WO2005060330A2

WO2005060330A2 - Freeze-dried vaccine adjuvant

Info

Publication number: WO2005060330A2
Application number: PCT/DK2004/000893
Authority: WO
Inventors: Else Marie Agger; Peter Andersen
Original assignee: Statens Serum Institut
Priority date: 2003-12-22
Filing date: 2004-12-21
Publication date: 2005-07-07
Also published as: WO2005060330A3

Abstract

The present invention discloses a new adjuvant component that 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 which can be freeze-dried and subsequently dissolved without heating. The adjuvant of the invention comprises a quaternary amine with a halogen counter ion of the formula NR1R2R3R4-hal, wherein R1 and R2 independently each is a short chain alkyl group containing to 1 to 3 carbon atoms, preferably methyl groups, R3 and R4 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.

Description

Freeze-dried Vaccine Adjuvant

The present invention relates to an adjuvant, which can be freeze-dried and subsequently dissolved without heating. In particular 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.

Background of the invention

Since the English doctor Edward Jenner in 1796 discovered that the infectious agency causing cowpox in cattle was able to produce immunity against smallpox in human beings without causing serious illness many efforts have been made in order to find other vaccines which can generate immunity against more or less severe diseases in animal and human beings without provoking the unpleasant, serious or fatal symptoms and reactions usually accompanying the ordinary diseases in question.

Thus, for example, tuberculosis in man has for many years been combated by vaccination with attenuated but living strains of Mycobacterium bovis (BCG vaccine). However, this procedure does not always provide satisfactory resistance to human tuberculosis in every population.

Therefore, attempts have been made to isolate and use fragments or subfragments of strains of human Mycobacterium tuberculosis instead as immunogenic agent which when injected intradermally, intramuscularly or subcutaneously in individuals would cause satisfactory immunity against infections with naturally occurring strains of human Mycobacterium tuberculosis. Thus, uncharacterized substances from culture filtrates as well as a few isolated molecules such as Ag85 and ESAT-6 of Mycobacterium tuberculosis have been shown to provide some degree of tuberculosis immunity. In the future it would be desirable to have vaccines based on well-defined substances which would always create strong immunity against tuberculosis and other diseases.

Unfortunately, many highly purified substances, e.g. purified recombinant proteins, are not very immunogenic and do not generate an effective immune response protective against the real infectious disease. This fact has been recognized since the beginning of this century and many attempts have been made to counteract the low immunogenicity by combining the substance in question with immunogenic response potentiating agents, so-called adjuvants. A large number of such adjuvants and kind of adjuvants have been suggested but in general without any being ideal in all respects.

A well known adjuvant is the quaternary hydrocarbon ammonium halogenide dimethyl dioctadecyl ammonium (DDA) 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. Administration of Arquad 2HT, which comprises DDA, in humans was promising and did not induce apparent side effects (Stanfield, 1973). An experimental vaccine based on culture filtrate proteins from M. 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). These studies demonstrate that, in contrast to e.g. alum, DDA-based adjuvants are able to induce a protective immune response against TB in mice. Moreover, 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).

A major disadvantage of DDA as an adjuvant is that it cannot be properly redispersed at room temperature after freeze-drying. This implies that 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. Thus DDA needs careful handling and storage with regard to temperature. This is an obvious disadvantage for producing and distributing vaccines. In many of the countries where vaccines are most needed, 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.

Summary of the invention

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.

Disclosure of the invention.

Thus, the present invention relates to an adjuvant comprising a quaternary amine with a halogen counter ion of the formula NR¹R²R³R -hal, wherein R¹ and R² independently each is a short chain alkyl group containing 1 to 3 carbon atoms, preferably methyl groups, R³ and R⁴ 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.

In the formula NR¹R²R³R -hal the R¹ and R² groups may e.g. be methyl, ethyl, propyl and isopropyl, whereas R³ and R⁴ may be dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl nonadecenyl and eicocenyl groups. However, also other C₁₂-C₂o alkenes are possible because even though the R³ and R⁴ groups usually and preferably are straight chain hydrocarbon groups they may in minor degree be branched having e.g. methyl and ethyl side chains.

