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WO2008037033A1 - Vaccin contre la grippe constitué d'un mélange de mannane et d'antigène de la grippe - Google Patents

Vaccin contre la grippe constitué d'un mélange de mannane et d'antigène de la grippe Download PDF

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Publication number
WO2008037033A1
WO2008037033A1 PCT/AU2007/001460 AU2007001460W WO2008037033A1 WO 2008037033 A1 WO2008037033 A1 WO 2008037033A1 AU 2007001460 W AU2007001460 W AU 2007001460W WO 2008037033 A1 WO2008037033 A1 WO 2008037033A1
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Prior art keywords
flu
mannan
virus
composition
antigen
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PCT/AU2007/001460
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English (en)
Inventor
Geoffrey Allan Pietersz
Sandra Elizabeth Esparon
Owen Proudfoot
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The Macfarlane Burnet Institute For Medical Research And Public Health Limited
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Publication date
Priority claimed from AU2006222717A external-priority patent/AU2006222717A1/en
Application filed by The Macfarlane Burnet Institute For Medical Research And Public Health Limited filed Critical The Macfarlane Burnet Institute For Medical Research And Public Health Limited
Priority to EP07815271A priority Critical patent/EP2066332A4/fr
Priority to AU2007302633A priority patent/AU2007302633A1/en
Priority to US12/443,048 priority patent/US8182821B2/en
Publication of WO2008037033A1 publication Critical patent/WO2008037033A1/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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • A61K2039/5252—Virus inactivated (killed)
    • 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
    • A61K2039/55583—Polysaccharides
    • 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

  • the present invention relates to a vaccine composition
  • a vaccine composition comprising a carbohydrate polymer comprising mannose and influenza virus (flu) antigen(s) (e.g. whole inactivated flu), and a method of immunising a subject comprising the step of administering the vaccine composition to a subject.
  • flu mannose and influenza virus
  • antigen(s) e.g. whole inactivated flu
  • the oral polio vaccine remains the only successful large-scale vaccine that gives protection at the mucosal level. It is a live vaccine whose attenuation was achieved empirically with the attendant risks of back-mutation.
  • immunisation with defined protein antigens would have many advantages, including safety in immunosuppressed individuals.
  • adjuvants and delivery systems to overcome the problems associated with mucosal vaccination using defined protein antigens, including poor immunogenicity or the induction of tolerance.
  • Bacterial enterotoxins such as cholera toxin (CT) produced by Vibrio cholerae, and labile toxins from Escherichia coli have been co-administered with antigens and act as adjuvants with antigens [5,6].
  • CT cholera toxin
  • labile toxins from Escherichia coli have been co-administered with antigens and act as adjuvants with antigens [5,6].
  • these adjuvants pose health risks due to their toxic properties, and for the most part, cannot be used in human trials, and have limited usefulness for human vaccination.
  • Vaccines containing DNA encoding an antigen and low-viscosity carboxymethylcellulose sodium salt (CMCS-L) as a vehicle to carry the DNA to a site of action have also been studied [7].
  • CMPL monophosphoryl lipid A
  • Adjuvants which have been administered with immunogens via the mucosa have therefore consisted of potentially dangerous bacterial or viral derivatives and display variable degrees of adjuvanticity.
  • Mannan a polymannose or polysaccharide derived from the cell wall of yeast, when oxidised and conjugated to human Mucin 1 (an overexpressed cancer antigen) has been used in mice to induce immune responses [10, H]. Intraperitoneal immunisation resulted in the induction of cellular immune responses as shown by production of cytotoxic T lymphocytes (CTLs) and their precursors, ThI cytokines IFN- ⁇ and IL-12. Antibody production was usually low. In tests in more than 100 patients, mannan has not shown any obvious toxicity or autoimmunity [12].
  • CTLs cytotoxic T lymphocytes
  • ThI cytokines IFN- ⁇ and IL-12 cytotoxic T lymphocytes
  • Mannose receptors which bind mannan have so far been identified on macrophages and dendritic cells. Oxidised mannan has been shown to stimulate production of IL-12 in macrophages, and also to stimulate T-cells, and to cause rapid trafficking of antigens to the class I pathway to produce cytotoxic T-cells in mice [13].
