influenza A virus antigen comprised in the vaccine composition by at least 25, 30, 40, 50, 60, 70, 80, 90 or even 100 amino acids. However, it may not differ by more than 120 or preferably 1 10 amino acids.
  • the variant of a subtype of an influenza B virus antigen against which cross- neutralization in an influenza microneutralization assay is effected differs from each variant of a subtype of an influenza B virus antigen comprised in the vaccine composition by at least 15 amino acids over their entire length.
  • Said variant of a subtype of an influenza B virus antigen against which cross-neutralization in an influenza microneutralization assay is effected may also differ from each variant of a subtype of an influenza A virus antigen comprised in the vaccine composition by at least 16, 18, 20, 25, 30, 35, 40 or even 50 amino acids. However, it may not differ by more than 70 or preferably 60 amino acids.
  • the "immune response” is preferably a "protective" immune response.
  • a “protective” immune response refers to the ability of a vaccine to elicit an immune response, either humoral or cell mediated or both, which serves to protect the subject from influenza.
  • the protection provided need not be absolute, i.e., influenza need not be totally prevented or influenza viruses be totally eradicated, if there is a statistically significant improvement compared with a control population of subjects. Protection may be limited to mitigating the severity or rapidity of onset of symptoms of influenza.
  • the immune response is preferably sufficient to treat and/or prevent influenza.
  • the terms “treating” or “preventing” influenza denote at least an inhibition of replication of the causative influenza virus, inhibition of influenza transmission, prevention of an influenza virus from establishing itself in a subject, and/or amelioration or alleviation of the symptoms of the influenza infection.
  • the present invention provides the use of at least three variants of a subtype of an influenza A virus antigen, wherein
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza A virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 35 amino acids over their entire length
  • a vaccine composition which effects cross-neutralization against at least one variant of said subtype of said influenza A virus antigen which is different from said variants comprised in said vaccine.
  • the present invention provides the use of at least three variants of a subtype of an influenza B virus antigen, wherein
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 20 amino acids over their entire length for the manufacture of a vaccine composition which effects cross-neutralization against at least one variant of said subtype of said influenza B virus antigen which is different from said variants comprised in said vaccine.
  • the present invention provides a method for producing a vaccine composition against influenza A virus, comprising
  • a first and second variant of a subtype of an influenza A virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza A virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza A virus antigen differ from each other by more than 35 amino acids over their entire length
  • the present invention provides a method for producing a vaccine composition against influenza B virus, comprising
  • a first and second variant of a subtype of an influenza B virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a second and third variant of a subtype of an influenza B virus antigen differ from each other by at least 10 amino acids over their entire length;
  • a first and third variant of a subtype of an influenza B virus antigen differ from each other by more than 20 amino acids over their entire length
  • the final gene combinations were then shuffled into a vector harbouring the recognition sites for the Tn7 bacterial transposase for generation of recombinant baculoviruses for expression in insect cells.
  • the whole shuffled cassette comprising one or more genes was inserted in the viral genome via recombination using the bacmid Tn7 site, as previously described (Luckow et al. (1993), J Virol. 67(8),:4566-4579).
  • A/Brisbane/59/2007 (SEQ ID No: 5).
  • HB variants were used for the generation of VLPs: B/Hong Kong/8/73 (SEQ ID No: 21 ),
  • H4 variants can be used for the generation of VLPs:
  • NA N1 variants can be used for the generation of VLPs:
  • A/Brisbane/59/2007 (SEQ ID No: 33).
  • Spodoptera frugiperda derived Sf9 were maintained in a protein-free liquid culture medium (Insect-XPRESSTM) and routinely re-inoculated every 3-4 days at 0.6 to 1.0 x 10 6 cells ml-1.
  • the recombinant bacmids were transfected into Spodoptera frugiperda (Sf9) host cells using the reagent ViaFectTM (Promega), according to the manufacturer protocol.
  • the viruses were harvested three days post infection and amplified to generate the master seed virus.
  • Parental or stable insect High Five cells were routinely sub-cultured to 0.3x10 6 cells/mL every 3-4 days when cell density reached 2-3x10 6 cells/mL in 125, 250 or 500 mL shake flasks (10 % working volume) in an Innova 44R incubator at 27 °C and 100 rpm (orbital motion diameter of 2.54 cm) using Insect XPRESSTM medium.
  • baculoviruses containing Influenza matrix M1 gene alone or in combination with one or multiple hemagglutinin (HA) genes were generated as described herein (see A).
  • Amplification of baculovirus stocks was performed as described in Vieira et al., J Biotechnol 2005;120:72-82. Briefly, Spodoptera frugiperda Sf-9 cells infected at 1 *10 6 cells/mL at a multiplicity of infection (MOI) of 0.1 infectious particles per cell (ip/cell). When cell viability reached 80-85 %, cultures were harvested and centrifuged at 200 g for 10 min at 4 °C. The pellet was discarded and the supernatant was centrifuged at 2000 g for 20 min at 4 °C. The resulting supernatant was stored at 4 °C until further use.
  • MOI multiplicity of infection
  • Influenza VLPs were produced in shake flaks (125, 250 or 500 mL with 10 % working volume), in glass stirred tank bioreactors or in single-use wave induced bioreactors. Briefly, shake flask cultures were infected at a cell concentration at infection (CCI) of 1 , 2, 3 or 4*10 6 cells/mL using a MOI of 0.1 , 1 or 10 ip/cell.
