WO2008040098A1 - Polypeptides immunogènes issus de la boucle de clivage de l'hémagglutinine précurseur d'un virus de la grippe - Google Patents

Polypeptides immunogènes issus de la boucle de clivage de l'hémagglutinine précurseur d'un virus de la grippe Download PDF

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WO2008040098A1
WO2008040098A1 PCT/AU2007/001539 AU2007001539W WO2008040098A1 WO 2008040098 A1 WO2008040098 A1 WO 2008040098A1 AU 2007001539 W AU2007001539 W AU 2007001539W WO 2008040098 A1 WO2008040098 A1 WO 2008040098A1
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peptide
influenza
vaccinating
seq
peptides
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PCT/AU2007/001539
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English (en)
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Peng Li
Tuckweng Kok
Darren Miller
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Medvet Science Pty Ltd
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Publication of WO2008040098A1 publication Critical patent/WO2008040098A1/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
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • 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
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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

Definitions

  • This invention relates to an immunogenic polypeptide suitable for inducing an antibody response targeting the cleavage loop of a hemagglutinin precursor of an influenza viruses, immunogenic composition of the polypeptide including a vaccine preparation, and a method of eliciting an immune response by immunising using such a composition and antibodies resulting therefrom.
  • Influenza is a condition that has a significant impact during winter months.
  • the very young and elderly are particularly at risk to acute symptoms that at times can lead to death.
  • influenza viruses are divided into types A, B and C based on antigenic differences.
  • Influenza A viruses are described by a nomenclature which includes the sub-type or type, geographic origin, strain number, and year of isolation, for example, A/Beijing/353/89.
  • Other subtypes of influenza A such as H7 and H9 are also know to sporadically infect humans with lesser morbidity.
  • influenza vaccines currently used are inactivated vaccines: they may be constituted of entire virions, or of virions subjected to treatment with agents which dissolve lipids ("split” vaccines), or else of purified glycoproteins ("sub-unit vaccines"). These inactivated vaccines mainly protect by causing synthesis of the receiver's antibodies directed against the hemagglutinin. Antigenic evolution of the influenza virus by mutation results in modifications in HA and NA. Accordingly, inactivated vaccines used at present only protect against strains having surface glycoproteins which comprise identical or cross-reactive epitopes. To provide a sufficient antigenic spectrum, conventional vaccines comprise components from several viral strains; they generally contain two type A strains and one type B strain. The choice of strains for use in vaccines is reviewed annually for each particular year and is predicated on WHO FDA recommendations. These recommendations reflect international epidemiological observations.
  • Antigenic drift and mutation mean that sometimes the approved vaccine will not afford sufficient protection against particular outbreaks, and more so if the outbreak is of a subtype different to that of the choice of strains approved usually some several months before.
  • Influenza A virus still causes significant morbidity and mortality worldwide particularly in high risk groups including the young, elderly and immuno-compromised individuals, resulting in 3-5 million serious cases and approximately 500,000 deaths worldwide.
  • the recent emergence of highly pathogenic avian influenza strains crossing to humans signals the rapid requirement for the development of novel, cheap and effective vaccines.
  • Vaccines comprised of synthetic peptide sequences are readily manufactured, easy to handle and are free from infectious agents therefore not requiring inactivation.
  • pandemic potential For the impending threat of avian H5N1 viruses, there is currently no pre-existing immunity in the human population.
  • H5N1 viruses The pathogenicity of H5N1 viruses, on the other hand, is extremely high with more than 50% mortality in humans reported in South East Asia (W.H.0. 2005). Although the pathogenicity of avian influenza virus is polygenic, the susceptibility of its hemagglutinin precursor glycoprotein HA 0 to cellular proteases is undoubtedly a major determinant (7, 9, 11, 13). For human influenza viruses and avirulent avian influenza viruses, the HA 0 is cleaved extra-cellularly by proteases enriched at the surface of mucosal tissues.
  • HA 0 is cleaved intra-cellularly by the cellular protease furin and related enzymes in the trans-golgi network to yield mature HA 1 and HA 2 , The resulting HA 1 and HA 2 are then displayed on the surface of the virions.
  • the N-terminus of the mature HA 2 contains the fusion peptide that plays a crucial role in the subsequent fusion between viral and cellular endosomal membranes. This process leads to the release of viral ribonucleoprotein (RNP) into the cell cytoplasm and subsequent transport to the nucleus for viral transcription.
  • RNP viral ribonucleoprotein
  • intracellular cleavage of HA 0 allows viral replication in multiple cell types - thus leading to systemic, infection, which correlates with high pathogenicity.
  • the highly conserved fusion peptide at the N-terminus of HA 2 is deeply buried in the cavity of the trimeric HA.
  • the connecting peptide sequence across the HA cleavage site is a prominent surface loop, as this sequence has to be exposed to proteases (3).
  • the connecting peptide sequence across the HA cleavage site is highly conserved, and for pathogenic avian isolates (including the H5N1 viruses) these sequences contain multiple basic residues (11, 13).
  • the 3-D structure of the HA is known from X-ray crystallography and the location of the receptor binding site and the five antigenic sites of HA have been mapped to this structure including the HA 0 loop.
  • the antigenic sites were identified from amino acid, sequence changes in naturally occurring variants as well as variants selected with monoclonal antibodies.
  • the HA 0 maturation cleavage loop is highly conserved and represent a highly prospective target for a so called universal antigen that provides antibody that can neutralize several sub types of Influenza A.
  • H1 to H3 are consensus sequences the remainder are as follows
  • H6 (A/chicken/California/9420/2001 (H6N2)) H7(A/mallard/Italy/4810-7/2004(H7N7))
  • Subtypes that commonly manifest transmissions between humans are H1, H2, and H3 and these subtypes tend to be susceptible to a narrower range of proteases and are limited to the respiratory and the intestinal organs.
  • Subtype H5 represents HPAI (High- pathogenicity avian influenza) possess multiple basic amino acids at the cleavage site (for example REKRRKKR (SEQ ID NO: 15)) which are cleaved by ubiquitous proteases in a wide range of organisms resulting in lethal systemic infection. Whilst human to human transmission of subtype H5 influenza virus has not been confirmed, when there is avian to human transmission this has a high morbidity.
  • H7 conjunctivitis and mild respiratory
  • H9 mild respiratory
  • mice were vaccinated with either linear peptide sequences across HA 0 or multiple copies of HA1 or HA2 specific peptides covalently linked by branched lysine residues.
  • Mice were vaccinated mice with 100ug of multi-peptide or linear peptide constructs adjuvanted in Freunds complete and incomplete adjuvants.