  • a carbohydrate polymer such as mannan is administered via a mucosal site (e.g. by intranasal administration), beneficial immunogenic effects are produced.
  • an antigen from an influenza virus (flu) such as a whole inactivated flu
  • influenza-specific mucosal IgA and/ or serum IgG immune repsonses can be produced.
  • intramuscularly i.m.
  • such an admixture of mannan and a flu antigen elicited a strong IgG immune response when administered intramuscularly (i.m.), such an admixture of mannan and a flu antigen elicited a strong IgG immune response.
  • the present invention provides a vaccine composition
  • a vaccine composition comprising a carbohydrate polymer comprising mannose and influenza virus (flu) antigen(s) in admixture.
  • the flu antigen(s) may be derived from human or animal influenza viruses (e.g. avian flu and equine flu).
  • the flu antigen (s) is preferably selected from whole inactivated influenza virus (e.g. whole inactivated strain HlNl flu virus).
  • the carbohydrate polymer comprising mannose is preferably a carbohydrate polymer comprising mannan, more preferably oxidised mannan.
  • the vaccine composition is adapted to be administered at a mucosal site.
  • the present invention provides a method of immunising a subject, comprising the step of administering a composition comprising a carbohydrate polymer comprising mannose and influenza virus antigen(s) in admixture.
  • the step of administering the composition involves administration through a mucosal site (e.g. intranasal (i.n.) administration), but other routes, and particularly intramuscular (i.m.) administration, are also suitable.
  • a mucosal site e.g. intranasal (i.n.) administration
  • other routes, and particularly intramuscular (i.m.) administration are also suitable.
  • the subject immunised may be a human or other animal.
  • the present invention resides in the use of a carbohydrate polymer comprising mannose and influenza virus antigen(s) in the preparation of an admixture vaccine composition for immunising a subject.
  • the vaccine composition comprises a carbohydrate polymer comprising mannose (preferably mannan) and a limited (i.e. antigen- or dosage-sparing) amount of flu antigen(s) (preferably, whole inactivated strain HlNl or H51N influenza virus) in admixture.
  • Figure 1 shows IgG levels in mice when intranasally administered 10 ⁇ g dose of strain HlNl influenza (flu) virus alone, flu virus and mannan admixture, and flu virus-mannan conjugate;
  • Figure 2 shows IgG levels in controls involving intramuscular administration of strain HlNl influenza (flu) virus alone, flu virus and mannan admixture, and flu virus-mannan conjugate;
  • Figure 3 shows IgA levels at 10 ⁇ g dose in mice when intranasally administered 10 ⁇ g dose of strain HlNl influenza (flu) virus alone, flu virus and mannan admixture, and flu virus-mannan conjugate;
  • Figure 4 shows IgA levels in controls involving intramuscular administration in the mice when intranasally administered 10 ⁇ g dose of strain HlNl influenza (flu) virus alone, flu virus and mannan admixture, and flu virus-mannan conjugate;
  • Figure 5 shows IgA levels in lung washes of mice administered intranasally with 10 ⁇ g dose of strain HlNl influenza (flu) virus alone, flu virus and mannan admixture, and flu virus-mannan conjugate;
  • Figure 6 shows serum IgGl levels of mice administered intranasally with 10 ⁇ g dose of strain HlNl influenza (flu) virus and mannan admixture 14 days apart;
  • Figure 7 shows IgG2a levels of mice administered intranasally with 10 ⁇ g dose of strain HlNl influenza (flu) virus and mannan admixture 14 days apart;
  • Figure 8 shows IgA levels of mice administered intranasally with 10 ⁇ g dose of strain HlNl influenza (flu) virus and mannan admixture 14 days apart;
  • Figure 9 shows results of IgA levels from lung washes of mice administered 3 times intranasally with 10 ⁇ g dose of strain HlNl influenza (flu) virus and mannan admixture 14 days apart;
  • Figure 10 shows serum HI after intramuscular injection of mice with strain HlNl influenza (flu) virus alone, flu virus and mannan admixture, and flu virus- mannan conjugate;