  • CCI cell concentration at infection
  • the culture medium was supplemented with a mixture containing insect medium supplement 10x, 5 mM glutamine, 10 mM asparagine and 20 mM glucose at a ratio of 10 % (v/v) regarding the final culture volume.
  • Bioreactor cultures were performed in computer-controlled BIOSTAT ® B-DCU 2L vessels equipped with two Rushton impellers, a sparger for gases supply, a water recirculation jacket for temperature control, and multiple ports for temperature, pH, p0 2 (partial pressure of oxygen) probes as well as for additions (e.g. culture medium) and sampling/harvesting of cell culture.
  • the p0 2 was set to 30 % of air saturation and maintained by varying the agitation rate from 70 to 250 rpm and the percentage of 0 2 in the gas mixture from 0 to 100 %.
  • the gas flow rate was set to 0.01 wm and temperature was kept at 27 °C.
  • the working volume was 2 L.
  • Bioreactor cultures were also performed in computer-controlled wave induced bioreactors, WaveTM 20/50 EH, equipped with multiple ports for temperature and p0 2 probes as well as for additions (e.g. culture medium) and sampling/harvesting of cell culture.
  • the p0 2 was set to 30 % of air saturation and controlled by varying the agitation rate and sequentially the percentage of N 2 and 0 2 in the gas mixture between 0-100 %.
  • the gas flow rate was set to 0.03 vvm.
  • the temperature was kept at 27 °C by using an in-situ heating plate supporting the wave bag.
  • the working volume was 10 L.
  • the downstream processing (DSP) scheme used for purification of VLPs consisted in a five- stage process that included a clarification step for cells removal, an UF/DF step for product concentration (TFF-membrane cassette), a chromatographic step for product purification (AEX or SEC), a second UF/DF step for final product concentration, and a final sterile filtration step (0.2 pm filter) before final formulation.
  • DSP downstream processing
  • Scheme 1 the supernatant was passed through two depth filters (5 pm and 0.65 pm), or Scheme 2 - culture broth was centrifuged at 200 g for 10 min at 4 °C, supernatant was collected, mixed with Benzonase (50 U/mL) for digestion of nucleic acids and then passed through two depth filters of 0.45 pm and 0.2 pm.
  • Scheme 1 - clarified bulk was passed through a cassette with 500 kDa of pore size keeping TMP within 0.8-1 bar and permeate flux between 25-35 LMH, or Scheme 2 - clarified bulk was passed through a cassette 300 kDa of composite regenerated cellulose keeping TMP within 0.8-1 bar, 24 LMH permeate flux and retention flux of 48 LMH membrane area.
  • Scheme 1 - retentate of UF/DF step was passed through an AEX chromatographic column using Buffer A (50 mM HEPES pH 7.5), Buffer B (Buffer A + 1 M NaCI), starting conductivity of 15 mS/cm, 25 CV gradient length and a flow rate of 2.5 CVs/mL, or Scheme 2 - retentate of UF/DF step was passed through a SEC column using a buffer containing 50 mM HEPES, pH 7.5 and 300 mM of NaCI, a volume injection of 5% CV and a flow rate 4 mL/min.
  • Buffer A 50 mM HEPES pH 7.5
  • Buffer B Buffer A + 1 M NaCI
  • Scheme 1 the purified product coming from AEX or SEC was concentrated using hollow fibers 750 kDa PES, 50 cm 2 , operated at TMP lower than 0.8 bar (measured with the two pressure sensors placed on the column valve of an AKTA york) and 30 mL/min recirculation flow rate
  • Scheme 2 the purified product coming from AEX or SEC was concentrated using 300kDa cassette regenerated cellulose of 50 cm 2 operated at TMP of 1.2 bar, 40 ml/min and 5DV - final formulation.
  • the final sterile filtration step was performed using two different filters, the Acrodisc from Pall Lifesciences and the Whatman cellulose regenerated membrane filter.
  • VLPs were formulated in a buffer consisting of 50 mM HEPES, 300 mM NaCI, pH 7.4 and trehalose 15% (w/v), and stored at -80 °C until further.
  • Glucose, glutamine, lactate and glutamate concentrations were determined with automated enzymatic assays using the YSI 7100 Multiparameter Bioanalytical System.
  • concentration of other metabolites was estimated by 1 H-NMR spectroscopy as described in Carinhas et al., Biotechnol Bioeng 2013; 1 10:3244-3257. Briefly, spectra were recorded in a 500 MHz Avance spectrometer equipped with a 5 mm QXI inversed probe, using a NOESY- based pulse sequence with water pre-saturation. DSS-d6 was used as internal standard for metabolite quantification in all samples. In order to obtain a similar pH between samples, they were mixed with phosphate buffer (pH 7.4) prepared in D 2 0 at a 2:1 ratio. Each spectrum was phased, baseline corrected and integrated using the Chenomx NMR Suite 8.0 software.
  • Negative staining TEM was used to assess the conformation and size of purified Influenza VLPs. Briefly, 10 ⁇ of purified VLP sample was fixed for 1 min in a copper grid coated with Formvar-carbon. Grids were washed with H 2 0 and then stained with 1 % uranyl acetate for 2 min and left to air dry. Samples were then observed in a Hitachi H-7650 Transmission Electron Microscope.
  • the NIBSC influenza anti- A/Johannesburg/33/1994 and anti-A/Nanchang/933/1995 HA sera were used to detect HA in monovalent and multivalent H3 VLPs.