The 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. In the literature there is some confusion concerning the abbreviation of dimethyl dioctadecyl ammonium and dimethyl dioctadecenyl ammonium where the former is sometimes referred to as DDA and sometimes to DODAC. In the present description DODAC is always referring to dimethyl dioctadecenyl ammonium and DDA is always referring to dimethyl dioctadecyl ammonium.

It is known from WO0180900, WO0203959, WO0287541 , WO0234236,

WO0339595, US5981501 and Cancer Gene Therapy (2000), 7(3): 353-359, to use DODAC together with nucleic acid as a transfection system or delivery vehicle. From WO0115726, 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.

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. The so-called 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). In one embodiment of the present invention the main phase transition of a quaternary ammonium adjuvant system is increased by incorporating quaternary amines of the formula NR1 R2R3R4-hal, wherein R¹ and R² groups may e.g. be methyl, ethyl, propyl and isopropyl and wherein at least one of the radicals R3 or R4 is a saturated alkyl chain containing from 12 to 20 carbon atoms, preferable from 14 to 18 carbon atoms. In one particular preferred embodiment the 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. Therefore it is possible to obtain a phase transition temperature of the final adjuvant system at any temperature from below 0 °C to 40°C, which makes it possible to make a freeze-dried formulation of the vaccine adjuvant, being conformational stable, but still readily dispersible at room temperature. Hence, the combination of these two lipids, both with known adjuvant effect, offers a unique technique to formulate freeze dried vaccines without compromising the adjuvant effect.

Another embodiment of the invention is any of above mentioned adjuvants additionally comprising a quaternary amine of the formula NR¹R²R³R⁴-hal, where R¹ and R² groups may e.g. be methyl, ethyl, propyl and isopropyl and wherein at least one of the radicals R³ or R⁴ 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 NR¹R²R³R⁴-hal, wherein R¹ and R² groups may e.g. be methyl, ethyl, propyl and isopropyl and wherein at least one of the radicals R³ or R⁴ 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.

Amongst the hydrophobic second adjuvant components, lipophilic adjuvants, such as trehalose dibehenate (TDB) or monophosphoryl lipid A (MPL-A) are preferred.

The monophosphoryl lipids (MPL) 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 Kabelitz, 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.

Also, the monophosphoryl lipids (MPL) derivable from the microbial polysaccharides and suitable for use in the adjuvant combinations of the present invention are described in more detail in the above references. The preferred MPL is MPL-A. The most preferred MPL-A is designated 3-O-deacylated monophosphoryl lipid A. However, also other derivatives of the MPL-A's may be applicable. 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.

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.

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 NR¹R²R³R⁴-hal, wherein R¹ and R² independently each is a short chain alkyl group containing 1 to 3 carbon atoms, R³ and R⁴ 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 NR¹R²R³R⁴-hal, wherein R¹ and R² groups may e.g. be methyl, ethyl, propyl and isopropyl and wherein at least one of the radicals R³ or R⁴ 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. c) an aqueous composition comprising the hydrophobic second adjuvant component such as TDB or MPL-A.

A preferred adjuvant combination is wherein the 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 1 mg MPL-A and about 2 μl triethylamine per ml of composition (b) and preferably combined into one single aqueous composition.

An other preferred adjuvant combination is wherein the 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. However, formation of useful possible micelle structures appears to be sensitive to anions, like phosphate and sulphate ions. Thus, it is preferred that the adjuvant compositions of the inventions are formed in the absence or low levels of such ions.

The 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.

If expedient, the different adjuvant compositions may be combined into one single composition either as a stock composition or immediately before use.

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, 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. However, vaccine formulations which induce mucosal immunity have been developed, typically by nasal or oral delivery, but also rectal delivery could be a route of choice. One of the most widely studies delivery systems for induction of mucosal immunity contains cholera toxin (CT) or its B subunit (CTB). This protein enhances mucosal immune responses and induce IgA production when administered in vaccine formulations. An advantage is the ease of delivery of nasal and oral vaccines. The use of mucosal vaccines furthermore removes the requirement to use needles, which reduces the cost of the vaccination process, and offers increased safety, especially in areas where HIV is endemic. 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.

