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WO2005060330A2 - Adjuvant de vaccin lyophilise - Google Patents

Adjuvant de vaccin lyophilise Download PDF

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Publication number
WO2005060330A2
WO2005060330A2 PCT/DK2004/000893 DK2004000893W WO2005060330A2 WO 2005060330 A2 WO2005060330 A2 WO 2005060330A2 DK 2004000893 W DK2004000893 W DK 2004000893W WO 2005060330 A2 WO2005060330 A2 WO 2005060330A2
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Prior art keywords
adjuvant
dodac
derivatives
carbon atoms
dda
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PCT/DK2004/000893
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English (en)
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WO2005060330A3 (fr
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Else Marie Agger
Peter Andersen
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Statens Serum Institut
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Publication of WO2005060330A2 publication Critical patent/WO2005060330A2/fr
Publication of WO2005060330A3 publication Critical patent/WO2005060330A3/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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/14—Quaternary ammonium compounds, e.g. edrophonium, choline
    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • 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
    • A61K2039/55544—Bacterial toxins
    • 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
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • 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
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A

Definitions

  • the present invention relates to an adjuvant, which can be freeze-dried and subsequently dissolved without heating.
  • the invention relates to vaccine adjuvants in aqueous media for immunization, where the final product can be freeze dried for storage and transport and later be dissolved without heating before use.
  • the invention also relates to vaccines and immunization combination kits comprising said adjuvant and an antigenic substance.
  • tuberculosis in man has for many years been combated by vaccination with attenuated but living strains of Mycobacterium bovis (BCG vaccine).
  • BCG vaccine Mycobacterium bovis
  • DDA quaternary hydrocarbon ammonium halogenide dimethyl dioctadecyl ammonium bromide or chloride.
  • DDA is a lipophilic quaternary ammonium compound, which forms cationic liposomes in aqueous solutions at temperatures above 40°C. It promotes cell mediated immunity (Hilgers & Snippe, 1992). Combinations of DDA and other immunomodulating agents have been described.
  • tuberculosis and DDA generated a protective immune response against TB in mice (Andersen, 1994).
  • Vaccination of mice with a fusion protein of M. tuberculosis proteins ESAT-6 and Ag85B, and DDA/MPL as adjuvant provides protection similar to that obtained by BCG vaccination (Olsen et al, 2001).
  • DDA-based adjuvants are able to induce a protective immune response against TB in mice.
  • DDA has been used as an adjuvant for a DNA vaccine against pseudorabies virus leading to enhanced T-cell responses and anti-viral immunity (van Rooij et al, 2002).
  • DDA as an adjuvant
  • the final vaccine containing the adjuvant has to be kept cold during transport and storage and DDA forms precipitates when it is stored at 4 degrees Celsius.
  • DDA needs careful handling and storage with regard to temperature.
  • the cold chains necessary for storage of such vaccines do not exist. This means at best that the vaccines are unavailable, at worst that improperly stored and potentially dangerous material can be distributed.
  • the cost of cold storage and distribution can be many times the cost of the vaccine, meaning that it may be economically unappealing for companies to make or distribute an effective vaccine. Even if this is not the case the extra cost of refrigeration at all steps of the vaccine distribution chain means that the price may thereby be placed out of reach of many consumers.
  • the present invention discloses a new adjuvant system based on cationic liposomes, which possesses the capacity to elicit a strong and long-persisting immune response, when administered in combination with an antigenic substance even though this antigenic substance may have only poor immunogenicity per se, and additionally can be freeze-dried and subsequently dissolved without heating.
  • the adjuvant system can be controlled in regard to particle size and main phase transition temperature, which is important parameters when developing stable freezedried liposome adjuvants, able to be dissolved.
  • the present invention relates to an adjuvant comprising a quaternary amine with a halogen counter ion of the formula NR 1 R 2 R 3 R -hal, wherein R 1 and R 2 independently each is a short chain alkyl group containing 1 to 3 carbon atoms, preferably methyl groups, R 3 and R 4 independently each is an alken containing from 12 to 20 carbon atoms, preferable from 14 to 18 carbon atoms, and hal is a halogen atom, not comprising any nucleic acid as adjuvant.
  • the R 1 and R 2 groups may e.g. be methyl, ethyl, propyl and isopropyl, whereas R 3 and R 4 may be dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl nonadecenyl and eicocenyl groups.