  • the NIBSC influenza anti-B/Hong Kong/8/73 and anti- B/Victoria/2/87 HA sera were used to detect HA in monovalent and multivalent B VLPs.
  • M1 protein identification a commercially available antibody was used. All primary antibodies were used at dilutions between 1 :1000 and 1 :2000.
  • anti-sheep, anti-mouse or anti-goat antibodies conjugated with either HRP-labeling or AP were used at dilutions between 1 :2000 and 1 :5000.
  • Protein band detection was performed using the enhanced chemiluminescence detection system or the 1-stepTM NBT/BCIP blotting detection reagents.
  • Single radial immunodiffusion (SRID) Single radial immunodiffusion
  • the SRID protocol was used to estimate the concentration of immunologically active HA in purified Influenza VLPs. This technique is based on the ability of antigen forming a precipitate ring when in contact with equal concentration of antiserum. Briefly, antiserum was added to an agarose solution of 1 % (w/v) before the gel was made. The quantity of antiserum to mix with agarose depended on the number of plates to perform and reagent specifications. After gels have set, wells were cut into the agar gel and plates were left at 4°C (humidified) until further use. Antigen (standard or purified VLP) was treated with Zwittergent 3-14 detergent 10 % (w/v) and the mixture was left at RT for 30 min.
  • each antigen solution was added into the wells cut into the agar gel and then the plates were incubated for a minimum of 18 h in a moist box at 20-25 °C.
  • the antigen was able to diffuse from wells; as the antigen diffused out of the well, its concentration still remained above that of antiserum thus forming relatively soluble antigen- antibody (Ag-As) adducts.
  • the plates were destained using a mixture of acetic acid (12 %) and methanol (29 %) for 15-30 min, allowed to dry in warm air, and the diameter of the Ag-As rings measured in different angles.
  • BCA Bicinchoninic acid
  • the principle of the BCA assay relies on the formation of a copper-protein complex under alkaline conditions, followed by reduction of Cu 2+ to Cu + .
  • the extent of copper ions reduction is proportional to the amount of protein present. Since BCA forms a purple-blue complex with Cu + in alkaline environments, it is possible to estimate the concentration of total protein in a sample by comparing the color development of that sample with a standard of known concentration.
  • Total protein content in purified VLP samples was determined using the BCA protein assay kit (96-well plate protocol) from Pierce Biotechnology following manufacturer's protocol. AccuBlue assay
  • the AccuBlueTM assay allows precise quantitation of purified dsDNA across a wide range of concentrations by using fluorescent DNA binding dyes that are highly sensitive and selective for dsDNA.
  • the AccuBlueTM Broad Range dsDNA Assay was used according to manufacturer's instructions for estimating the content of residual DNA in purified VLPs samples.
  • Endotoxin content was quantified using Endosafe @ -PTS TM following manufacturer's instructions.
  • the Endosafe ® -PTS TM is a rapid, point-of-use test system that uses LAL reagents in an FDA-licensed test cartridge with a handled spectrophotometer.
  • the PTSTM uses the LAL kinetic chromogenic methodology to measure color intensity (color development take up to 15 min) that is directly correlated with the concentration of endotoxins in a sample. By comparing sample's color with that of a control standard endotoxin, it is possible to estimate the concentration of endotoxins. Using this analytical tool the concentration of endotoxins in purified VLPs samples was estimated.
  • the fluid thioglycollate medium (a general-purpose medium for the cultivation of anaerobes, microaerophiles and aerobes) and the soybean casein digest broth (a general-purpose medium for the cultivation of bacteria and fungi) are formulations adopted by the United States Pharmacopeia and the European Pharmacopeia as sterility test media. Therefore, they were herein used to confirm the sterility of purified VLPs samples. Briefly, 10-300 ⁇ of purified samples was added to 5 ml of both media and the mixture allowed to incubate at 27 or 37 °C for a maximum of 3 or 5 days. After incubation, growth of contaminants is evidenced by the presence of turbidity in the tubes. The pH of in-process and purified VLP samples was assessed using the Crison Micro pH 2002 system. Master baculovirus seed stocks were screened for the presence of mycoplasmas using qPCR.
  • NanoSight NS500 was used for nanoparticle tracking analysis throughout the DSP of Influenza VLPs, and thus estimate total particle concentration in purified VLPs samples. It utilizes the properties of both light scattering and Brownian motion to obtain the size distribution and concentration measurement of particles in solution. By laser beaming particles in solution when these pass through a sample chamber, particles scatter light in such a manner that they can be visualized via a magnification microscope onto which a camera is mounted. Then, a software analysis the video file of the particles moving under Brownian motion recorded by this camera and using the Stokes-Einstein equation calculates their hydrodynamic diameters.
  • samples were diluted in D-PBS to a concentration of 10 s — 10 9 particles ml_ "1 , the instrument's linear range. All measurements were performed at room temperature (22 °C). Sample videos were analyzed with the Nanoparticle Tracking Analysis (NTA) 2.3 Analytical software, release version build 0025. Capture settings (shutter and gain) were adjusted manually. For each sample 60-seconds videos were acquired and particles between 70 and 150 nm were considered.
  • NTA Nanoparticle Tracking Analysis
  • MTT MTT
  • NAD(P)H-dependent cellular oxidoreductase enzymes NAD(P)H-dependent cellular oxidoreductase enzymes.
  • Cells are then solubilised with dimethyl sulfoxide and the released, solubilized formazan crystals are measured spectrophotometrically. Since only metabolically active cells can reduce MTT, the development of purple color is directly correlated with the number of viable cells. Because the baculovirus is a lytic virus, infection will reduce cell growth.