  • Vaccination inducted high- rates of peptide specific antibodies and also provided significant but sub-optimal protection following 2 LD 50 H1N1 (A/PR8/35) influenza infection (10).
  • a peptide conjugate vaccine homologous to the highly conserved HA 0 cleavage site of influenza B elicited conjugated to the outer membrane protein complex of Neisseria meningitidis.
  • WO 2004/080403 discloses the use of a peptide conjugate of the maturation cleavage loop of HA 0 and the M2 protein as a means of providing vaccination.
  • Cross protection is shown against intranasal challenge by an H1 subtype following intramuscular immunization with HA 0 H3 peptide conjugates.
  • the administration is by injection either intravenous, intraperitoneal, subcutaneous or intramuscular, with intramuscular being preferable. There is no disclosure of mucosal administration.
  • WO 2007/051036 discloses a combinatorial peptide vaccine with peptides taken, from differing regions of the HA including the maturation cleavage loop of HA 0 as well as NA.
  • the present invention has developed immunogenic polypeptides, compositions containing the immunogenic polypeptides and methods of delivery that provide an alternative approach to vaccinating against influenza infection.
  • the invention could be said to reside in an isolated immunogenic polypeptide, of 16 amino acids of longer selected from within the cleavage loop of a hemagglutinin (HA), the polypeptide being immunogenic.
  • HA hemagglutinin
  • the invention is particularly useful where the hemagglutinin is a subtype known to have adverse health effects in humans, and in particular one of the subtypes of an influenza A virus.
  • the HA subtype is selected from the groups consisting of H1, H2, H3, H5 H7 and H9.
  • the immunogenic polypeptide may be of 16 amino acids or longer selected from within the H5 cleavage loop sequence, and in one form the cleavage loop amino acid sequence is NTPQRERRRKKRGLFGAlAGFIEGGW (SEQ ID NO 16).
  • the sequence may be varied from the consensus H5 cleavage loop sequence in line with known, or yet to emerge cleavage loop sequences, including furin recognition motifs.
  • the immunogenic polypeptide may be of 16 amino acids or longer selected from within the H3 cleavage loop sequence, and in one form the cleavage loop amino acid sequence is TGMRNVPEKQTRGIFGAIAGFIENGWE (SEQ ID 17).
  • the sequence may be varied from the consensus H3 cleavage loop sequence in line with known, or yet to emerge cleavage loop sequences, including furin recognition motifs.
  • the invention could be said to reside in an immunogenic composition comprising an immunologically effective amount of at least one immunogenic polypeptide of the first aspect of the invention.
  • the invention could be said to comprise a nasal dispenser, or a container adapted to be fitted to a nasal dispenser, said container or dispenser containing the composition of this invention.
  • the invention could be said to reside in an antibody preparation that specifically binds the immunogenic polypeptide of the first aspect of this invention.
  • the invention could be said to reside in a method for stimulating the immune system of an individual to produce a protective immune response against influenza virus, the method comprising administering to the individual an immunologically effective amount of me immunogenic preparation of this invention.
  • the invention could be said to reside in a method of inhibiting influenza viral propagation in a human or non human animal, the method comprising the step of administering to the animal an immunologically effective amount of the immunogenic preparation of this invention.
  • Figure 2 shows the percentage of original weight of the subjects of the studies set out in figure 1 being indicative of the degree to which the subjects were affected by challenge with influenza virus
  • Figure 3 are graphs showing the "survival' of subjects of the two studies referred to above,
  • Figure 4 are representative histological sections of subject lungs after immunoperoxidase staining four days after challenge with influenza virus
  • Figure 5 shows the level of Influenza RNA in subject lungs at four different time points
  • Figure 6 shows the number of different H3 peptides according to this invention and two CD4 helper peptides useful for biasing the immune response toward being cellular, it is to be noted that the two helper peptides referred to as TH1 and TH2 also referred to as THLp A and THLp B.
  • the sequence listing numbers are sequentially top to bottom as follows SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28,
  • Figures 7 and 8 tabulates the treatment of 6 groups of subjects in a second study supporting the present invention
  • Figure 9 shows the percentage of original weight of the subject of the treatments set out in figures 7 and 8
  • Figure 10 shows total anti-peptide immunoglobulin produced following vaccination with control (A) and with peptides of H3 HA 0 cleavage loop over a total of 35 days
  • Figure 11 shows the body weight loss following challenge with a H3N2 influenza type A strain
  • Figure 12 shows the isotype specific anti-peptide immunoglobulin produced following immunization
  • Figure 13 shows the body weight loss following challenge with a H3N2 influenza type A strain following intranasal vaccination with peptides of H3 HA 0 cleavage loop
  • Figure 14 shows real-time RT PCR analysis of mouse lung tissue following challenge with a H3N2 influenza type A strain
  • Figure 15 shows immunoperoxidase staining for influenza A antigens follow challenge with a H3N2 influenza type A strain after intranasal vaccination with peptides of H3 HA 0 cleavage loop
  • Figure 16 shows the phenomenon of "cuffing" found in vaccinated and recovered mice.
  • the cellular composition of these cuffs is predominantly CD3+ cT-cells. Additionally significant numbers of cells are also shown to be Asialo GM1+,
  • Figure 17 shows the distribution of Influenza infected MDCK
  • Figure 18 shows the peptides used in experiments of the experiments conducted in the examples, with the following sequence listing numbers
  • Figure 19 shows the heterotypic influenza A protection afforded to H3N2 influenza type A virus following intranasal vaccination with peptides of H5 HA 0 cleavage loop
  • Figure 20 shows the protection afforded to mice challenged by H5N1 influenza type A virus following intranasal vaccination with peptides of H5 HA 0 cleavage loop.
  • immunogenically effective amount has its usual meaning in the art, that is, an amount of an immunogen which is capable of inducing an immune response which significantly engages agents (e.g. pathogenic agents) which share immunological features with the immunogen.
  • Immunogenic refers to the ability to elicit an immune response (for example, cellular) in a patient, such as a human, and/or in a biological sample.
  • antigens that are immunogenic are capable of stimulating cell proliferation, interleukin-12 production and/or interferon- ⁇ production in biological samples comprising one or more cells selected from the group of T cells, NK cells, B cells and macrophages, where the cells are derived from an influenza immune individual.
  • adjuvant has its usual meaning in the art of vaccine technology, that is a substance or a composition of matter which alone does not provide an immune response against the immunogen, vaccination with the immunogen may or may not give rise to an immune response against the immunogen, but the combination of vaccination with immunogen and adjuvant induces an immune response against the immunogen which is stronger than that induced by the immunogen alone.
  • the adjuvant of the present invention is selected as being capable of acting on a mucosal surface.
  • an "isolated” biological component is one that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, that is other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles.
  • Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, proteins, polypeptides or antibodies prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • the invention could be said to reside in an isolated immunogenic polypeptide, of 16 amino acids or longer selected from within the cleavage loop of a hemagglutinin (HA), the polypeptide being immunogenic.
  • HA hemagglutinin
  • the invention is particularly useful where the hemagglutinin is a subtype known to have adverse health effects in humans, and in particular one of the subtypes of an influenza A virus.
  • the HA subtype is selected from the groups consisting of H1, H2, H3, H5, H7 and H9, and more preferably H1-3 and H5..
  • the invention also has applicability to non-human, animals and thus is applicable to all subtypes particularly for avian animals.
  • the immunogenic polypeptide may be of 16 amino acids or longer selected from within the H5 cleavage loop sequence, and in one form the cleavage loop amino add sequence is NTPQRERKRKKRGLFGAIAGFIEGGW (SEQ ID NO: 55).
  • the sequence may be varied from the consensus H5 cleavage loop sequence in line with known, or yet to emerge cleavage loop sequences, including furin recognition motifs.
  • the immunogenic polypeptide may be of 16 amino acids or longer selected from within, the H5 cleavage loop sequence, and in one form the.
  • cleavage loop amino acid sequence is TGMRNVPEKQTRGIFGAIAGFIENGWE (SEQ ID NO: 56).
  • the sequence may be varied from the consensus H3 cleavage loop sequence in line with known, or yet to emerge cleavage loop sequences, including furin recognition motifs.
  • the immunogenic polypeptide might selected from 16, 17, or more amino acids from a corresponding region of another HA subtype, including those of particular relevance to humans, in particular this might include H1 , and H2, although it will be understood that the invention may also encompass other sub types of HA.
  • H1 this might include at least a majority of the amino acid sequence
  • NIPSIQSRGLFGAIAGFIE SEQ ID NO: 57.
  • the invention will also encompass immunogenic peptides that are specific for the cleavage loop of influenza B, the later in particular with regards to the preferred embodiments of the immunogenic polypeptide and compositions provided below.
  • the immunogenic polypeptide may be coupled or conjugated to a display particle, such as a protein, or synthetic carrier, either a bead or other solid support, so that more than one immunogenic polypeptide is coupled thereto by known means to enhance the immunogenicity of the polypeptide.
  • a display particle such as a protein, or synthetic carrier, either a bead or other solid support
  • Inert beads such as latex beads or other resinous particle may also act as a display particle.
  • the display particle may alternatively be a carbohydrate.
  • the display particle may be selected to also act as an adjuvant to further enhance the immunogenicity of the polypeptide. Where nasal delivery is contemplated the display particle will be of a suitable nature to permit access to respiratory mucosal surfaces.
  • the display particle is a protein this may comprise a so called fusion protein.
  • the display particle to be coupled with the immunogenic polypeptide may be chosen for a number of purposes in addition to simply display.
  • a display particle may, for example, serve as an immunological fusion partner by assisting in the provision of T helper epitopes, preferably T helper epitopes recognized by humans.
  • the display particle protein is a fusion partner it may serve as an expression enhancer, assisting in expressing the protein at higher yields than the native recombinant protein.
  • Certain preferred fusion partners are both immunological and expression enhancing fusion partners.
  • Other fusion partners may be selected so as to increase the solubility of the protein or to enable the protein to be targeted to desired intracellular compartments.
  • Still further fusion partners include affinity tags, which facilitate purification of the protein.
  • Attachment of the immunogenic polypeptides to protein display particles may generally be prepared using standard techniques, including chemical conjugation, and this is preferred.
  • the fusion protein is expressed as a recombinant protein, allowing the production of increased levels, relative to a non-fused protein, in an expression system.
  • Protein display particles are also provided that comprise a immunogenic polypeptide of the present invention together with an unrelated immunogenic protein.
  • the immunogenic protein is capable of eliciting a memory response.
  • proteins include tetanus, tuberculosis and hepatitis proteins for example this may derived from protein D, a surface protein of the gram-negative bacterium Hemophilus influenza B, or from the outer membrane protein complex of Neisseria meningitidis or diphtheria toxin.
  • the immunogenic polypeptide comprise a fusion protein
  • the chimeric protein when formed being for example transported to the surface of the micro- organism to display the immunogenic polypeptide.
  • phage display systems are known for this purpose.
  • the micro-organism may be a non-harmful bacterium.
  • the invention could be said to reside in an immunogenic composition comprising an immunologically effective amount of at least one immunogenic polypeptide of this invention.
  • the composition could comprise a mixture of two or more of the immunogenic polypeptides of the first aspect of this invention.
  • the two or more immunogenic polypeptides may each be specific for one HA subtype overlapping but not coterminus with each other, preferably however a first of the two or more immunogenic polypeptides is selected to be specific for a first HA sub type and a second immunogenic polypeptide selected to be specific for a second HA type.
  • the first and second subtype are preferably are H1 and H3.
  • the composition may additionally comprise a third immunogenic polypeptide, selected to be specific for the H2 subtype.
  • the composition may further comprise a fourth immunogenic polypeptide selected to be specific for the H5 subtype.
  • immunogenic polypeptide can be provided for yet further of the subtypes or combinations of two or more of them, and additionally two or more immunogenic polypeptide may be specific for one HA subtype. It is preferred however that any mixture of peptides is specific for one subtype only.
  • the display particles have two or more immunogenic polypeptides coupled thereto.
  • the composition may comprise just one display particle having all immunogenic polypeptides of the composition coupled thereto.
  • the composition may comprise two or more display particles.
  • the composition may further comprising a T helper epitope.
  • the T helper epitope could be a universal T helper epitope, for example (P2, P30, pan DR epitope peptide PADRE) or a CD4 T helper cell peptide.
  • the TH epitope is preferably a promiscuous TH epitope.
  • the promiscuous epitope can according to the invention be a naturally occurring human T H epitope such as epitopes from tetanus toxoid (e.g.
  • the epitope can be any artificial T-cell epitope which is capable of binding a large proportion of haplotypes.
  • PADRE pan DR epitope peptides
  • T-helper epitopes are preferred and the T-helper epitope may be any T-helper epitope known to the skilled artisan for enhancing an immune response in a particular target subject (i.e. a human subject, or a specific non-human animal subject such as, for example, a rat, mouse, guinea pig, dog, horse, pig, or goat). Synthetic or small peptides are preferred because unlike T-helper epitopes provided by carrier proteins these do not introduce irrelevant immunities, that may or may not mask the immune responses mounted against the HA 0 peptides, and may or may not modify the immunogenic determinant during the coupling process.
  • Preferred T-helper epitopes comprise at least about 10-24 amino acids in length, more generally about 15 to about 20 amino acids in length.