For the subunit vaccine, 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 NR¹R²R³R⁴-hal, wherein R¹ and R² independently each is a short chain alkyl group containing 1 to 3 carbon atoms, preferably methyl groups, R³ and R⁴ 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 NR¹R²R³R⁴-hal, wherein R¹ and R² groups may e.g. be methyl, ethyl, propyl and isopropyl and wherein at least one of the radicals R³ or R⁴ 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.

Such 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.

In principle 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.

Specifically 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. Particulary 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. Their immunogenecity per se is low, but in combination with the adjuvant combinations of the present invention it has turned out to be potent candidates for provoking high and persisting immunity against tuberculosis as is demonstrated in the following detailed part of this specification. 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.

In addition to provide immunity to diseases 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.

Without being bound by theory it is believed that 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.

Preferred embodiments of the adjuvant combination and the immunization combination kit of the present invention are set forth in the dependent claims in the accompanying set of claims attached.

Definitions

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. In the context of vaccination, 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. In order to identify relevant T-cell epitopes which are recognized during an immune response, it is possible to use a "brute force" method: Since 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. to the IFN-gamma assay described herein. 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.

Although the minimum length of a T-cell epitope has been shown to be at least 6 amino acids, it is normal that such epitopes are constituted of longer stretches of amino acids. Hence, it is preferred that 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. Hence, in important embodiments of the inventive method, it is preferred that 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 μg to 1000 μg, such as in the range from about 1 μg to 300 μg, and especially in the range from about 10 μg to 50 μg. 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. For 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.

Live recombinant vaccines

Expression of the relevant antigen in a vaccine, in a non-pathogenic microorganism or virus. Well-known examples of such microorganisms are Mycobacterium bovis BCG, Salmonella and Pseudomonas and examples of viruses are Vaccinia Virus and Adenovirus.

Dendritic cells as antigen delivery vehicles

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.

For all of these vaccine constructs, the addition of a suitable adjuvant has resulted in enhanced vaccine efficacies (Brandt, 2000; van Rooij, 2002; Wang, 2002; Eriksson, 2003). FIGURE LEGENDS

Figure 1.

Release of IFN-gamma from inguinal lymph nodes isolated from C57BI/6J mice immunized with 2 micrograms of Ag85B-ESAT6 in 250 micrograms of DDA (panel A), 2 micrograms Ag85B-ESAT6 in 250 micrograms of DODAC (stored at room temperature, panel B), 2 micrograms of Ag85B-ESAT6 in 250 micrograms of DDA and 25 micrograms of Lipid-A (panel C), 2 micrograms of Ag85B-ESAT6 in 250 micrograms of DODAC (stored at room temperature) and 25 micrograms of Lipid-A (panel D) or 2 micrograms of Ag85B-ESAT6 in 250 micrograms of DODAC (stored at 4 degrees Celsius, panel E) or from naϊ^'ve C57BI/6J mice (panel F). 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.

Figure 2.

Logio resistance in the lungs of mice (n= 6) immunized with Ag85B-ESAT6 in DODAC/TDB, or BCG. * Vaccines inducing significant protection compared to naϊve, un-immunized mice (p< 0.05).

Figure 3.

Number of IFN-gamma producing cells in spleens of mice immunized with 5 microgram of Ag85B-ESAT-6 in 0-75 microgram of DODAC administered i.n. Mice were immunized with the vaccine three times with a two weeks interval. Two weeks after the last immunization, spleens cells were re-stimulated in vitro with 5 microgram of Ag85B-ESAT6 and the number of antigen-specific cells determined by ELISPOT. EXAMPLES

Materials and Methods

ANIMALS

Female C57BL/6 mice, 8 to 12 weeks old, were obtained from Bomholtgaard (Ry, Denmark).

ADJUVANTS AND VACCINES

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. The 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. Dimethyldioctadecenyl ammonium chloride (DODAC, Mw=582,48), Dimethyldioctadecyl ammonium Bromide (DDA, Mw=630,97) and Trehalose 6,6'- dibehenate (TDB, Mw=987.5) (Avanti Polar Lipids, Alabaster, Al) were dissolved separately in chloroform methanol (9:1). The solvent were evaporated using a gentle stream of N₂ and the lipid films were dried over night under low pressure to remove trace amounts of solvent. The dried lipid films were hydrated in TRIS-buffer (10mM, pH=7.4) and placed on a 70°C water bath for 20 min. The samples were vigorously shaken every 5. min. The preparations were freeze-dried in a CHRIST Alfa 2-4 lyophilizer (Martin Christ GmBH, DE) and Water for Injection (WFI, Statens Serum Institut) was used to resuspend the formulations

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.