  • Such a reduction is dose-dependent and can be estimated by measuring the viable cell concentration using MTT and can be correlated to the viral titer (i.e. concentration of infectious baculovirus) using a set of mathematical regressions(Roldao et al., J Virol Methods 2009; 159:69-80). Briefly, 100 ⁇ of 5x10 5 cell/ml of Sf-9 cells were seeded onto a 96-well tissue culture plate and allowed to settle for 1 h at 27 °C. Supernatant was removed and 100 ⁇ of viral stocks diluted 10 ⁇ 1 to 10 ⁇ 10 times was added per well and plates incubated 6 days at 27 °C.
  • MTT was added (10 % (v/v) of total volume per well at 5 mg/ml) and plates incubated at 27 °C for additional 4 h. The supernatant was removed and formazan crystals solubilised by adding 150 ⁇ per well of DMSO. Plates were agitated for 10-20 min in a wellmix shaker WM-506 and absorbance (570/690 nm wavelength) measured using Infinite ® 200 PRO NanoQuant microplate reader. Collected data was analyzed using Prism 5 for Windows to determine the tissue culture lethal dose 50 (TCLD 50 ). The conversion of TCLD 50 to viral titers (pfu/ml) was carried out using the mathematical regressions reported elsewhere 4 .
  • ELISA is a plate-based assay technique designed to detect the presence of an antigen and estimate its concentration.
  • the ELISA herein used for detection of HA protein was an indirect ELISA and consisted in: (1 ) immobilization of the antigen to a solid surface, (2) incubation with a primary antibody followed by a secondary antibody that is linked to horseradish peroxidase, (3) incubation with the TMB substrate (3,3',5,5'- Tetramethylbenzidine) to produce a measureable product, and (4) signal-detection using a spectrophotometer. Briefly, cell culture samples were centrifuged at 200 g, 4 °C for 10 min and supernatants collected for ELISA analysis.
  • a mixture of 1 :10 or 1 :100 of culture supernatant in coating buffer (0.1 M Na 2 HP0 4 ) was added to a 96 well Nunc-Maxisorp plate (100 ⁇ /well) and allowed to incubate overnight at 4 °C. Plate was washed 3x with wash buffer (PBS with 0.05 % Tween-20) and blocked with 200 ⁇ /well blocking buffer (3 % BSA in PBS) for 1 h at room temperature. Then, 100 ⁇ /well of primary antibody in dilution buffer (3 % BSA in PBS with 0.05 % Tween-20) was added and plate incubated for 1 h at RT.
  • the plate was washed 3x with wash buffer and 100 ⁇ /well of secondary HRP-coupled antibody in dilution buffer was added. After 1 h at RT, plate was washed 3x with wash buffer and developer TMB Substrate (100 ⁇ /well) was added. Reaction was stopped with 100 ⁇ 1 M HCI as soon as positive wells were blue and the plate was subsequently read at 450nm using a Infinite ® 200 PRO NanoQuant microplate reader. Hemagglutination assay
  • Hemagglutination assay is based on the process of hemagglutination, in which sialic acid receptors on the surface of red blood cells (RBCs) bind to HA localized on the surface of influenza viruses, creating a network (lattice structure) of interconnected RBC and viral particles (Hirst (1942),. J Exp Med 75: 49-64).
  • the formation of this lattice structure is correlated with the concentration of viral particles in a specific sample. For example, if the virus concentration is too low, there is not enough HA to bind to RBC and thus RBC settle to the bottom of the well.
  • the RBC used in this assay are typically from chickens, turkeys, horses, guinea pigs and humans but are highly dependent on the selectivity of the targeted virus and the associated surface receptors on the RBC.
  • the hemagglutination assay herein used is a plate-based assay in which the concentration of HA in bulk and purified VLP samples is determined by comparing the hemagglutination profile of these samples with that of a standard of known HA concentration. Briefly, samples were 2-fold serially diluted in PBS and incubated for 30 min at 4 °C with 25 ⁇ of 1 % chicken RBC. Hemagglutination of RBC was identified visually by the formation of a network (lattice structure) of interconnected RBC and HA (positive results).
  • protein deglycosylation can be very useful to improve between-laboratory calibration of influenza antigen standards (Harvey et al. (2012), Biologicals 40(1 ): 96-99.).
  • the protein deglycosylation protocol was kindly provided by NIBSC. Briefly, supernatant of culture samples or purified VLPs samples were mixed with 2 ⁇ of 10x denaturing buffer (provided with enzyme) up to a total volume of 14 ⁇ (add water if needed) and incubated at 95-100 °C for 10 min.
  • Mass spectrometry was used for identification of HA and M1 proteins in bulk and purified VLP samples. Briefly, samples were run in SDS-PAGE gels, stained with coomassie blue and gel bands corresponding to HA or M1 proteins (according to Western blot results) were excised, digested (trypsin), desalted and concentrated using C18 microcolumns. Eluates were directly spotted on a MALDI plate (matrix used was a-Cyano-4-hydroxycinnamic acid) and analysed on a 4800 Plus MALDI-TOF/TOF analyser. Raw data were generated by the 4000 Series Explorer Software v3.0 RC1 . Database search for protein identification was performed using the algorithm MOWSE (version 2.2). Swissprot database and a custom database extracted from SwissProt containing extra HA entries (to include all HA and M1 protein sequences herein used) were used.