  • Figure 11 shows serum HI after intranasal administration of mice with strain HlNl influenza (flu) virus alone, flu virus and mannan admixture, and flu virus- mannan conjugate;
  • Figure 12 shows a Western blot of gels showing that antibodies produced to strain HlNl influenza (flu) virus are cross-reactive with strain H5N1 influenza (flu) virus;
  • Figure 13 shows HI titres in mice given two immunisations (7 days apart) with with strain HlNl influenza (flu) virus alone, flu virus and mannan admixture, and flu virus-mannan conjugate;
  • Figure 14 shows the results of serum IgG from mice intranasally administered with flu virus mannan admixtures with various amounts of mannan and a low (i.e. 1 ⁇ g) amount of strain HlNl influenza (flu) virus;
  • Figure 15 shows serum HI after intranasal administration with flu virus mannan admixtures with various amounts of mannan and a low amount of strain HlNl influenza (flu) virus;
  • Figure 16 shows serum IgGl levels in mice following intranasal administration of various amounts of strain H5N1 influenza (flu) virus in admixture with mannan (amount of mannan to flu virus ratio was 1:10);
  • Figure 17 shows lung IgA levels in mice following intranasal administration of various amounts of strain H5N1 influenza (flu) virus in admixture with mannan (amount of mannan to flu virus ratio was 1:10).
  • the present invention provides a vaccine composition comprising a carbohydrate polymer comprising mannose and influenza virus (flu) antigen(s) in admixture.
  • composition simply comprises a mixture such that there is no covalent linkage between the carbohydrate polymer and the antigen(s).
  • the flu antigen(s) may be derived from human or animal influenza viruses (e.g. avian flu and equine flu).
  • the flu antigen (s) is preferably selected from whole inactivated influenza (flu) virus, attenuated flu preparations, and specific antigenic flu polypeptides (e.g. for "subunit” vaccine compositions), or antigenic or epitope-containing fragments thereof.
  • Particular whole inactivated influenza viruses suitable for use in the present invention include whole inactivated strain HlNl influenza virus and whole inactivated strain H5N1 influenza virus.
  • Particular antigenic flu polypeptides suitable for use in the present invention include haemagglutinin (Ha) polypeptides (e.g. Ha from human, avian or equine flu).
  • Ha haemagglutinin
  • An antigenic flu polypeptide, or antigenic or epitope-containing fragment thereof may form part of a fusion protein or polypeptide in order to, for example, act as a carrier and/ or facilitate expression and purification upon production of the fusion protein in recombinant host cells.
  • the non-antigen portion of the fusion protein would generally represent the N-terminal region of the fusion potein or polypeptide with the C- terminal sequences comprising the flu sequences.
  • Fusion proteins and polypeptides may be selected from glutathione- S-transferase, ⁇ -galactosidase, or any other protein or part thereof, particularly those which enable affinity purification utilising the binding or other affinity characteristics of the protein (or part thereof) to purify the resultant fusion protein or polypeptide.
  • the nature of the fusion protein or polypeptide will depend upon the vector system in which fusion proteins are produced.
  • An example of a bacterial expression vector is pGEX which upon subcloning of a flu sequence of interest, produces a fusion protein consisting of glutathione-S- transferase with the flu polypeptide or peptide. Examples of other vector systems which give rise to fusion proteins are described in Sambrook et ah, "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Press, United States of America, 1989, incorporated herein in its entirety by reference.
  • the carbohydrate polymer comprising mannose is most preferably a carbohydrate polymer comprising mannan.
  • the carbohydrate polymer is oxidised mannan (as described in WO 95/18145, which is hereby incorporated in its entirety by reference).
  • the mannan is preferably isolated from the cell wall of yeast, and may be oxidised using reagents such as sodium periodate to produce a polyaldehyde.