  • T-helper epitopes are particularly preferred as these are readily synthesized chemically and obviate the need to use longer polypeptides comprising multiple T-helper epitopes.
  • promiscuous or permissive T-helper epitopes that may be suitable for use in connection with HA 0 peptides of the present invention include: i) a rodent or human T-helper epitope of tetanus toxoid peptide (TTP), such as, for example amino acids 830-843 of TTP; ii) a rodent or human T-helper epitope of Plasmodium falciparum pfg27; iii) a rodent or human T-helper epitope of lactate dehydrogenase; iv) a rodent or human T-helper epitope of the envelope protein of HIV or HIVgp120; v) a synthetic human T-helper epitope (PADRE) predicted from the amino acid sequence of known anchor proteins; vi) a rodent or human T-helper epitope of measles virus fusion protein; vii) a T-helper epitope comprising at least about 10 amino acid
  • a T-helper epitope may be recognised by one or more mammals of different species. Accordingly, the designation of any T-helper epitope herein is not to be considered restrictive with respect to the immune system of the species in which the epitope is recognised.
  • a rodent T-helper epitope can be recognised by the immune system of a mouse, rat, rabbit, guinea pig, or other rodent, or a human or dog.
  • T-helper epitopes disclosed herein are included for the purposes of exemplification, only. Using standard peptide synthesis techniques known to the skilled artisan, the T- helper epitopes referred to herein are readily substituted for a different T-helper epitope to adapt the lipopeptide of the invention for use in a different species. Accordingly, additional T-helper epitopes known to the skilled person to be useful in eliciting or enhancing an immune response in a target species are not to be excluded.
  • T-helper epitopes may be identified by a detailed analysis, using in vitro T- cell stimulation techniques of component proteins, protein fragments and peptides to identify appropriate sequences (Goodman and Sercarz, Ann. Rev. Immunol, 1, 465, (1983); Berzofsky, In: "The Year in Immunology, Vol. 2" page 151, Karger, Basel, 1986; and Livingstone and Fathman, Ann. Rev. Immunol.. 5, 477, (1987)).
  • composition comprises a CD4 T helper epitope being one or both OfTHLp A: PRYVKQNTLKLAT, (SEQ ID NO: 27) and THLp B: QYIKANSKFIGITELKK (SEQ ID NO: 28)17mer.
  • the T helper epitopes may be coupled to the display particle, and are preferably coupled to the same display particle as the immunogenic polypeptides.
  • composition of the invention include liquid preparations for orifice, e.g., oral, nasal, anal, vaginal, peroral, intragastric, mucosal (e.g., per lingual, alveolar, gingival olfactory or respiratory mucosa) etc., administration such as suspensions, syrups or elixirs; and preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions.
  • orifice e.g., oral, nasal, anal, vaginal, peroral, intragastric, mucosal (e.g., per lingual, alveolar, gingival olfactory or respiratory mucosa) etc.
  • administration such as suspensions, syrups or elixirs
  • parenteral, subcutaneous, intradermal, intramuscular or intravenous administration e.g., injectable administration
  • compositions for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration may induce a systemic response
  • compositions for orifice or mucosal administration may induce a local response.
  • Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like.
  • the compositions can also be lyophilized.
  • the compositions can contain auxiliary substances such as wetting or emulsifying agents, Ph buffering agents, gelling or viscosity enhancing additives, preservatives, flavouring agents, colours, and the like, depending upon the route administration and the preparation desired. Standard texts, such as "Remington's Pharmaceutical Science", 19th edition, 1995, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.
  • Solutions, suspensions and gels normally contain a major amount of water (preferably purified water) in addition to the antigens, and optional adjuvant. Minor amounts of other ingredients such as Ph adjusters (e.g., a base such as NaOH), emulsifiers or dispersing agents, buffering agents, preservative, wetting agents, gelling agents, (e.g., methylcellulose), colours and/or flavours may also be present.
  • Ph adjusters e.g., a base such as NaOH
  • emulsifiers or dispersing agents e.g., a base such as NaOH
  • emulsifiers or dispersing agents e.g., buffering agents, preservative, wetting agents, gelling agents, (e.g., methylcellulose), colours and/or flavours
  • the inventive compositions can be isotonic, i.e., it can have the same osmotic pressure as blood and lacrimal fluid.
  • compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes.
  • sodium chloride is preferred particularly for buffers containing sodium ions.
  • a pharmaceutically acceptable preservative can be employed to increase the shelf-life of the compositions.
  • Benzyl alcohol may be suitable, although a variety of preservatives including for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride may also be employed.
  • a suitable concentration of the preservative will be from 0.02% to 2% based on the total weight although there may be appreciable variation depending upon the agent selected.
  • Preferably administration is to a mucosal surface and more preferably intranasaly.
  • the nasal mucosa has a high density of B and T cells and has the potential of eliciting a rapid immune response.
  • This has a number of advantages, in that the site of initial infection with influenza is principally in the respiratory apparatus of the affected person, and there is a stronger prospect of eliciting local immunity in the respiratory tract.
  • Mucosal administration of immunogenic compositions is of particular interest since this route is able to stimulate locally-produced antibodies (secretory IgA antibodies) which are preferred particularly for their intracellular effect.
  • this route of administration comprises an adjuvant and a T helper epitope or other immunomodulatory compound such as a cytokine.
  • the immunogenic polypeptides of this invention can be self administered, by for example a puffer, or other similar dosing device. Whilst enteric delivery for effecting mucosal immunity is also known, this route has the difficulty that preparations in particular peptides must be presented in an acid resistant format so that they can traverse the stomach without being degraded by stomach acids. Additionally providing the immunogenic polypeptide as an injectable is also not preferred because in part this requires attendance of medical personnel.
  • the intranasal administration, of the composition can be formulated, for example, in liquid form as nose drops, spray, or suitable for inhalation, as powder, as cream, or as emulsion.
  • compositions may be in a form and dispensed by a squeeze spray dispenser, pump dispenser or aerosol dispenser.
  • Pump dispensers can preferably dispense a metered dose or, a dose having a particular particle size.
  • Pump spray dispensers are commercially available, e.g., from Valois of America, Inc., Connecticut. Pump spray dispensers are commonly fabricated from a flexible material such as plastic and cause a spray to dispense in response to being squeezed.
  • Aerosol spray preparations can be in a pressurized container with a suitable propellant such as a hydrocarbon propellant.
  • composition also may also comprise a propellant preferably a hydrocarbon propellant that for environmental reasons is not a chlorofluorocarbon, preferably the propellant is ahydrofluoroalkane propellant.