  • MOWSE version 2.2
  • Isotope Dilution Mass Spectrometry (IDMS) method was herein developed for quantitation of HA in the purified monovalent and multivalent VLPs. Briefly, samples were denatured and digested with trypsin. Isotopically labelled peptides were added in the digest as internal standards (IS). The level of HA peptides was determined following peptide standard curves, where the peptide fragment ion intensity was used and expressed as the ratio of the native peptide to the heavy peptide (IS). The protein amount was calculated based on the molarity of peptides multiplying by HA molecular mass.
  • Preliminary mouse studies focused on the generation of high titre reference sera from monovalent H1 , H3, NA and B strains VLPs for use in the project and for harmonisation purposes.
  • pilot mouse studies have been conducted on monovalent H1 , H3, NA and B strains VLPs, as well as with a mixture of all variants per (sub)type, to asses assay conditions and verify the vaccine dose and immunisation regimen.
  • the VLPs generated as described in A and B have been evaluated in outbred (Swiss) mice.
  • the total HA dose per vaccination was kept at 1 .5 ⁇ g (1/10th of a human dose per (sub) type), and 0.5 ⁇ g for the NA, both irrespective of the VLPs valence.
  • the mice are influenza naive, in contrast to the human target population, we vaccinated three times at a four weekly intervals to allow the humoral immune response to mature. Generation of reference mouse sera
  • mice In order to obtain sufficient quantities of mouse high titer sera from each of the 20 selected monovalent HAs (H1 , H3, B) and 3 NAs, groups of four outbred Swiss mice, six-weeks old female, have been immunised subcutaneously three times with 1.5 ⁇ g HA or 0.5 ⁇ g NA adjuvanted with Montanide ISA 51 VG, Seppic, France, at four week intervals. All vaccine formulations have been performed in a laminar flow cabinet mixing antigen solution and ISA 51 VG in 1 :1 ratio. Homogeneous water in oil emulsion was reached passing antigen solution and adjuvant 20 times back and forth through a 22-gauge couple piece. Emulsion formed and viscosity increased during process.
  • Formulations were inspected under phase contrast microscopy, at 1000 x magnification. Three bleeding points, at day 0 to confirm flu sera negativity, at day 42 for intermediate evaluation and at day 70 for the final bleeding have been performed. On average, 1.2 mL serum/pool has been obtained for each strain. Pooled sera from mice immunised with the five selected HA variants from each subtype (H1 , H3, B) and with the three selected NA variants, have been analysed for specific HA / NA IgG quantity using ADAMSEL FPL, a free-software for non-commercial users. This application converts the Optical Density (OD) readings obtained from ELISA plate readers into concentrations by four-parameter fitting. This software is designed to provide an auditable system that minimises data handling, thereby reducing the chances of error. With this system, a yield of 1 OD over background has been considered as 1 AU/mL.
  • OD Optical Density
  • the selected monovalent strains used for the mixture are the following: A Texas/36/1991 (H1 variant 3), A/Sichuan/2/1987 (H3 variant 3) and B/Yamagata/16/1988 (B variant 3).
  • mice For HA/NA competition studies, eight mice have been immunised with 0.5 pg of the trivalent NA VLPs (N1 -2-3) and eight mice with the mix of trivalent NA / H3 VLPs. Three bleeding points, at day 0 to confirm flu sera negativity, at day 42 for intermediate evaluation and at day 70 for the final bleeding have been performed. The sera generated were individually collected (300 ⁇ sera/mouse) and used for ELISA testing.
  • Mouse vaccination study hexavalent group 2 HA vaccine
  • Hexavalent group 2HAs VLPs H3, H4, H7, H10, H14, H15
  • H3, H4, H7, H10, H14, H15 can be similarly assessed, separately and in combination, to investigate the potential to induce heterosubtypic neutralising antibodies.
  • Three bleeding points at day 0 to confirm flu sera negativity, at day 42 for intermediate evaluation and at day 70 for the final bleeding can be performed.
  • the sera generated can be individually collected (300 ⁇ sera/mouse) and used for ELISA testing.
  • antigens from a different expression system were produced to coat the ELISA plates.
  • Egg-derived completely purified viruses have been produced for the five variants of each sub type H1 , H3 and B to be used as coating antigens for all planned ELISA assay.
  • Half area microplate (Greiner Bio On) allows up to a 50% sample and reagent reduction
  • dilute animal sera in dilution buffer start with 1 : 1000 dilution.
  • ADAMSEL FPL free-software for non-commercial users.
  • Micro-neutralisation assays MNA
  • Micro neutralisation assays were carried out to assess VLP-immunised mouse sera for anti-influenza antibodies.
  • virus stocks were titrated. A series of Iog10 dilutions were made and 0.1 ml/well (10 wells per dilution) was added into flat-bottomed 96-well plates containing a monolayer of confluent MDCK cells. Plates were incubated at room temperature for 30 minutes before replacing inoculum with infection medium (DMEM containing 2mM glutamine, sodium bicarbonate , penicillin-streptomycin 1/100, amphotericin B and 0.0025 ⁇ g/ml TPCK trypsin). Plates were further incubated for 72 hours at 35°C. 50 ⁇ per well supernatants were harvested and run in HA assays using 0.7% turkey red blood cells. 50% Tissue culture infectious doses (TCID50) was calculated using the Spearman-Karber formula.
  • Sera samples were heat treated at 56°C for 50 minutes then added in duplicate into flat- bottomed 96-well plates using a starting dilution of 1/20, followed by a further seven doubling dilutions.