  • C 12 -C 2 o alkenes are possible because even though the R 3 and R 4 groups usually and preferably are straight chain hydrocarbon groups they may in minor degree be branched having e.g. methyl and ethyl side chains.
  • halogen atom "hal" is preferably bromine or chlorine because the other halogens, fluorine and iodine, may have undesirable biochemical, physiological and injurious effects, but for some experimental purposes, where such effects can be accepted, they may also be selected.
  • a preferred embodiment is wherein a quaternary amine with a halogen counter ion is dimethyl dioctadecenyl ammonium bromide or chloride (DODAC-Br or DODAC-CI.
  • DODAC-Br or DODAC-CI dimethyl dioctadecenyl ammonium bromide or chloride
  • DODAC DODAC
  • nucleic acid e.g. nucleic acid
  • WO0394828 and US2003125292 it is also known to use DODAC together with immunomodulating particles of nucleic acid e.g. CpG-motifs and an antigen to induce an immune response.
  • CpG-motifs are known ligands of the Tolllike receptor-9 whereas the underlying mechanism for the immune stimulatory actions of DODAC and DDA is unknown.
  • DDA In contrast to DODAC, DDA has a long history of proven adjuvant effect and promotes a combined humoral and cell- mediated immune response (Hilgers and Snippe, 1992). Moreover, investigations have shown that the use of DDA is not related to any kind of toxicity. (Larsen, ST. et al, 2004). Combinations of DDA and other immunomodulating agents have been described. DDA/MPL as adjuvant provides protection similar to that obtained by BCG, when combined with a fusion protein of M. Tuberculosis proteins ESAT-6 and Ag85B (WO0069458 and Olsen et al, 2001).
  • the above-mentioned quaternary amines have amphiphilic properties i.e. two long hydrophobic carbon-chains attached to a hydrophilic quaternary ammonium head- group. When dispersed in water these amphiphilic molecules self-organize into closed-vesicle structures e.g. liposomes.
  • main phase transition temperature of the vesicles that takes the lipid bilayer from a low-temperature gel- phase, characterized by ordered alkyl chains (a solid-ordered phase) to a high- temperature fluid-phase in which the alkyl chains have a high degree of conformational disorder (a liquid-disordered phase) has been reported to influence the immunological response of phospholipid adjuvants (Kersten, G.F.A, 1995).
  • the main phase transition of a quaternary ammonium adjuvant system is increased by incorporating quaternary amines of the formula NR1 R2R3R4-hal, wherein R 1 and R 2 groups may e.g.
  • phase transition temperature of an adjuvant system composed of DODAC is increased by adding 0 to 95 mol% DDA.
  • the phase transition temperature of DODAC is below 0 °C whereas the phase transition temperature of DDA is about 40°C.
  • Another embodiment of the invention is any of above mentioned adjuvants additionally comprising a quaternary amine of the formula NR 1 R 2 R 3 R 4 -hal, where R 1 and R 2 groups may e.g. be methyl, ethyl, propyl and isopropyl and wherein at least one of the radicals R 3 or R 4 is a saturated alkyl chain containing from 12 to : carbon atoms, preferable from 14 to 18 carbon atoms e.g. DDA-CI or DDA-Br.
  • R 1 and R 2 groups may e.g. be methyl, ethyl, propyl and isopropyl and wherein at least one of the radicals R 3 or R 4 is a saturated alkyl chain containing from 12 to : carbon atoms, preferable from 14 to 18 carbon atoms e.g. DDA-CI or DDA-Br.
  • a most preferred embodiment is wherein the quaternary amine with a halogen counter ion is dimethyl dioctadecenyl ammonium bromide or chloride (DODAC-Br or DODAC-CI) ) is mixed with a quaternary amine of the formula NR 1 R 2 R 3 R 4 -hal, wherein R 1 and R 2 groups may e.g. be methyl, ethyl, propyl and isopropyl and wherein at least one of the radicals R 3 or R 4 is a saturated alkyl chain containing from 12 to 20 carbon atoms, preferable from 14 to 18 carbon atoms e.g. DDA-CI or DDA-Br.
  • a preferred combination of DDA and DODAC is a ratio of from 19:1 to 1:19 by weight.