  • a propellant preferably a hydrocarbon propellant that for environmental reasons is not a chlorofluorocarbon, preferably the propellant is ahydrofluoroalkane propellant.
  • the composition may be housed in a pump spray apparatus.
  • the invention might thus also be said to comprise a nasal dispenser, or a container adapted to be fitted to a nasal dispenser, said container or dispenser containing the composition of this invention.
  • composition particularly suited for nasal delivery may also comprise a surfactant to facilitate penetration at least through the mucous layer of the respiratory mucosa and perhaps one or more non-ionic surface-active agents as an absorption enhancer, chelating agents which enhance absorption across respiratory mucous membranes.
  • the composition for nasal administration comprises immunostimulatory compounds in particular effective for inducing mucosal immunity, in particular adjuvants suited for nasal mucosal action.
  • the adjuvants might be selected from the group consisting, mixture of polyethylene glycol substituted caprylic/captic acid glycerides and Tween 20®, a triglyceride, certain di or triglycerides as disclosed in US patent 6514503, cholera toxin, ISCOMs, aluminum preferably in the form of aluminium hydroxide (Al(OH) 3 ) or aluminum phosphate (AlPO 4 ), ethanol, isopropyl alcohol and polyethylene glycol, certain galactoceramides in particular alpha-galactoceramide. Preferred are aluminium and alpha galactoceramide.
  • the composition may also be for oral delivery, and may thus include aspect for protection against degradation in the stomach.
  • peptides may be contained with capsules, caplets or other acid resistant outer housing.
  • the composition may be coated with a resistant coating, or may be configured as a nanoparticle.
  • composition might also comprise an injectable further comprising a suitable adjuvant, for example Freund's Adjuvant, ISA-51, aluminium-based adjuvants (aluminium phosphate or aluminium hydroxide, e.g. from Danfoss), Calcium Phosphate, QS21 (Antigenic), MF59 (Chiron Corp.), and Ribi (Glaxo SmithKline).
  • a suitable adjuvant for example Freund's Adjuvant, ISA-51, aluminium-based adjuvants (aluminium phosphate or aluminium hydroxide, e.g. from Danfoss), Calcium Phosphate, QS21 (Antigenic), MF59 (Chiron Corp.), and Ribi (Glaxo SmithKline).
  • composition whether as an injectable or otherwise may comprise other immune system stimulating moieties.
  • Typical examples of such moieties are cytokines, heat-shock proteins, and hormones, as well as effective parts thereof.
  • Suitable cytokines to be used according to the invention are those which will normally also function as adjuvants in a vaccine composition, e.g. interferon ⁇ .
  • the immune stimulating moiety can be a toxin, such as listeriolycin (LLO), lipid A and heat-labile enterotoxin.
  • compositions of this invention are prepared by mixing the ingredients following generally accepted procedures.
  • the selected components may be simply mixed in a blender, or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration.
  • Compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the particular route of administration, intranasal, oral, peroral, mucosal, intragastric, rectal etc.
  • the invention could be said to reside in an antibody preparation that specifically bind the immunogenic polypeptide of the first aspect of this invention.
  • an antibody preparation may comprise a polyclonal preparation, and the preparation may have been elicited against two or more different of the immunogenic polypeptides of this invention.
  • the antibody preparation may have result from an immune response in an animal immunised by the Immunogenic composition of this invention.
  • the invention may reside in an isolated antibody that binds specifically to one or more of the immunogenic polypeptides of this invention.
  • the isolated antibody may be a monoclonal antibody produced by a hybridoma, that has been developed by methods well known in the art.
  • the antibody is of an IgA isotype, although another isotype such as IgG may also be useful.
  • antibody includes reference to an immunoglobulin, molecule immunologically reactive with a particular antigen, and includes both polyclonal and monoclonal antibodies.
  • the term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies).
  • antibody also includes antigen binding forms of antibodies, including fragments with antigen-binding capability, for example, Fab', F(ab')2, Fab, Fv and rlgG.
  • the term also refers to recombinant single chain Fv fragments (scFv).
  • the term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies.
  • An antibody immunologically reactive with a particular antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors (Vaughan et al, Nature Biotech. 14:309-314 (1996)), or by immunizing an animal with the antigen or with DNA encoding the antigen.
  • an immunoglobulin typically has a heavy and light chain. Each heavy and light chain contains a constant region and a variable region. Light and heavy chain variable regions contain four "framework" regions interrupted by three hypervariable regions, also called complementarity-determining regions (CDRs). The extent of the framework regions and CDRs have been defined. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three dimensional space.
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • the CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially Starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located.
  • a V H CDR3 is located in the variable domain of the heavy chain of the antibody in, which it is found
  • a V L CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.
  • References to V H or a VH refer to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv , or Fab.
  • V L or a "VL” refer to the variable region of an immunoglobulin light chain, including the light chain of an Fv, scFv , dsFv or Fab.
  • the phrase single chain Fv (scFv) refers to an antibody in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form one chain.
  • a linker peptide is inserted between the two chains to allow for proper folding and creation of an active binding site.
  • An antibody having a constant region substantially identical to a naturally occulting class IgA antibody constant region refers to an antibody in which any constant region present is substantially identical, that is, at least about 85-90%, and preferably at least 95% identical, to the amino acid sequence of the naturally occurring class IgA antibody's constant region.
  • monoclonal antibody as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • Monoclonal antibodies useful with the present invention may be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow and Lane, “Antibodies: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, New York (1988); Hammerling et al., in; “Monoclonal Antibodies and T-CeIl Hybridomas,” Elsevier, New York (1981), pp. 563-681 (both of which are incorporated herein by reference in their entireties).
  • the antibodies of he present invention including in vivo use of the HA 0 peptide antibodies in humans for and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies.
  • a “chimeric antibody” is an immunoglobulin molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e,g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • Methods for producing chimeric antibodies are known in the art.
  • humanized antibody or “humanized immunoglobulin” refers to an immunoglobulin comprising a human framework, at least one and preferably all complementarity determining regions (CDRs) from a non-human antibody, and to which any constant region present is substantially identical to a human immunoglobulin constant region, i.e., at least about 85-90%, and preferably at least 95% identical.
  • CDRs complementarity determining regions
  • all parts of a humanized immunoglobulin, except possibly the CDRs are substantially identical to corresponding parts of one or more native human immunoglobulin sequences.
  • such humanized antibodies are chimeric antibodies wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identity framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions.
  • Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting, veneering or resurfacing and chain shuffling (U.S. Pat. No. 5,565,332)
  • Completely "human” antibodies may be desirable for therapeutic treatment of human patients.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. Commercal providers can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
  • Completely human antibodies that recognize a selected epitope can be generated using a technique referred to as "guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope.