  • Reduced mannan may also be used, and a composition comprising reduced mannan may be prepared by adding sodium borohydride to an oxidised mannan.
  • the amount (by weight) of the mannan relative to the flu antigen(s) may be in the range of 3:1 to 250:1, preferably 5:1 to 120:1, and more preferably 10:1 to 100:1.
  • the vaccine composition is adapted to be administered at a mucosal site.
  • the vaccine composition may be formulated for administration at a mucosal site by inhalation, through spraying the vaccine composition (e.g. an aerosol formulation) into the nasal region.
  • the vaccine composition may also be formulated as droplets or fluid placed onto, or applied to, one or more mucosal sites such as regions of the mouth, tongue, throat, the gut including the stomach, the nasal and respiratory passages including the lungs, the reproductive tract including the vagina and cervix, and the rectum.
  • the vaccine composition may also be by formulated as a cream or balm for administration through rubbing or massaging onto one or more accessible mucosal sites.
  • the vaccine composition may also be formulated as a slow- or time-release device such as a capsule or a suppository (e.g. those based on alginate microspheres, poly(lactide-co-glycolide) or collagen polymers) which may be inserted, for example, in the rectum.
  • the vaccine composition may also be formulated for administration through injection into a mucosal site.
  • the vaccine composition may further comprise other adjuvants, pharmaceutically acceptable carriers, diluents or auxiliaries which may enhance the immunogenicity or effectiveness of the vaccine composition.
  • the vaccine composition may be provided in accordance with the manner in which it is to be administered.
  • the vaccine composition may be provided in a spray-container, aerosol can or nebuliser for intranasal administration by inhalation.
  • the vaccine composition may be provided in a dropper bottle to enable dropwise administration of a solution or liquid formulation of the composition (e.g. to the oral cavity or throat for absorption).
  • the present invention provides a method of immunising a subject, comprising the step of administering a composition comprising a carbohydrate polymer comprising mannose and influenza virus antigen(s) in admixture.
  • the step of administering the composition involves administration through a mucosal site (e.g. intranasal (i.n.) administration), but other routes, and particularly intramuscular (i.m.) administration, are also suitable.
  • a mucosal site e.g. intranasal (i.n.) administration
  • other routes, and particularly intramuscular (i.m.) administration are also suitable.
  • the immunisation may result in a cellular immune response against influenza virus, but preferably results in a humoral immune response against influenza virus. More preferably, the immunisation results in a humoral response wherein influenza-specific IgA antibody production (e.g. an IgA immune response) is stimulated, such that the titre of IgA at a selected mucosal area, and/ or in the serum, is increased.
  • influenza-specific IgA antibody production e.g. an IgA immune response
  • the IgA production following immunisation is greater when compared with production of IgG, IgM and IgE.
  • the immunisation results in greater production of IgA relative to the increase in IgGl and/ or IgG2a production.
  • the levels of antibody may be increased in serum or at mucosal sites, or both.
  • a secretory IgA immune response against influenza virus at one or more mucosal sites can be particularly advantageous, because this can assist in the neutralisation of influenza virus upon entry into the body of the subject through mucosal membranes.
  • Administration of the vaccine composition at a mucosal site may involve administration at a region, or any area, of any mucosal surface (e.g. those lining the oral cavity or tissues, including the teeth and gingivae, those lining the gastrointestinal tract, or those lining the nasal passages and lungs, and the reproductive tract/ tissues).
  • the conjunctiva of the eyes also provides a suitable mucosal site for the administration of the vaccine composition.
  • preferred examples of mucosal sites for administration of the vaccine composition are the respiratory tract such as the nasal region (i.e. intranasal (i.n.) administration), the trachea, bronchi and the lungs, the buccal or oral tissues including the oral (e.g. the mouth and gingivae) and oro-pharyngeal cavities, the throat including the tonsils, the gastrointestinal tract (e.g. oesophagus, stomach, duodenum, small and large intestines, colon and rectum).