  • DDA also has a long history of proven adjuvant effect, and promotes a combined humoral and cell- mediated immune response (Hilgers and Snippe, 1992). Combinations of DDA and other immunomodulating agents have been described. DDA/MPL as adjuvant provides protection similar to that obtained by BCG, when combined with a fusion protein of M. Tuberculosis proteins ESAT-6 and Ag85B (WO0069458 and Olsen et al, 2001).
  • Another preferred embodiment is an adjuvant comprising the above mentioned quaternary amines with a halogen counter ion and a second adjuvant component.
  • the second component is preferably selected from i.e. the group of Toll-Like Receptor (TLR) ligands derived from microbes, comprising trehalose dimycolate and trehalose 6,6'-dibehenate (TDB) and derivatives or synthetic analogs thereof, lipopolysaccharides (LPS) and derivatives thereof (such as monophosphoryl lipids), heat-labile toxins and derivatives thereof, and derivatives and synthetic analogs thereof, and mycobacterial lipids, derivatives or synthetic analogs thereof.
  • TLR Toll-Like Receptor
  • lipophilic adjuvants such as trehalose dibehenate (TDB) or monophosphoryl lipid A (MPL-A) are preferred.
  • the monophosphoryl lipids are e.g. obtainable from microbial lipopolysaccharide (LPS) and are usually prepared from bacteria even though other microbial sources like viruses, moulds, fungi, yeasts and algae may yield similar phosphoryl lipids.
  • Suitable bacterial moities are e.g. described in "The Theory and Practical Applications of adjuvants", chapter thirteen, pp. 287-313, Ed. by D.E.S. Stewart-Tull, 1995, John Wiley Sons Ltd., in "Methods in Microbiology", Vol. 25, pp. 471-502, Ed. Stefan AE Kaufmann and Dieter Kunststoffitz, 1998, Academic Press, San Diego, California, USA and London, UK, and in “Vaccine", vol. 15, No. 3, pp. 248- 256, 1997, Elsevier Science Ltd., GB.
  • MPL monophosphoryl lipids
  • the preferred MPL is MPL-A.
  • the most preferred MPL-A is designated 3-O-deacylated monophosphoryl lipid A.
  • a preferred combination of [DODAC/DDA] and MPL-A is a ratio of from 30:1 to 4:1 by weight, preferably from 20:1 to 5:1 by weight, more preferably in a ratio of about 10:1 by weight.
  • TDB is a pure synthetic analogue of TDM also known as cord factor, which is one of the most important immunomodulatory components of the mycobacterial cell wall (Yamagami, H. et al, 2001). TDB in combination with DDA is known to promote a strong protective immune response against tuberculosis using Ag85b-ESAT-6 as antigen (Holten-Andersen, L. et al, 2004).
  • a preferred combination of [DODAC/DDA] and TDB is a ratio of from 20:1 to 2:1 by weight.
  • the main phase transition temperature By being able to control the main phase transition temperature by adjusting the proportion between the two cationic lipids (e.g. DODAC and DDA) it is possible to formulate the optimal freezedried adjuvant, no matter which influence the second adjuvant component eg. MPL-A or TDB, should have on the main phase transition temperature.
  • DODAC cationic lipids
  • the adjuvant combination of the present invention may preferably be in the form of: a) an aqueous composition comprising the quaternary amine with a halogen counter ion of the formula NR 1 R 2 R 3 R 4 -hal, wherein R 1 and R 2 independently each is a short chain alkyl group containing 1 to 3 carbon atoms, R 3 and R 4 independently each is a medium chain length alken containing 12 to 20 carbon atoms and hal is a halogen atom, and b) a quaternary amine of the formula NR 1 R 2 R 3 R 4 -hal, wherein R 1 and R 2 groups may e.g.
  • an aqueous composition comprising the hydrophobic second adjuvant component such as TDB or MPL-A.
  • composition (a) and composition (b) in total comprises about 2.5 mg DODAC-Br or DODAC-CI and DDA-Br or DDA-CI per ml
  • composition (c) comprises about 1 mg MPL-A and about 2 ⁇ l triethylamine per ml of composition (b) and preferably combined into one single aqueous composition.
  • composition (a) and composition (b) in total comprises about 2.5 mg DODAC-Br or DODAC-CI and DDA- Br or DDA-CI per ml, and the composition (c) comprises about 0.5 mg TDB and preferably combined into one single aqueous composition.
  • the aqueous media in these aqueous compositions may be any suitable aqueous solvent.