  • the invention could be said to reside in a method for stimulating the immune system of an individual to produce a protective immune response against influenza virus, the method comprising administering to the individual an immunologically effective amount of the immunogenic preparation of this invention.
  • Such antibodies may be particularly useful to administer to a human or animal recipient to impart passive immunity to the HA 0 peptide and thus Influenza A.
  • the invention could be said to reside in a method of inhibiting influenza viral propagation in a human or non human animal, the method comprising the step of administering to the animal an immunologically effective amount of the immunogenic preparation of this invention.
  • the method comprises an initial immunization, followed by at least one booster. Most preferably the immunization is by nasal delivery, although oral delivery is also a preferred route.
  • the invention is of particular benefit to human subjects, however it is also applicable to animals, for example sporting animals such as horses, agricultural animals particularly pigs, and fowl, such as chicken duck, geese, turkeys and the like, and perhaps also domestic animals such as cats. It will be appreciated that the range of subtypes applicable to animals will vary from that for humans, thus for example avian animals are likely to be affected by all 16 currently known subtypes and vaccination for all of these
  • mice 6- week-old female Balb/C mice were obtained from the Animal Care Facility at the Institute of Medical and Veterinary Science, (Adelaide, Australia). Mice were weighed on a daily basis and assessed for visual signs of infection. Observations were recorded on clinical record sheets. Water and food was provided at ground level in all experiments. Mice were voluntarily euthanased at either significant deterioration of condition or at 20% body weight loss. All animal handling procedures and protocols were assessed, approved and carried out in accordance with the guidelines of the Institute of Medical and Veterinary Science animal ethics committee and the National Health and Medical Research Council (NHMRC) of Australia.
  • NHMRC National Health and Medical Research Council
  • X31 (H3N2) influenza A a recombined virus of A/Aichi/68 and A/PR/8/34 influenza virus, was grown, in the allantoic cavity of 10 day-old embryonated chicken eggs at 34°C for 48 hrs.
  • the haemagglutinin ability end-point of the resultant virus was 1/1028
  • tissue culture infectious dose (TCID50) assessed by plaque-titration on MDCK cells was 9x10 6 /ml mouse lethal infectious dose (MED 50 ) of 10 -1.5
  • CD4 T helper peptides were also synthesised (Table 1).
  • the first is an influenza HA peptide (THIp 1) upstream of the HA cleavage site, known to be a human CD4 T helper cell epitope (4, 8).
  • the second is a tetanus toxoid peptide (THIp 2) which has been used successfully to stimulate CD4 T helper cell function in raising peptide antibodies in rabbits and mice (Prof. Shaun. McCoIl, University of Sydney, personal communication).
  • Table 1 An influenza HA peptide upstream of the HA cleavage site, known to be a human CD4 T helper cell epitope (4, 8).
  • the second is a tetanus toxoid peptide (THIp 2) which has been used successfully to stimulate CD4 T helper cell function in raising peptide antibodies in rabbits and mice (Prof. Shaun. McCoIl, University of Sydney, personal communication).
  • mice were lightly anaesthetised with 0.1ml/10g body weight of ketamine:xylazine by intraperitoneal injection. A volume of 35uL containing 5 or 6 MID 50 was delivered into the left nostril. Mice were monitored and weighed daily. Observations such as loss of coat condition, reluctance to move or consume food and water were recorded on clinical record sheets.
  • mice Two groups of 20 six- week-old female Balb/C mice were immunised IP with either saline or 100ug (combined equimolar amounts of all peptides, as measured by effective peptide content) in CFA. A booster immunisation of the same amounts of immunogen was administered two weeks later IP in IFA. Mice were further boosted by intranasal administration of 10ug of combined peptides and 0.15 ug of GalCer.
  • mice In addition to these two groups of mice, an non-blinded small-scale study was concurrently conducted to investigate the efficacy of the peptide vaccines administered via the " intranasal" (IN) route only. This vaccination protocol was particularly attractive as it alleviated the requirement for the use of Freunds or similar adjuvants and also did not require needles.
  • Two groups of 20x 6-week-oId female BALB/c were immunised with either saline or with 50ug (combined equimolar amounts of all peptides, as measured by effective peptide content) in. A single booster immunisation of the same amounts of immunogen was administered two weeks later IP in IFA.
  • mice were further boosted by intranasal administration of 10ug of combined peptides to the presence of GalCer (0.15ug). Blood samples were collected from the orbital sinus for analysis of levels of anti-peptide antibodies prior to influenza A challenge. Seven days later, all mice were challenged IN with 6 MID 50 . Mice were monitored daily for clinical symptoms, body weight and survival for a 9 day period. Eight mice from each group were euthanased on day 4 post- infection to assess immunopathology, viral RNA and antigen expression within the lung. All other mice were euthanased following 30% weight loss or day 8 p.i.
  • mice Two groups of 20 six- week-old female BALB/c mice were immunised IN with 50ug of peptide (combined equiraolar amounts of all peptides, as measured by effective peptide content) ia. the presence of GalCer (0.3ug). Two booster IN immunisations containing the same amounts of immunogen were then administered at seven day intervals. Serum was collected from the orbital sinus for analysis of levels of anti-peptide antibodies prior to influenza A challenge. Seven days after the final immunisation, mice were challenged IN with 6 MID 50 . Animals were monitored daily for clinical symptoms, body weight and survival for a 5 day period. On day 3 post-infection eight mice from each group were autopsied and lung tissue collected to assess immunopathology, viral RNA and antigen expression within the lung. All remaining mice were euthanased on day 5 post-infection.
  • Histopathology of the respiratory tract and lung of the challenged animals were examined by immuno-peroxidase staining for influenza A antigens and T-cell markers. Viral replication in the lung was measured by quantitative real-time RT-PCR.
  • Immunoperoxidase of formalin fixed lung tissue Lung tissue was fixed for 24 hours in 10% buffered formalin prior to paraffin embedding. Serial 8uM sections were cut onto polylysine (Menzel-Glaser) coated microscope slides. For the influenza and NKT specific staining, antigen was retrieved with citrate buffer retrieval. In the case of CD3 staining, antigen was retrieved by with the use of Target retrieval solution (Dako Cat # S-1699) Followed by try trypsin type II digestion (Sigma Cat # T-8128) Following retrieval endogenous peroxidise was blocked with 0.05% H202 in PBS for 15 minutes. Slides were then blocked with a 1/30 dilution of normal giat serum in PBS.