  • the respiratory tract such as the nasal region
  • the step of administering the vaccine composition may be conducted by a single event or multiple events, or may be part of a prime-boost protocol, a combination of these, or each of these with other, conventional methods of administration of a vaccine composition.
  • the prime-boost protocol may, for example, comprise priming by intramuscular administration, and boosting by intranasal administration.
  • the prime-boost may, in addition to the vaccine composition of the present invention, include DNA, viruses (e.g. vaccinia) or other immunogenic peptides or molecules, including the antigens described above.
  • One or both of the priming and boosting composition may include the vaccine composition of the present invention.
  • one of the events may include the vaccine composition in accordance with the present invention, while the other may omit the carbohydrate polymer comprising mannose.
  • the amount and frequency that the vaccine composition is given to a subject may be routinely determined by an attending physician or veterinarian. However, by way of example, l ⁇ g to 10,000 ⁇ g/kg may be administered to a subject, preferably 5 ⁇ g to 5000 ⁇ g/kg, more preferably 8-1000 ⁇ g/kg and most preferably 400- 600 ⁇ g/kg. Even more preferably, a dose of 100-200 ⁇ g/kg is contemplated, particularly for a human subject.
  • the vaccine composition may also be administered to a subject in conjunction with other immune regulators that lend themselves to efficacious administration via the mucosa. Further, the vaccine composition may also be administered with other adjuvants, pharmaceutically acceptable carriers, diluents or auxiliaries which may enhance the immunogenicity or effectiveness of the vaccine composition, or the vaccine composition may also be included therein, or be coadministered therewith. For example, lipofectamine may be co-administered with the vaccine composition of the invention.
  • the subject immunised may be a human or other animal.
  • the invention may be used to prevent influenza in humans or other animals, especially mammals (e.g. livestock and companion animals).
  • the mannan not only acts as an adjuvant, but is also a potent mucosal adjuvant by virtue of increased or efficient uptake via the mucosa.
  • the present invention resides in the use of a carbohydrate polymer comprising mannose and influenza virus antigen(s) in the preparation of an admixture vaccine composition for immunising a subject.
  • the vaccine composition comprises a carbohydrate polymer comprising mannose (preferably mannan) and a limited (i.e. antigen- or dosage-sparing) amount of flu antigen(s) (preferably, whole inactivated strain H51N influenza virus) in admixture. That is, it has been found that the vaccine composition of the present invention may comprise surprisingly low amounts of flu antigen(s) (e.g.
  • the vaccine composition of the particularly preferred embodiment is preferably adapted to be administered intranasally.
  • the vaccine composition of the particularly preferred embodiment also preferably comprises an amount (by weight) of the mannan relative to the flu antigen(s) in the range of 50:1 to 200:1, preferably 80:1 to 120:1, and more preferably about 100:1.
  • mannan admixtures were prepared by simply mixing the whole inactivated flu virus with an amount of mannan in solution.
  • the amount of mannan to flu virus is typically 10:1, but preferably admixtures are prepared to provide a doasge unit comprising 100 ⁇ g of mannose regardless of the amount of flu virus.
  • mice were lightly anaesthetised with penthrane and 50 ⁇ l of vaccine composition (i.e. flu antigen(s) mannan admixture or mannan-antigen conjugate) (12 ⁇ g antigen/ mouse in bicarbonate buffer pH9.0, unless otherwise specified), placed onto the nares to be inhaled by the mouse. Unless stated otherwise, this procedure was performed on days 0, 10 and 17 of the experiments.
  • vaccine composition i.e. flu antigen(s) mannan admixture or mannan-antigen conjugate
  • mice Serum samples were collected after mice were bled by cardiac puncture following euthanasia at the end of each experiment. For time-course experiments, mice were placed on a heat box, a small incision made in a lateral vein and 200 ⁇ l of blood collected with a micropippeter. The serum was subsequently separated by centrifugation. Lung washings were obtained after mice were euthanased with CO 2 . Lungs were washed in situ with 0.5ml PBS through an opening in the trachea. All samples were stored at -20 0 C prior to assay.