  • formation of useful possible micelle structures appears to be sensitive to anions, like phosphate and sulphate ions.
  • the adjuvant compositions of the inventions are formed in the absence or low levels of such ions.
  • aqueous adjuvant compositions may be prepared by any suitable process or procedure, e.g. as described further on in the detailed part of this specification.
  • the different adjuvant compositions may be combined into one single composition either as a stock composition or immediately before use.
  • any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral, nasal or mucosal application in either a solid form containing the active ingredients (such as a pill, suppository, capsule or plaster) or in a physiologically acceptable dispersion, such as a spray, powder or liquid, or parenterally, by injection, for example, subcutaneously, intradermally or intramuscularly.
  • the dosage of the vaccine will depend on the route of administration and will vary according to the age of the per- son to be vaccinated and, to a lesser degree, the size of the person to be vaccinated. Currently, most vaccines are administered intramuscularly by needle injection and this is likely to continue as the standard route.
  • Mucosal vaccines typically can be administered by healthcare workers instead of medically trained staff, which means they are far more readily accessible in low- income areas where medical staff may not be readily found. Finally, since mucosal vaccines are non-invasive, typical parenteral vaccination problems such as irritation, bruising or infection at the site of infection are avoided.
  • the stability of the adjuvant is crucial.
  • the ideal formulation of a subunit vaccine should be stable, easy to administer, cheap and induce the desired type of immune response.
  • Single vial vaccines that do not require frozen storage and that are ready to use or which just need to re-dissolved are preferred because of the ease of administration. Lyophilization or freeze-drying of vaccines offer significant benefits in the context of storage and shipping.
  • the invention concerns also a kit for immunization, said kit comprising: a) a first adjuvant component which is a combination of a quaternary amine with a halogen counter ion of the formula NR 1 R 2 R 3 R 4 -hal, wherein R 1 and R 2 independently each is a short chain alkyl group containing 1 to 3 carbon atoms, preferably methyl groups, R 3 and R 4 independently each is a alken containing from 12 to 20 carbon atoms, preferably from 14 to 18 carbon atoms, and hal is a halogen atom, and a quaternary amine of the formula NR 1 R 2 R 3 R 4 -hal, wherein R 1 and R 2 groups may e.g.
  • R 3 or R 4 be methyl, ethyl, propyl and isopropyl and wherein at least one of the radicals R 3 or R 4 is a saturated alkyl chain containing from 12 to 20 carbon atoms, preferable from 14 to 18 carbon atoms e.g. DDA-CI or DDA-Br, b) a hydrophobic second adjuvant component such as TDB or MPL-A, c) an antigenic substance.
  • kit may be presented in the form of individual containers or compartments containing the different adjuvants and the antigenic substance and any solvent necessary for assuring the immunization procedure as well as any necessary device for the performance thereof. If appropriate the adjuvants and the antigenic substance may also be combined and stocked in one single container. If the adjuvants and the antigenic substance each are contained in a separate container they may be mixed in any order before use. For some applications it may be advantageous, however, to mix the adjuvants and the antigenic substances in a particular order for obtaining optimum results.
  • the antigenic substance may be any pure chemical species such as a protein or a fragment thereof or artificial mixtures prepared of such species. But it can also be any naturally occurring mixture of chemical species such as e.g. a cell homogenate or fractions thereof, a culture filtrate from microorganisms or cell tissues from multicellular organisms, e.g. higher animals.
  • the antigenic substance may be derived from a culture of metabolising Mycobacterium tuberculosis, Mycobacterium bovis and other environmental mycobacteria such as e.g. Mycobacterium avium.
  • Mycobacterium avium particularly interesting substances from the filtrate of such mycobacteria is the ESAT-6 gene family proteins (such as ESAT6, Ag85A, Ag85B, TB10.4, ORF2c, Rv1036 and Rv0285) which are dominant targets for cell mediated immunity in the early phase of tuberculosis in TB patients and in different animal models.
  • ESAT-6 gene family proteins as well as many other antigens applicable in combination with the adjuvant combinations of the present invention, today can be produced artificially, e.g. synthetically or by genetic recombinant techniques.
  • the adjuvant combinations of the present invention can also be used for producing antibodies against compounds which are poor immunogenic substances per se and such antibodies can be used for the detection and quantification of the compounds in question, e.g. in medicine and analytical chemistry.