The 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.

ANTIGEN

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.

IMMUNIZATION

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. Alternatively, mice were immunized by the intranasal (i.n.) route with 5 μg 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.

LYMPHOCYTE CULTURES

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 10⁵ 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).

ELISPOT ANALYSES

Filters plates were pre-coated at room temperature over night with 0.8 μg/well of rat anti-mouse IFN-γ mABS (Pharmingen, USA) in sterile PBS using ELISPOT Filter Plates (Invitrogen, MAHA S45 10; cellulose ester). Plates were washed extensively using PBS and blocked with complete RPMI and 10% FCS for 1¹/₂ hour before addition of splenocytes. After 48 hours of stimulation, plates were washed and added 0.125 μg/well of biotin-labelled rat anti-mouse IFN-γ mAbs (Pharmingen, USA) for 2 hours at room temperature. IFN-γ 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. EXPERIMENTAL INFECTIONS

For evaluation of the efficacy of selected vaccines, 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. As a positive control, 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 μl 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.

STATISTICAL METHODS

Differences in number of colonies between infected mice and control mice were tested by analysis of variance. When significant effects were indicated, differences between means were assessed by Dunnetts test.

Example 1

DODAC CAN BE AUTOCLAVED 62 mg DODAC (Dimethyldioctadecenylammonium chloride) was hydrated at room temperature for 30 minutes by adding HEPES-buffer (25 mM; pH 7.4) to a concentration of 3.1 mg/ml. Due to their amphiphilic properties i.e. two long hydrophobic acyl chains attached to a hydrophilic quarternary ammonium head- group, DODAC forms liposomal structures when dispersed in aqueous buffer. The particle size of the vesicles was measured by dynamic light scattering (Zetasizer 4), which provides an accurate measurement of average size and size distribution of suspended particles. The z-average particle size of the DODAC vesicles was 195 nm. The liposomal suspension of DODAC was autoclaved at 121°C for 21 minutes; the visual appearance of the autoclaved DODAC was not changed. DODAC CAN BE FREEZE-DRIED

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.

0.8 ml autoclaved DDA 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. When an attempt was made to redissolve the freeze-dried DDA-antigen mixture the DDA could not be dissolved which resulted in a 'milky suspension' and a quite significant change in visual appearance.

PREPARATION OF LARGE DODAC LIPOSOMES CONTAINING A SECOND ADJUVANT COMPONENT

Large unilamellar vesicles were prepared by the chloroform vaporization method (Ribeiro and Chaimovich, 1983; Deamer and Bangham, 1976). D-(+)-Trehalose 6,6'- dibehenate (TDB) (2 mg) and DODAC (5 mg) were dissolved in 0.5 ml chloroform and injected (0.5 ml/min) into 2 ml aqueous phase maintained at 70 degrees Celsius, in nitrogen atmosphere. After injection the solution was maintained at the same temperature for 5 minutes in nitrogen atmosphere for complete vaporization of the solvent. The final DODAC and TDB concentration were 2.5 and 1 mg/ml, respectively. The preparation appeared as a stable homogeneous suspension.

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.

PREPARATION OF LARGE DODAC LIPOSOMES CONTAINING A SECOND

ADJUVANT COMPONENT MIXED WITH ANTIGEN

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.

Example 2

PREPARATION OF LIPOSOMES WITH A COMBINATION OF DODAC, DDA AND A SECOND ADJUVANT COMPONENT

Specified volumes of each individual compound (see Table 1 ) were mixed in glass test tubes. The solvent were evaporated using a gentle stream of N₂ and the lipid films were dried over night under low pressure to remove trace amounts of solvent. The dried lipid films were hydrated in TRIS-buffer (10mM, pH=7.4) to the concentrations specified in Table 1 , and placed on a 70°C water bath for 20 min. The samples were vigorously shaken every 5. min.