  • 10 2 TCID50 (100 ⁇ ) virus was then added into each well. Plates were incubated at room temperature for 1 hour before adding the mixtures to flat-bottomed 96-well plates containing a monolayer of confluent MDCK cells. After 30 minutes incubation at room temperature the serum-virus mixture was replaced with 100 ⁇ of infection media and incubated for 72 hours at 35°C. Supernatants were screened using an HA assay, as above. Serum neutralisation titres were expressed as the reciprocal of the highest dilution whereby 50% infection was prevented.
  • Titres were the average of duplicate samples. Each assay run included a back -titration of the viruses used and validation criteria of 10 2 +/- 10° 5 /100 ⁇ . Values above threshold of detection in this assay of >2560 are reported as 5120; values below threshold of detection in this assay of ⁇ 20 are reported as 10. F. Results
  • mice Six groups of five mice were immunised with five monovalent VLP or with a mixture of five VLPs on days 0, 28 and 56. Sera for analysis were collected on day 70. The total HA dose was constant at 1.5 thus the mix of five group received 0.3 ⁇ 9 HA per strain.
  • Four groups of five mice were immunised on days 0, 28 and 56 with N1 VLPs, either monovalent or a mixture of three VLPs. Sera for analysis were collected on day 70. The total antigen (NA) dose was constant at 0.5 ⁇ 9.
  • NA antigen
  • mice were immunised with monovalent and polyvalent VLPs as well as mixtures of H1 , H3 and B VLPs. Mice were immunised on days 0, 28 and 56 with 1 .5 ⁇ g of VLPs or 4.5 ⁇ g for the H1 , H3 and B mixture groups. Two groups of 8 mice were immunised on days 0, 28 and 56 with polyvalent N1 VLP expressing three N1 variants on each VLP or combined with H1 polyvalent VLP expressing three H1 antigens. The total antigen (NA) dose was constant at 0.5 ⁇ g. Blood samples for analysis were collected on day 70.
  • NA total antigen
  • mice immunised with the high dose had about twice the IgG levels (2.1 95% CI: 0.0 to 4.6) as mice immunised with the high dose.
  • the microneutralisation (MN) and ELISA IgG results from both mouse studies are presented in Figures 1-6. Immune responses were evaluated using MN with cytopathic and Haemagglutination read out using the procedures as described in E. ELISA was performed using egg-grown whole influenza virus as coating antigen.
  • ELISA IgG titres are expressed as Arbitrary Units (AU), where 1 AU/mL yields an OD value of 1 over blank. Thus a titre of 100 AU/mL indicates that a serum can be diluted 100 fold and yield an OD of 1 over blank.
  • Table 1 and Figure 1 show the MN titres for H1 N1 strains.
  • the data show that immunisation with a monovalent VLP only yields MN responses to the homologous antigen.
  • the NC99 and Bris07 components show significant cross-reactivity in MN.
  • the vaccines with multiple components do show broader reactivity, albeit at the cost of MN titre.
  • PR8 1832 (503 to 6663) 10 (10to 10) 10 (10to 10) 10 (10to 10) 10 (10to 10) 10 (10to 10) 10 (10to 10)
  • HIT 230 (140to 378) 10 (10to 10) 19 (10to 35) 44 (22 to 91) 16 (9 to 28) 10 (10to 10)
  • HIP 342 (237to 492) 32 (8to 125) 37 (17to 84) 115 (65 to 202) 25 (10to 63) 10 (10to 10)
  • PR8 monovalent VLP of HA variant A/Puerto Rico/8/1934
  • USSR77 monovalent VLP of HA variant A/USSR/92/1977
  • Tex91 monovalent VLP of HA variant A/Texas/36/1991
  • NC99 monovalent VLP of HA variant A/New Caledonia/20/1999
  • Bris07 monovalent VLP of HA variant A/Brisbane/59/2007
  • Mix 5 mixture of all five monovalent VLPs (PR8+USSR77+Tex91 +NC99+Bris07)
  • H1 T polyvalent VLP of HA variants A/Puerto Rico/8/1934 + A/Texas/36/1991 + A/Brisbane/59/2007
  • H1 P polyvalent VLP of HA variants A/Puerto Rico/8/1934 + A/USSR/92/1977 + A/Texas/36/1991 + A/New Caledonia/20/1999 + A Brisbane/59/2007
  • Cal09 A/Californ
  • PR8 5142 (975 to 27125) 2452 (852 to 7056) 2068 (624 to 6857) 3504(1036 to 11843) 1144 (531 to 2467) 1622 (882 to 2983)
  • TX91 277 (88 to 871) 2484 (659to 9358) 68661 (39105 to 120556) 8759 (4044 to 18971) 3366 (1254 to 9038) 725 (113 to 4662)
  • NC99 356 (96 to 1313) 3692 (1013 to 13457) 7811 (2523 to 24175) 71162 (23105 to 219175) 44994 (10710to 189030) 1482 (342 to 6419)
  • MoMx 4641 (1897 to 11355) 13064 (8938 to 19094) 23450(13296 to 41357) 16465 (10618to 25532) 19822 (11352 to 34610) 1259 (672 to 2360)
  • HIT 22161 (15477to 31731) 2990 (778 to 11489) 46979 (36745 to 60062) 13137 (9029 to 19114) 21671 (13732 to 34200) 1308 (562 to 3047)
  • HITMx 13030 (9434 to 17997) 3222 (1475 to 7041) 31528 (20787 to 47819) 9260 (6387 to 13426) 12278 (8078 to 18661) 923 (442 to 1930)
  • HIP 24760 (18137 tD 33803) 8993 (5931 to 13635) 37688 (28890 to 49165) 15103 (9910 to 23017) 25085 (15331 to 41047) 904 (543 to 1505)
  • HIPMx 6501 (3972 to 10641) 3366 (1766 to 6416) 11214 (6882 to 18274) 10419 (8432 to 12874) 14950 (11370 to 18672) 419 (113 to 1554)
  • PR8 monovalent VLP of HA variant A/Puerto Rico/8/1934
  • USSR77 monovalent VLP of HA variant A/USSR/92/1977
  • Tex91 monovalent VLP of HA variant A/Texas/36/1991
  • NC99 monovalent VLP of HA variant A/New Caledonia/20/1999
  • BrisOJ monovalent VLP of HA variant A/Brisbane/59/2007;
  • TX91s monovalent VLP of HA variant ATexas/36/1991 ;
  • HmMx mixture of monovalent VLPs of H1 (Tex91 ), H3 (SI87) and B (Yam88) subtypes (4.5ug total dose);
  • H1 T polyvalent VLP of HA variants A/Puerto Rico/8/1934 + A/Texas/36/1991 +
  • HI TMx mixture of polyvalent VLPs of H1 (H1 T), H3 (H3T) and B (HBT) subtypes (4.5ug total dose);
  • H1 P polyvalent VLP of HA variants A/Puerto Rico/8/1934 +
  • Table 3 and Figure 3 show the MN titres for H3N2 strains.