  • an adjuvant such as DODAC, which induces strong CMI (cell mediated Immune) responses, has the ability to form micelles, liposomes or lipid bilayers in aqueous solutions.
  • the lipid portion of this structure provides a matrix for the inclusion of other lipophilic compounds and the formation of composite micelles with increased adjuvant activity.
  • An adjuvant is defined as a substance that non-specifically enhances the immune response to an antigen. Depending on the nature of the adjuvant it can promote either a cell-mediated immune response, a humoral immune response or a mixture of the two. Since the enhancement of the immune response is non-specific, it is well understood in the field that the same adjuvant can be used with different antigens to promote responses against different targets e.g. with an antigen from M. tuberculosis to promote immunity against M. tuberculosis or with an antigen derived from a tumor, to promote immunity against tumors of that specific kind.
  • An antigenic component or substance is a molecule, which reacts with preformed antibody and/or the specific receptors on T and B cells.
  • a molecule that can stimulate the development of specific T or B cells leading to the formation of a memory population of immune cells that will promote a faster "memory" response if the antigen is encountered a second time by immune cells. Since memory populations are rarely clonal, in practice this means that an antigen is any molecule or collection of molecules, which can stimulate an increase in immune responses when it is re-encountered by immune cells from an individual who has previously been exposed to it.
  • the antigenic component or substance can be a polypeptide or a part of the polypeptide, which elicits an immune response in an animal or a human being, and/or in a biological sample determined by any of the biological assays described herein.
  • the immunogenic portion of a polypeptide may be a T-cell epitope or a B-cell epitope.
  • T-cell epitopes are linear, deletion mutants of the polypeptide will, if constructed systematically, reveal what regions of the polypeptide are essential in immune recognition, e.g. by subjecting these deletion mutants e.g.
  • Another method utilizes overlapping oligopeptides (preferably synthetic having a length of e.g. 20 amino acid residues) derived from the polypeptide. These peptides can be tested in biological assays (e.g. the IFN-gamma assay as described herein) and some of these will give a positive response (and thereby be immunogenic) as evidence for the presence of a T cell epitope in the peptide.
  • B-cell epitopes can be determined by analyzing the B cell recognition to overlapping peptides covering the polypeptide of interest as e.g. described in Harboe et al, 1998.
  • the polypeptide fragment of the invention has a length of at least 7 amino acid residues, such as at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, and at least 30 amino acid residues.
  • the polypeptide fragment has a length of at most 50 amino acid residues, such as at most 40, 35, 30, 25, and 20 amino acid residues. It is expected that the peptides having a length of between 10 and 20 amino acid residues will prove to be most efficient as diagnostic tools, and therefore especially preferred lengths of the polypeptide fragment used in the inventive method are 18, such as 15, 14, 13, 12 and even 11 amino acids.
  • a vaccine is defined as a suspension of dead, attenuated, or otherwise modified microorganisms (bacteria, viruses, or rickettsiae) or parts thereof for inoculation to produce immunity to a disease.
  • the vaccine can be administered either prophylactic to prevent disease or as a therapeutic vaccine to combat already existing diseases such as cancer or latent infectious diseases but also in connection with allergy and autoimmune diseases.
  • the vaccine can be emulsified in a suitable adjuvant for potentiating the immune response.
  • the vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to mount an immune response, and the degree of protection desired.
  • Suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a preferred range from about 0.1 ⁇ g to 1000 ⁇ g, such as in the range from about 1 ⁇ g to 300 ⁇ g, and especially in the range from about 10 ⁇ g to 50 ⁇ g.
  • Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations.
  • the manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral or mucosal application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like.
  • the dosage of the vaccine will depend on the route of administration and will vary according to the age of the person to be vaccinated and, to a lesser degree, the size of the person to be vaccinated.
  • the vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral or mucosal formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and advantageously contain 10-95% of active ingredient, preferably 25-70%.
  • the vaccine of choice can e.g. be: ⁇ Protein Vaccine ⁇ A vaccine composition comprising a polypeptide (or at least one immunogenic portion thereof) or fusion polypeptide.
  • Loading of antigen to antigen-presenting cells, such as dendritic cells, have shown to be an effective method for generating active T-cells with a role in antitumor immunity.
  • Lymph nodes were removed 6 days after the immunization and the lymphocytes were stimulated with medium containing no antigen (negative control), ConA (positive control) and the antigen Ag85B-ESAT6 (1.0, 0.2 and 0.04 micrograms/millilitres) abbreviated as 85B- E6.