  • the primary antibodies polyclonal rabbit anti-H3N2 (12) diluted 1/1000 in 10% foetal calf serum (FCS)/PBS, monoclonal mouse anti-CD3 (DAKO ##) diluted 1/6000 in 3% normal horse serum (NHS) and rabbit anti-mouse Asialo GM1 (NK antibody) diluted 1/2000 in 10% FCS and incubated at 37°C for 1 hr then 4oC overnight. Slides were washed in PBS followed by addition of goat anti-rabbit HRP (KPL) diluted 1/200 in 10% FCS/PBS or sheep anti-mouse HRP diluted 1/200 in 3% NHS/PBS and incubated for 1 hr at 37°C . Slides were further washed in PBS and then developed by addition, of substrate 3,3' di-aminobenzidine (DAB)(Sigma) for 15 minutes in the dark then counterstained with haematoxylin and mounted.
  • DAB substrate 3,3' di-aminobenzidine
  • Plates were coated with 100ng (total peptide concentration) of pooled non-conjugated peptides (3A3, 3A4, 3B3 and 3B4) in PBS and incubated at 37° C overnight. Following washing, plates were blocked with 200uL/welI with SMPBST.
  • Plates were coated with 100ng (total peptide concentration) of pooled non-conjugated peptides (3A3, 3A4, 3B3 and 3B4) in PBS and incubated at 37° C overnight. Following washing, plates were blocked with 200uL/well with SMPBST. Samples were added to the plate at 1/1000 then farther diluted 10-fold across three wells of the plate. Following washing, sheep anti-mouse Ig HRP (GE Healthcare #NA9310V) diluted 1/5000 in SMPBST was added. Finally, substrate was added and absorbances read.
  • the matrix gene of influenza A was selected as the target for amplification as it is highly conserved amongst influenza A isolates as previously described (14).
  • PCR primers were: AMPfor1 : 5' GACCAA TCC TGT CAC CTC TGA3' (SEQ ID NO: 33) and AMPrev1 : 5'GTA TAT GAG GCC CAT RCA ACT3' (SEQ ID NQ: 34).
  • RTPCR was performed using a QuantiTect SYBR Green RT-PCR (Qiagen) with 0.5 uM of each primer added in a final volume of 25 uL/reaction. The RT- PCR was performed on a Rotorgene PCR machine.
  • cycling parameters were, reverse transcription, 50oC for 30 min, PCR activation 95°C for 15 min, cycling 94°C for 15 sec, 55°C , 30 sec, 72°C 30 sec for 45 cycles.
  • a standard curve was constructed from normal lung tissue spiked with 10uL of stock virus prior to extraction. Normal lung RNA was added to the standard curve to standardise for competitive cellular RNA at the concentration of which the test sample was diluted.
  • RNA added to the reaction was performed by a separate real-time RT-PCR under the same condition as the influenza specific reaction using primers spanning the intron/exon junctions of the mouse B-actin gene,
  • the sequence of the B-actin primers was BActF 5' CCT TCT TGG GTA TGG AAT C 3' (SEQ ID NO: 35) and BactR 5' GGA GCA ATG ATC TTG ATC T 3' (SEQ ID NO: 36).
  • Single melt curves and agarose gel electrophoresis revealed a single 200 bp product in both DNAse treated and untreated samples confirming the specificity of the primers (not shown). Normalisation was performed with the aid of a standard curve and by adjusting for relative fold increases to a normal mouse lung RNA control.
  • mice were vaccinated via the intraperitoneal route on day -35 with 100ug of peptide emulsified in Freunds Complete adjuvant. On Day -21 mice received a booster vaccination also delivered intraperitoneally in Freunds incomplete adjuvant.
  • mice were vaccinated with 5ug of peptides adjuvanted with GalCer, delivered as three doses into the nasal passages only. Control mice received saline only delivered under identical conditions. Following three doses of vaccine, significant litres of peptide specific total immunoglobulins were detected including both IgA and IgG subtypes ( Figure. 12). Following influenza A challenge all peptide and GalCer vaccinated mice rapidly recovered from infection whereas mock vaccinated mice rapidly fell below 80% of their original body weight and were euthanased (Figure 13).
  • DT3.1 TGMRNVPEKQTR/GIFGAIAG-C-DT 21mer (SEQ ID NO: 46) DT3.2 MRNVPEKQTR/GIFGAIAGF-C-DT 20mer (SEQ ID NO: 47) DT3.3: RNVPEKQTR/GIFGAIAGFIG-C-DT 20mer (SEQ ID NO: 48) DT3.4: DT-C-TGMRNVPEKQTR/GIFGAIAG 21mer (SEQ ID NO: 49) DT3.5 DT-C-MRNVPEKQTR/GIFGAIAGF 20mer (SBQ ID NO: 50) DT3.6 DT-C-RNVPEKQTR/GIFGAIAGFIG 21mer (SEQ ID NO: 51) F3.1: TGMRNVPEKQTR/GIFGAIAG 20mer (SEQ ID NO: 52) F3.2 MRNVPEKQTR/GIFGAIAGF 19mer (SEQ ID NO: 53) F3.3 RNVPEKQTR/GIFGAIAGFIG 20mer (S
  • THLp A PRYVKQNTLKLAT 13mer (SEQ ID NO: 27)
  • THLp B QYIKANSKFIGITELKK 17mer (SEQ ID NO: 28)
  • the first is an influenza HA peptide (designated THLp A here) upstream of the HA cleavage site, known to be a human CD4 T helper cell epitope. It impossible that the class II restricted recognition of the THLp A epitope is conserved in mice.
  • the second is a tetanus toxoid peptide (designated THLp B here) which has been used successfully to stimulate CD4 T helper cell function in raising peptide antibodies in rabbits and mice (Prof. Shaun McCoIl, University of Sydney, personal communication). The use of these two peptides enhance the T helper cell function needed for the generation of strong and lasting antibody responses.
  • THLp A and THLp B An equimolar mixture of THLp A and THLp B, termed TH, will be used as CD4 T helper cell peptides. Equimolar amounts of peptides, as measured by effective peptide content, will be selectively combined to form 4 groups of immunogens designated
  • Virus stocks were grown in the allantoic cavities of embryonated eggs at 37°C for 24 hrs (H5N1 virus) or at 34°C for 48 hrs (H3N2 virus) and plaque-titrated on MDCK cells. 10- week-old female BALB/c mice were used to establish the 50% mouse infectious dose (MID50), calibrated to PFU titer.
  • MID50 50% mouse infectious dose
  • Virus propagation was carried out under appropriate biosafety conditions at the IMVS (PC2, H3N2) or at the AAHL (PC3+, H5N1).