  • the data show that immunisation with a monovalent VLP only yields MN responses to the homologous antigen.
  • the vaccines with multiple components do show broader reactivity, albeit at the cost of MN titre.
  • the only vaccine capable of inducing neutralisation titres to all vaccine components was the pentavalent H3 vaccine (H3P).
  • the pentavalent H3 vaccine also shows broadening as reflected in the H3N2 MNA titres to 2013 (SW13) and 2014 (HK14) H3N2 variants.
  • Table 3 H3N2 micro-neutralisation assay titres
  • H3T 263 (214 to 324) 22 (12 to 42) 17 (10 to 28) 30 (10 to 91) 1438 (667 to 3099) 10 (10 to 10) 10 (10 to 10)
  • H3P 165 (107 to 253) 153 (112 to 209) 20 (9 to 45) 392 (126 to 1224) 1182 (405 to 3445) 26 (12 to 56) 26 (12 to 56)
  • HK68 monovalent VLP of HA variant A/Hong Kong/1/1968
  • EN77 monovalent VLP of HA variant A/England/321/1977
  • SI87 monovalent VLP of HA variant A/Sichuan/2/1987
  • J094 monovalent VLP of HA variant A/Johannesburg/33/1994
  • FU03 monovalent VLP of HA variant A/Wyoming/3/2003
  • Mix 5 mixture of all five monovalent VLPs (HK68+EN77+SI87+JO94+FU03)
  • H3T polyvalent VLP of HA variants A/Hong Kong/1/1968 + A/Sichuan/2/1987 + A/Wyoming/3/2003
  • H3P polyvalent VLP of HA variants A/Hong_Kong/1/1968 + A/England/321/1977 + A/Sichuan/2/1987 + A/Johannesburg/33/1994 + A/Wyoming/3/2003
  • the H3N2 IgG data show similar trends as MNA (Table 4 and Figure 4). Again monovalent vaccines mainly induce strain-specific IgG.
  • the vaccines with multiple components incl. the pentavalent H3 vaccine - H3P show increased breadth.
  • H3T 11406 (8500 to 15306) 14957 (12095 to 18497) 1366 (914 to 2043) 9579 (6566 to 13974) 34428 (23993 to 49403)
  • H3TMx 6341 (4162 to 9661) 8186 (4587 to 14610) 1130 (580 to 2205) 4178 (2189 to 7973) 23232 (12998 to 41526)
  • H3P 9857 (5631 to 17255) 33331 (20848 to 53289) 1891 (886 to 4037) 21227 (10633 to 42375) 35456 (17591 to 71464)
  • H3PMx 11101 (6605 to 18658) 21427 (12667 to 36247) 1134 (810 to 1590) 15948 (10205 to 24922) 33765 (19894 to 57306)
  • HK68 monovalent VLP of HA variant A/Hong Kong/1/1968
  • EN77 monovalent VLP of HA variant A/England/321/1977
  • SI87 monovalent VLP of HA variant A/Sichuan/2/1987
  • J094 monovalent VLP of HA variant A Africa/33/1994
  • FU03 monovalent VLP of HA variant A Wyoming/3/2003
  • Mix 5 mixture of all five monovalent VLPs (HK68+EN77+SI87+JO94+FU03);
  • SI87s monovalent VLP of HA variant A/Sichuan/2/1987 (bridging group);
  • MoMx mixture of monovalent VLPs of H1 (Tex91 ), H3 (SI87) and B (Yam88) subtypes (4.5ug total dose);
  • H3T polyvalent VLP of HA variants A/Hong Kong/1/1968 + A/Sichuan/2/1987 + A/Wyoming/3/2003;
  • H3TMx mixture of polyvalent V
  • MNA responses following vaccination with monovalent or multiple component formulations again show a strong homologous response for the monovalent vaccines, whereas responses to the multicomponent compositions are broadened (Table 5 and Figure 5).