  • mice Female C57BL/6 mice, 8 to 12 weeks old, were obtained from Bomholtgaard (Ry, Denmark).
  • Dimethyldioctadecenylammonium chloride (in the following abbreviated to DODAC, Avanti Polar Lipids, Alabaster, Alabama) was hydrated by addition of sterile water or 25 mM hepes, pH 7.4 (2.5 - 3.1 milligrams DODAC/milliliter) at room temperature for 30 minutes.
  • DODAC liposomes were stored either at room temperature or at 4 degrees Celsius.
  • Dimethyldeoctadecylammonium-bromide (In the following abbreviated to DDA, Avanti Polar Lipids, Alabaster, Alabama) was added to sterile water (2.5 milligrams/milliliter) and heated to 80 °C while stirring continuously on a magnetic stirring hotplate for 20 min and allowed to cool before use. The DDA liposomes were stored at room temperature for no more than seven days where after they were discarded. An aqueous solution of Lipid-A (Avanti Polar Lipids, Alabaster, Alabama) was prepared as previously described for MPL (Brandt et al, 2000).
  • Vaccines were prepared as previously described (Brandt et al, 2000). One hour before immunization, the antigen was mixed with 0.9% saline and added to the adjuvant preparation. In some instances, Lipid-A was also added. The final suspension was mixed using a vortex mixer.
  • DODAC/DDA vesicles containing TDB as the second adjuvant were made using the thin film method.
  • the particlesize were measured with dynamic light scattering using a Malvern ZetaSizer 4 with a ZET 5110 cell (Malvern Instruments Ltd. UK) using Malvern PCS v.1.52 software.
  • the sample temperature was set to 25°C, the viscosity was set to 1 and the software was set to multimodal measurement.
  • transition state temperature were measured with a MicroCal VP-DSC MicroCalorimeter, using VPViewer2000 software for VP-DSC to define the parameters of of the experiment, and Origin®7 scientific plotting software to analyze the calorimetric data. 40psi pressure was applied to the cells. A scan at 30°C/hour over the temperature range 20-70°C was performed.
  • the fusion protein of Ag85B and ESAT-6 (in the following abbreviated to Ag85B- ESAT6) was produced recombinantly as previously described (Olsen et al, 2001).
  • the LPS content was measured by the Limulus amoebocyte lysate test and shown to be below 0.125 EU/millilitre - a concentration having no influence on cellular activity.
  • mice were immunized subcutaneously (sc) at the base of the tail 1-3 times with a two weeks interval between each immunization.
  • the vaccines (0.2 millilitre/mice) consisted of 2 micrograms of the fusion protein Ag85B-ESAT6 emulsified in 250 micrograms of DDA or DODAC and in some cases also 25 micrograms of Lipid-A or 100 microgram of TDB.
  • mice were immunized by the intranasal (i.n.) route with 5 ⁇ g of Ag85B- ESAT6 emulsified in different doses of DODAC. The mice received 25 microlitres in each nostril administered tree times with a two weeks interval between immunizations.
  • Lymphocytes from lymph nodes and spleens were obtained as previously described (Andersen et al, 1991). Cell cultures were performed in triplicate in round-bottomed microtiter wells containing 2 x 10 5 cells in a volume of 200 microlitres RPMI supplemented with 2-mercaptoethanol, glutamine, penicillin-streptomycin, hepes, and 10% foetal calf serum. Mycobacterial antigen Ag85B-ESAT6 was used in concentrations ranging from 1 to 0.04 micrograms/millilitre. Wells containing medium only and 5 micrograms/millilitre of ConA were included as negative and positive controls, respectively. Culture supernatants were harvested from parallel cultures after 72 hours of incubation in the presence of antigen, and the amount of IFN- gamma was determined by enzyme-linked immunosorbent assay (Brandt et al, 2000).
  • IFN- ⁇ positive cells were detected by 1 hour 15 mins incubation with AP-conjugated streptavidin (Zymed, USA) followed by staining with Sigma Fast BCIP-NBT substrate according to the manufacturer ' s manual (Sigma, USA). The reaction was stopped 10-30 minutes later by extensive washing using deinized water. Spots were counted using an AID Elispot Reader.