  • mice All immunisation will be carried out at the IMVS and additional immunised mice for possible necessary repeats in virus challenge experiments will be included. Control mice were immunised and boosted with adjuvants only. Two to three weeks after the second boost, mice were challenged intranasally with respective viruses at a dose of 10 MID50 under biosafety conditions at the IMVS or the AAHL as appropriate. Two different viral challenge protocols were needed. In the first challenge, animals will be monitored for clinical symptoms, body weight and survival during a 2-3 weeks period. In the second challenge experiment, mice will be sacrificed on day 4 and day 7 post virus challenge.
  • the histopathology of the respiratory tract and lung of the challenged animals will be examined in detail with immuno-florescence and /or immuno-peroxidase staining for H5N1 and H3N2 vital antigens, and where appropriate ⁇ ltra-structurally by electron microscopy.
  • Virus replication in the lung of immunised animals will be measured by quantitative teal-time RT-PCR.
  • immunohistochemistry analysis for the co-staining of an internal influenza viral antigen (NP) and mouse IgA in cells of the challenged animals were performed using lung bronchial epithelial and subepithelial tissues 4 days post virus challenge.
  • Influenza A virus X-31 (H3N2) was obtained from University of Melbourne and experimental stock was propagated in embryonated chicken eggs with a haemagglutinating titre of 1/1028 and a tissue culture infectious dose titre of 9x10 6 TCID50/ml .
  • mice were challenged by administration of 50 ⁇ l of serially diluted X31 virus stock into the nasal passages of groups often 8 week old Balb/C mice. Challenged mice were weighed on a daily basis and assessed visually for signs of disease. The onset of symptoms coincided well with the dose of the inoculum. The physical conditions of the mice that received the two highest doses of Influenza A virus (neat and 10 -1 dilution) rapidly deteriorated with clinical signs observed (ruffled coats, inactivity and weight loss) as early as days 1 and 2 post-infection respectively. Three of the mice in these two groups were found dead in their cages upon examination on the 4 th and 5th morning.
  • mice in the "neat" and 10 -1 dilution groups were euthanased at day 6 post-infection as their body weights were at the 30% weight loss threshold set by IMVS animal ethics committee. All virus challenged mice eventually showed a loss of the normally shiny and smooth coat condition, became inactive with a decrease in body weight. Surviving mice showed signs of recovery (increased activity, . water/food intake and increases in body weight) by day 8 post-infection. Control mice that received saline maintained a constant weight and normal appearance throughout the study. From the titration experiments we were able to establish a mouse infectious dose (MID50) of 10 -1.5 using our X31 virus stock. Further experiments, to optimise the doses and volume of inoculum, have since been performed and we have established the proposed challenge dose as 10 MID50 administered in a total volume of 35uL.
  • MID50 mouse infectious dose
  • mice receiving the highest doses of virus were well developed and attended by severe multifocal haemorrhage in mice receiving the highest doses of virus. Lesions were not as severe in mice challenged with lower doses of virus ranging from mild to large hemorrhrgic foci. Lesions were absent in the saline-controlled mice.
  • H3N2 peptides were synthesized first.
  • the peptide synthesis company (Mimotopes) experienced difficulties in the three C-terminal conjugated peptides, which were subsequently abandoned.
  • IP Intraperitoneal
  • a second administration was made 14 days before challenge followed by a smaller administration intranasaly, perhaps to boost any local immunity component.
  • the preliminary study provided for administration of peptides solely intra nasally. The results of these can be seen in the four graphs shown in Figure 2. Appreciable improvement ofhe experimental groups (the two right hand graphs) can be seen over the control subjects, as measured by relative weight loss over eight days. These data can be translated to survival as shown in figure 3 for the two studies. Immuno peroxidase staining for Influenza A antigens at day 4 post challenge are shown in figure 4.
  • the top two slides represent the experiment, the top left slide being representative of the major study and the top right slide being representative of the preliminary group.
  • the two control slides have appreciably more influenza A antigen present
  • Figure 5 shows the amount of Influenza A RNA levels estimates to be in 10mg of lung tissue.
  • mice were immunized intranasaly with the H5 peptides shown in Figure 18 together with the two T helper epitopes THLp A and THLpB together with alph galactoceramide substantially as set out for H3 peptides in Example 1.
  • Vaccines were administered intranasaly at Day 0, Day 14 and Day 21 , and mice were challenged with 6 LMID 50 H3N2 (X31).
  • H5N1 challenge 10 mice each were immunized as above, but challenged with 5 LMID 50 A/Vietnam/1203/04. It can be seen that two of the experimental animals survived challenge but none of the control animals survived. It is to be noted that this is a preliminary study.

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Abstract

L'invention concerne des préparations de vaccin peptidique et un procédé d'immunisation contre la grippe A au moyen du peptide subHA0 provenant d'un sous-type du virus de la grippe A. Le peptide HA0 a une longueur de 16 acides aminés ou davantage, choisis à partir de l'intérieur de la boucle de clivage de maturation d'une hémagglutinine HA0 du virus de la grippe A, ou d'une séquence consensus de celui-ci. Le peptide HA0 est administré à la muqueuse, de préférence par voie intranasale, avec un adjuvant et un peptide T helper qui sollicite la réponse immunitaire à une immunité à médiation par Th2.
PCT/AU2007/001539 2006-10-04 2007-10-04 Polypeptides immunogènes issus de la boucle de clivage de l'hémagglutinine précurseur d'un virus de la grippe WO2008040098A1 (fr)

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AU2006905461A AU2006905461A0 (en) 2006-10-04 Immunogenic Polypeptides Derived from the Cleavage Loop of Precursor Hemagglutinin of an Influenza Virus
AU2006905461 2006-10-04

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WO2011062263A1 (fr) * 2009-11-20 2011-05-26 財団法人東京都医学研究機構 Composition pharmaceutique
CN110003314A (zh) * 2019-04-11 2019-07-12 上海市计划生育科学研究所 H1n1流感病毒血凝素可诱发广谱保护性抗体的表位肽及其应用

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011062263A1 (fr) * 2009-11-20 2011-05-26 財団法人東京都医学研究機構 Composition pharmaceutique
EP2502995A1 (fr) * 2009-11-20 2012-09-26 Tokyo Metropolitan Institute of Medical Science Composition pharmaceutique
CN102782129A (zh) * 2009-11-20 2012-11-14 公益财团法人东京都医学综合研究所 药物组合物
EP2502995A4 (fr) * 2009-11-20 2013-05-01 Tokyo Metropolitan Inst Medical Science Composition pharmaceutique
CN110003314A (zh) * 2019-04-11 2019-07-12 上海市计划生育科学研究所 H1n1流感病毒血凝素可诱发广谱保护性抗体的表位肽及其应用
CN110003314B (zh) * 2019-04-11 2023-06-09 上海市计划生育科学研究所 H1n1流感病毒血凝素可诱发广谱保护性抗体的表位肽及其应用

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