  • the pentavalent formulation (HBP) again induced MNA titres to all vaccine component.
  • the pentavalent formulation also induced MNA titres to recent B strain variants of both Victoria (Bris08) and Yamagata lineages (Phu13).
  • HBT 67 (36 to 125) 10 (10 to 10) 10 (10 to 10) 26 (20 to 34) 16 (8 to 33) 14 (8 to 27) 14 (8 to 25)
  • HBP 43 (18 to 99) 41 (21 to 80) 26 (15 to 44) 13 (8 to 20) 98 (43 to 221) 28 (13 to 57) 26 (13 to 55)
  • HK73 monovalent VLP of HA variant B/Hong Kong/8/1973
  • Vic87 monovalent VLP of HA variant B/Victoria/02/1987
  • Yam88 monovalent VLP of HA variant B/Yamagata/16/1988
  • Jian03 monovalent VLP of HA variant B/Jiangsu710/2003
  • Mal04 monovalent VLP of HA variant B/Malaysia/2506/2004;
  • Bris08 B/Brisbane/60/2008, Vic; Phu13: B/Phuket/3073/2013, Yam; Mix 5: mixture of all five monovalent VLPs (HK73+Vic87+Yam88+Jian03+Mal04); HBT: polyvalent VLP of HA variants B/Hong Kong/8/1973 + B/Jiangsu710/2003 + B/Malaysia/2506/2004; HBP: polyvalent VLP of HA variants B/Hong Kong/8/1973 + B/Victoria/02/1987 + B/Yamagata/16/1988 + B/Jiangsu/10/2003 + B/Malaysia/2506/2004.
  • HK73 24500 (11237 to 53419) 31945 (11731 to 86986) 276 (63 to 1214) 10020 (4232 to 23724) 3375 (202 to 56505) 1545 (248 to 9622)
  • HB3s 8231 (5555 to 12197) 24644 (13479 to 45057) 852 (305 to 2382) 6396 (3988 to 10257) 480 (189 to 1218) 4855 (2770 to 8508)
  • MoMx 3854 (2321 to 6400) 10129 (6447 to 15915) 460 (195 to 1088) 3750 (1641 to 8569) 221 (87 to 564) 1601 (765 to 3350)
  • HBT 51281 (33450 to 78618) 14015 (9598 to 20464) 5747 (3789 to 8716) 97706 (54836 to 174088) 10372 (6469 to 16628) 3458 (1962 to 6094)
  • HBP 85288 (55132 to 131940) 20830 (13164 to 32959) 4439 (3352 to 5880) 151183 (111484 to 205018) 11949 (5929 to 24081) 4139 (2470 to 6935)
  • HBPMx 11420 (5036 to 25900) 2936 (1882 to 4581) 708 (461 to 1088) 37007 (24936 to 54922) 1638 (610 to 4395) 893 (425 to 1877)
  • HK73 monovalent VLP of HA variant B/Hong Kong/8/1973
  • Vic87 monovalent VLP of HA variant B/Victoria/02/1987
  • Yam88 monovalent VLP of HA variant B/Yamagata/16/1988
  • Jian03 monovalent VLP of HA variant B/Jiangsu710/2003
  • Mal04 monovalent VLP of HA variant B/Malaysia/2506/2004
  • Bris08 B/Brisbane/60/2008, Vic
  • Phu13 B/Phuket/3073/2013, Yam
  • Mix 5 mixture of all five monovalent VLPs (HK73+Vic87+Yam88+Jian03+Mal04);
  • HBT polyvalent VLP of HA variants B
  • H1 N1 the mix of 5 as well as the pentavalent covered all vaccine components (i.e. MNA and IgG titres induced). A broadening could not yet be measured. It is assumed that the MN assay lacks sensitivity to detect A/California/7/2009 responses in mouse serum as has been previously observed with mini HA stem antigen constructs (Impagliazzo et al. (2015), Science 349(6254), 101 -106).

Abstract

La présente invention concerne des compositions de vaccin comprenant au moins trois variants d'un sous-type d'un antigène du virus de la grippe A et/ou B, lesdits trois variants étant sélectionnés sur la base de différences entre leurs séquences d'acides aminés. La présente invention concerne en outre lesdites compositions de vaccin destinées à être utilisées pour une méthode de génération d'une réponse immunitaire contre des virus de la grippe. L'invention concerne de plus une méthode de production desdites compositions de vaccin.
PCT/EP2017/076705 2016-10-19 2017-10-19 Vaccin contre le virus de la grippe WO2018073340A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018157028A1 (fr) * 2017-02-27 2018-08-30 Flugen, Inc. Compositions immunogènes contre la la grippe
US11344616B2 (en) 2017-02-27 2022-05-31 Flugen, Inc. Immunogenic compositions against influenza
WO2020000100A1 (fr) * 2018-06-27 2020-01-02 Medicago Inc. Mutants d'hémagglutinine du virus de la grippe
WO2020000101A1 (fr) * 2018-06-27 2020-01-02 Medicago Inc. Mutants d'hémagglutinine du virus de la grippe
CN112534056A (zh) * 2018-06-27 2021-03-19 麦迪卡格公司 流感病毒血凝素突变体
WO2020172635A1 (fr) * 2019-02-21 2020-08-27 Distributed Bio, Inc. Compositions optimisées de vaccins et leurs procédés de préparation
WO2023187366A1 (fr) * 2022-03-29 2023-10-05 Oxford University Innovation Limited Compositions immunogènes pour la prévention de la grippe a

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