  • mice were challenged 10 weeks after the first immunisation by the aerosol route in a Glas-Col inhalation exposure system calibrated to deposit 25 CFU of virulent M. tuberculosis Erdman in the lungs.
  • a single dose of BCG Danish 1331 was injected s.c. at the base of the tail.
  • the bacterial load in lungs were determined six weeks later by plating serial dilutions onto Middlebrook 7H11 agar supplemented with 2 ⁇ l 2- thiphene-carboxylic acid hydrazide per millilitre to selectively inhibit the growth of BCG. Colonies were counted after 2-3 weeks of incubation at 37 °C.
  • Zetasizer 4 dynamic light scattering
  • DODAC liposomes 0.8 ml autoclaved DODAC liposomes (3.1 mg/ml) were mixed with 0.2 ml antigen (Ag85bESAT6; 0,506 mg/ml containing 10% glycerol).
  • the liposomal suspension were frozen at -80 °C and placed in a lyophilizer for 12 hours until all water was removed.
  • the freeze-dried DODAC-antigen mix was easily re-suspended by replacing the removed water.
  • the re-suspended mixture of DODAC and Ag was stable for several days showing no change of visual appearance.
  • the liposomal preparation were frozen at -80 °C and placed in a freeze-dryer for 12 hours until all water was removed.
  • the freeze-dried DODAC/TDB mix was easily re- suspended by replacing the removed water.
  • the re-suspended mixture of DODAC and TDB were stable for several days showing no change in particle size or visual appearance.
  • Preformed large liposomes prepared by the chloroform vaporization method composed of DODAC and TDB (0.5 ml; 2.5 mg DODAC/ml) were mixed with 0.1 ml antigen (Ag85bESAT6; 0.506 mg/ml containing 10% glycerol).
  • the antigen containing suspension were frozen at -80 degrees Celsius and placed in a lyophilizer for 12 hours until all water was removed.
  • the freeze-dried DODAC/TDB/antigen mix was easily re-suspended by replacing the removed water.
  • the re-suspended mixture of DODAC and Ag was stable for several days showing no change in visual appearance.
  • Specified volumes of each individual compound were mixed in glass test tubes.
  • the solvent were evaporated using a gentle stream of N 2 and the lipid films were dried over night under low pressure to remove trace amounts of solvent.
  • Table 1 List of a range of adjuvant formulations prepared in accordance with the present invention
  • DODAC:DDA:TDB ratio DODAC (mg/ml) DDA (mg/ml) TDB (mg/ml) 0:10:2 0 1.25 0,25 2:8:2 0.25 1.0 0.25 5:5:2 0.625 0.625 0.25 8:2:2 1.0 0.25 0.25 10:0:2 1.25 0 0.25
  • Table 2 schedule of the freeze-drying of adjuvant formulations prepared in accordance with the present invention Phase I Time (hours) Pressure (mbar) Shelf temp. (°C) Pre-freezing 1 :00 Atm -20 Main drying 21 :00 1.030 -10 Final Drying 2:00 0.001 +20
  • the particle size of the formulations listed in Table 1 was measured before and after freeze-drying by dynamic light scattering measurements.
  • Table 4 Particle size of the formulations before and after freeze-drying DODAC:DDA:TDB Ave size before Ave. size after ⁇ Ave. size ratio freeze-drying freeze-drying (before ⁇ after) 0:10:2 615.3 Not measurable - 2:8:2 538.5 1201.0 +662.5 5:5:2 463.9 933.9 +470.0 8:2:2 462.7 466.4 +3.7 10:0:2 456.8 412.2 -44.6
  • Formulations with high concentration of DODAC relative to DDA had similar average particle size before and after freeze-drying, in contrast to formulations with high and equal concentrations of DDA relative to DODAC, where the average particle size were markedly bigger after freeze-drying.
  • transition state temperatures (T m ) values and the transition state ranges ( ⁇ T 1 / 2 ) were measured on the formulations listed in Table 5Fejl! Henvisningskilde ikke fundet.
  • Table 5 Transition state -temperatures and -ranges of liposomes consisting of DODAC:DDA: TDB DODAC:DDA:TDB Ratio T m (°C) ⁇ T 1 / 2 (°C) 0:10:2 42,4 3,97 1 :9:2 42,6 5,66 2:8:2 41 ,8 6,75 10:0:2 ⁇ 0 -
  • mice were immunized with experimental vaccines consisting of 2 micrograms of fusion protein Ag85B-ESAT6 adjuvanted with DDA, DDA/Lipid-A, DODAC (stored either at room temperature or at 4 degrees Celsius) or DODAC/ ⁇ pid-A.