  • Microtitre plates (Nunc Roskilde, Denmark) were coated overnight at 4 0 C with 5 ⁇ g/ml antigen in carbonate buffer pH 9.1. The wells were then blocked with 2% foetal calf serum (FCS) (Trace Biosciences, Castle Hill, NSW, Australia) in PBS for 1 hour at 37 0 C. The plates were washed 3 times with 0.08% Tween 20 (BDH Laboratory Supplies, Poole, England) PBS and appropriately diluted samples in 50 ⁇ l added and incubated for 2 hours at room temperature.
  • FCS foetal calf serum
  • antigen-specific IgA was detected by the addition of an anti-mouse IgA affinity purified horse radish peroxidase (HRP) conjugate (Southern Biotechnology Associates Inc., Birmingham, United States of America) diluted 1/1000 in 0.1% bovine serum albumin (BSA) (CSL, Melbourne, Australia) for 1 hour at room temperature.
  • HRP horse radish peroxidase
  • BSA bovine serum albumin
  • Antigen specific IgGl or IgG2a was detected with the addition of a biotinylated anti-mouse IgGl or IgG2a conjugate (Caltag
  • the antibody titres of all the subclasses tested were determined when the substrates containing either 0.4 g/13, 3', 5, 5'-tetramethylbenzidine (TMB) (Kirkgaard and Perry Laboratories, Gaithersburg, MD, United States of America) and 0.02% H 2 O 2 or 2,2'-azino-bis(3- ethylbenthiazoline-6-sulphonic acid) (ABTS) (Sigma Chemical Co., St Louis, MO, United States of America) and 0.03% H2O2 were added (50 ⁇ l /well). Plates were left 10 minutes for colour to develop and the reaction stopped with 2M H2SO4 for the TMB substrate or 0.2M citric acid for ABTS.
  • TMB trimethylbenzidine
  • ABTS 2,2'-azino-bis(3- ethylbenthiazoline-6-sulphonic acid)
  • OD at 450 ran (TMB) or 405nm (ABTS) was read in an ELISA reader (Labsy stems, Helsinki, Finland).
  • Antibody titres were presented as the highest dilution which yielded an optical density at 450 run or 405 nm >0.1 OD units higher than normal serum at 1/100 dilution. For calculation of means, the titre was converted to logw and a geometric mean was derived.
  • HlNl was prepared along the lines set forth in Example 1. Inactivated HlNl was obtained from the WHO collaborative centre, Melbourne (Victroia, Australia), and prepared according to Davenport et al. [44]. Briefly, the virus was grown in embryonated hen eggs and purified using standard conditions. The virus was inactivated with gamma-butyrolactone.
  • Table 1 The various doses and modes of administration are shown below in Table 1: Table 1
  • Antibody levels were measured. At the 400 ng dose there was no enhancement of immunogenicity by mannan (admixture or conjugate) as shown by IgG levels. At the 5,000 ng dose the admixed flu virus mannan ("mannan mixed”) showed unexpected enhancement in IgG. This was more pronounced at the 10,000 ng dose (i.e. 10 ⁇ g) as shown in Figure 1.
  • Figure 2 shows results from positive controls involving intramuscular administration. The observation was confirmed when serum IgG was tested after two immunisations 10 days apart (data not shown).
  • lung washes were performed (mice were culled using drugs not CO 2 or cervical dislocation) and 700 to 1,000 ⁇ l of PBS was introduced then carefully recovered and assayed as described in Example 1.
  • the flu virus mannan admixture showed superior results as seen in Figure 5.
  • immunisation by intranasal administration of the flu virus and mannan admixture preferably involved two immunisations (two weeks apart), whereas high levels of serum IgG could be produced after a single intramuscular administration with the flu virus mannan admixture (although this did not result in any mucosal IgA).
  • the single intramuscular administration could use as little as 0.1 ⁇ g of flu virus (strain H5N1 tested) to induce a strong serum IgG immune response.