  • a group of na ⁇ ve mice was included as negative control. 6 days after the first immunization, the IFN-gamma release was evaluated after in vitro re-stimulation of inguinal lymph node lymphocytes with different concentrations of Ag85B-ESAT6 (Fig. 1).
  • mice vaccinated with Ag85B-ESAT6 adjuvanted with DODAC stored either at room temperature or at 4 degrees Celsius
  • DODAC did not have responses at the same high levels as mice immunized with Ag85B-ESAT6 adjuvanted with DDA.
  • Addition of Lipid-A increased the IFN-gamma release in mice immunized with Ag85B-ESAT6/DODAC as well as Ag85B-ESAT6/DDA.
  • DODAC has the same powerful adjuvant activity as previously observed for DDA
  • mice were immunized with Ag85B-ESAT6 in different doses of DODAC administered intranasally. Two weeks after the last immunization, immune responses determined as the number of IFN-gamma producing cells in the spleen was measured by ELISPOT. As shown in Fig. 3, Ag85B-ESAT6 in DODAC give rise to a high number of IFN-gamma positive cells even at doses as low as 5 microgram of DODAC. Together these results demonstrate that DODAC as an adjuvant has potential for both parenteral and mucosal administration. Furthermore, DODAC can be used in various adjuvant formulations comprising i.e. the well-known adjuvant DDA as well as various immunemodulators due to the ability of DODAC of being freeze-dried..
  • mice Protection of mice with a tuberculosis subunit vaccine based on a fusion protein of antigen 85B and ESAT-6. Infect. Immun. 69:2773-2778.

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Abstract

L'invention concerne un nouvel adjuvant apte à provoquer une réponse immunitaire forte et persistante s'il est administré en combinaison avec un antigène, même si cet antigène n'a qu'une faible immunogénicité en soi, cet adjuvant pouvant être lyophilisé, puis dissout sans être chauffé. L'adjuvant de l'invention comporte une amine quaternaire avec un contre-ion halogène de formule NR1R2R3R4-hal, dans laquelle R1 et R2 représentent indépendamment une groupe alkyle à chaîne courte contenant 1 à 3 atomes de carbone, de préférence des groupes méthyle, R3 et R4 signifient indépendamment un alcène contenant 12 à 20 atomes de carbone, de préférence 14 à 18 atomes de carbone, et hal désigne un atome halogène.
PCT/DK2004/000893 2003-12-22 2004-12-21 Adjuvant de vaccin lyophilise WO2005060330A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010130897A1 (fr) * 2009-05-14 2010-11-18 Sanofi Pasteur Procédé de détoxification du lipopolysaccharide (lps) ou du lipide a des bactéries à gram-négatif
EP3454891A4 (fr) * 2016-05-10 2019-12-25 The Regents of The University of Michigan Adjuvant en émulsion pour administration intramusculaire, intradermique et sous-cutanée

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US6649170B1 (en) * 1999-05-12 2003-11-18 Statens Serum Institut Adjuvant combinations for immunization composition and vaccines
WO2001015726A2 (fr) * 1999-08-27 2001-03-08 Inex Pharmaceuticals Corp. Compositions stimulant la secretion de cytokine et provoquant une reaction immunitaire
WO2002071944A1 (fr) * 2001-03-09 2002-09-19 Saavedra Scott S Membrane lipidique supportee biocompatible stabilisee
AU2002340662B2 (en) * 2001-11-07 2008-07-03 Tekmira Pharmaceuticals Corporation Mucosal adjuvants comprising an oligonucleotide and a cationic lipid

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010130897A1 (fr) * 2009-05-14 2010-11-18 Sanofi Pasteur Procédé de détoxification du lipopolysaccharide (lps) ou du lipide a des bactéries à gram-négatif
EP3454891A4 (fr) * 2016-05-10 2019-12-25 The Regents of The University of Michigan Adjuvant en émulsion pour administration intramusculaire, intradermique et sous-cutanée
US12076397B2 (en) 2016-05-10 2024-09-03 The Regents Of The University Of Michigan Emulsion adjuvant for intramuscular, intradermal and subcutaneous administration

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