  • Example 3 Further experiments with 10 ⁇ g admixed flu virus and mannan
  • mice received three immunisations 14 days apart (day 0, day 14 and day 28). The mice were bled on days 12, 26 and 35 and an analysis of serum antibody and IgA profiles were conducted including tests for IgGl and IgG2a. At the conclusion, mice were sacrificed and a lung fluid wash was conducted to determine levels of secretory IgA. Results are shown in Figures 6 to 9.
  • This example demonstrates the extended time frame between doses yields multiple benefits in antibody responses.
  • admixed flu virus and mannan administered intranasally at three 14 days intervals to mice results in reasonable IgGl and IgG2a titres more conveniently than the day 0, 10, 17 protocol.
  • IgGl was boosted to 10 5 after the second immunisation which was greater than the "total" IgG in the 0, 10, 17 day protocol.
  • IgG2a was boosted to around 5 x 10 4 after the second immunisation and 10 5 after the third immunisation.
  • intranasal administration at three 14 day intervals of the admixed flu virus and mannan results in reasonable IgA titres and is a marked improvement over the shorter immunisation protocol.
  • Similar end titres of IgA antibodies may be obtained by three immunisations at shorter intervals or by two immunisations at 14 days.
  • the 14 day protocol resulted in vastly improved IgA in lung wash fluid than the 0, 10, 17 day protocol.
  • HI assays were conducted. This is a well known assay and followed the protocol using turkey or chicken red blood cells. The assay measures the ability of the sera from immunised mice to inhibit the virus binding to sugar residues on the red blood cells. A final dilution of sera (across a two-fold series dilution starting at 10 fold) that inhibits agglutination denotes the "HI titre" of the sample. A "HI titre" of 40 is considered protective.
  • H5N1 Sera from the mice of Example 2 were used. Inactivated H5N1 was from the WHO collaborative centre, Melbourne. This virus was made by reverse genetics so that it can be grown in chicken eggs and inactivated as above. For the H5N1 in HI assays, live virus was used. The H5N1 is a modified H5N1 rather than the virulent H5N1 in that the polybasic site of the virulent strain has been removed. This also means that BL3 containment conditions do not have to be used with this virus.
  • Sera was collected from mice separately immunised with bicarbonate buffer alone, 10 ⁇ g HlNl alone, 10 ⁇ g HlNl -mannan admixture, 10 ⁇ g HlNl conjugated with oxidised mannan and, as a positive control, rabbit anti-HINl polyclonal antibody (Burnet Institute, Melbourne, Victoria, Australia) similar to Example 2.
  • Inactivated HlNl, H3N2 from the WHO collaborative centre,
  • H5N1 were run on an SDS-PAGE gel transferred to nitrocellulose and probed with the above anti-sera.
  • ferrets groups of 4 animals are lightly anaethetised and immunised intranasally with mannan conjugated to inactivated H5N1 virus, admixture of virus and mannan, virus alone and saline alone. Ferrets are observed daily for signs of illness and nasal washings collected every 3 days for 15 days post infection. To collect nasal washings ferrets are lightly anaesthetised and 0.5 ml PBS instilled into each nostril. The expelled liquid is collected, centrifuged and the supernatant used to assess viral titres and protein concentration.
  • mice are challenged with different doses of inactivated virus which are administered intranasally to groups of 5 to 8 mice on day 0, 14 and 28. 10 days after each immunisation mice are bled and sera collected and assayed for IgG2a, IgGl, IgM and IgA antibody against the H5N1 virus. If satisfactory titres are obtained some mice are euthanased after a further 10 days and lung washings isolated and tested for mucosal IgA. In addition other mice are challenged with live virus and survival rates, live weights and nasal washings are analysed.
  • Example 7 Use of "Split" H5N1 in admixture with mannan in intranasal vaccination
  • split H5N1 virus (i.e. fragmented virus) is used as above.
  • the split virus is virus that has been dissociated into various subunits by sodium deoxycholate.
  • the analysis of antibody titres is as above.
  • Example 8 Vaccination with H5N1
  • Vietnam. H " ⁇ 10" means no Hl observed at all, even at dilution factor 10.
  • Hl "actual 10" means Hl observed at dilution factor 10, but not beyond. Note that the only "single immunisation" HlNl trial sera tested were samples 33 to 36. The results are shown in Table 2 below:
  • Example 9 Dosage sparing amounts of HlNl and H5N1 Experiments were conducted to determine whether increasing the amount of mannan in admixed flu virus mannan compositions could reduce the dosage amount of the flu virus.

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Abstract

La présente invention concerne une composition de vaccin qui contient un polymère d'hydrate de carbone constitué d'un mélange de mannose et d'un ou de plusieurs antigènes contre la grippe (par exemple le virus entier de la grippe inactivé), ainsi qu'un procédé permettant d'immuniser un sujet et consistant à administrer la composition de vaccin à un sujet.
PCT/AU2007/001460 2000-06-06 2007-10-02 Vaccin contre la grippe constitué d'un mélange de mannane et d'antigène de la grippe WO2008037033A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP07815271A EP2066332A4 (fr) 2000-06-06 2007-10-02 Vaccin contre la grippe constitué d'un mélange de mannane et d'antigène de la grippe
AU2007302633A AU2007302633A1 (en) 2000-06-06 2007-10-02 Flu vaccine admixture of mannan and flu antigen
US12/443,048 US8182821B2 (en) 2000-06-06 2007-10-02 Flu vaccine admixture of mannan and flu antigen

Applications Claiming Priority (2)

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AU2006222717 2006-09-29
AU2006222717A AU2006222717A1 (en) 2000-06-06 2006-09-29 Improved vaccine

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WO2008037033A1 true WO2008037033A1 (fr) 2008-04-03

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013131898A1 (fr) 2012-03-06 2013-09-12 Crucell Holland B.V. Vaccination améliorée contre la grippe
RU2578420C2 (ru) * 2010-05-10 2016-03-27 Эсенд Байофармасьютикалз Пти Лтд Иммуностимулирующие и вакцинные композиции
RU2715674C1 (ru) * 2019-09-02 2020-03-02 Федеральное государственное бюджетное научное учреждение "Институт экспериментальной медицины" (ФГБНУ "ИЭМ") Вакцинный штамм вируса гриппа А/17/Швейцария/2017/51 (H3N2) для производства живой гриппозной интраназальной вакцины для взрослых и для детей

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000050078A1 (fr) * 1999-02-26 2000-08-31 Chiron Corporation Utilisation de systemes bioadhesifs et d'adjuvants pour l'administration mucosale d'antigenes
WO2001093912A1 (fr) * 2000-06-06 2001-12-13 The Austin Research Institute Vaccin

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000050078A1 (fr) * 1999-02-26 2000-08-31 Chiron Corporation Utilisation de systemes bioadhesifs et d'adjuvants pour l'administration mucosale d'antigenes
WO2001093912A1 (fr) * 2000-06-06 2001-12-13 The Austin Research Institute Vaccin

Non-Patent Citations (1)

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Title
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2578420C2 (ru) * 2010-05-10 2016-03-27 Эсенд Байофармасьютикалз Пти Лтд Иммуностимулирующие и вакцинные композиции
US9597392B2 (en) 2010-05-10 2017-03-21 Ascend Biopharmaceuticals Pty Ltd. Use of high molecular weight mannan for inducing and/or enhancing an immune response
WO2013131898A1 (fr) 2012-03-06 2013-09-12 Crucell Holland B.V. Vaccination améliorée contre la grippe
RU2715674C1 (ru) * 2019-09-02 2020-03-02 Федеральное государственное бюджетное научное учреждение "Институт экспериментальной медицины" (ФГБНУ "ИЭМ") Вакцинный штамм вируса гриппа А/17/Швейцария/2017/51 (H3N2) для производства живой гриппозной интраназальной вакцины для взрослых и для детей

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