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

FREEZE-DRYING OF LIPOSOMES COVERED IN THE PRESENT INVENTION

1 ml of each of the formulations listed in Table 1 was freeze-dried according to the schedule in Table 2. 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

All preparations containing DODAC (Table 1) were resuspended by replacing the removed water by WFI. The preparation without DODAC could not be resuspended. As listed in Table 3 formulations with high concentrations of DODAC were resuspended to a homogenous preparation almost immediately.

Table 3: Observations after rel lydration of formulation DODAC:DDA:TDB ratio Observation during resuspension in WFI 0:10:2 Not possible to resuspended 2:8:2 Possible to resuspend within 5 min. 5:5:2 Possible to resuspend within 2 min. 8:2:2 Resuspended almost immediately 10:0:2 Resuspended almost immediately

PARTICLE SIZE OF LIPOSOMES COVERED IN THE PRESENT INVENTION, COMPARED TO FREEZE-DRIED LIPOSOMES OF SAME INVENTION

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 ΔAve. 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.

TRANSITIONSTATE OF LIPOSOMES COVERED IN THE PRESENT INVENTION

The transition state temperatures (T_m) values and the transition state ranges (ΔT¹/₂) 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) ΔT¹/₂ (°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 -

The results show that with increased concentration of DODAC relative to DDA, the T_m is lowered and the ΔT¹/₂ is broadened. This demonstrates that with higher concentrations of DODAC relative to DDA the transition state temperature is spanning over a broader but lower temperature range.

Together these results demonstrate that by combining DODAC with DDA, the main transition state temperature is lowered compared to DDA alone and that the particle size after rehydration of the freeze-dried formulations is lowered with higher concentrations of DODAC. Therefore it is possible to use DODAC in a formulation together with DDA and other adjuvants so that a freeze-dried preparation would always be homogenous and readily dispersible at room temperature

Example 3

USE OF DODAC AS ADJUVANT FOR A TB SUBUNIT VACCINE

In this example studying the adjuvant activity of DDA and DODAC, 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/ϋpid-A. A group of naϊve 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). The groups of mice vaccinated with Ag85B-ESAT6 adjuvanted with DODAC (stored either at room temperature or at 4 degrees Celsius) gave 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. In conclusion, the experiment demonstrates that DODAC has the same powerful adjuvant activity as previously observed for DDA

Subsequently, the protective efficacy of DODAC administered in combination with TDB previously shown to possess significant adjuvant activity (Holten-Andersen 2004) was tested. Mice were given 3 immunizations of Ag85B-ESAT6 in DODAC/TDB and challenged by the aerosol route with virulent M. tuberculosis Erdman 6 weeks after the last immunization. Protective efficacies are expressed as the log-io reduction in bacterial counts in immunized mice in comparison with a group of un-immunized naϊve animals. As shown in Fig. 2, Ag85B-ESAT6 administered in DODAC/TDB give rise to significant protection (P<0.05) close to that afforded by the standard vaccine, BCG.

Example 4

USE OF DODAC AS A MUCOSAL ADJUVANT

In order to assess the ability of DODAC to act as a mucosal adjuvant, 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..

References

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Andersen, P., 1994, Effective vaccination of Mice against Mycobacterium tuberculosis Infection with a soluble Mixture of secreted Mycobacterial Proteins, Infect. Immun. ,62: 2536-2544.

Brandt, L, M. Elhay, I. Rosenkrands, E. B. Lindblad, and P. Andersen. 2000. ESAT- 6 subunit vaccination against Mycobacterium tuberculosis. Infect. Immun.68:791- 795.

Deamer, D. and A.D. Bangham. 1976. Large Volume Liposomes by an Ether Vaporization Method. Biochimica Et Biophysica Acta. 443: 629-634.

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Hilgers. L.A.T and H. Snippe, 1992. DDA as an immunological adjuvant. Res. Immunol. 143:494-503; discussion 574-6.

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Ribeiro, A.M.C. and H. Chaimovich. 1983. Preparation and Characterization of Large Dioctadecyldimethylammonium Chloride Liposomes and Comparison with Small Sonicated Vesicles. Biochimica Et Biophysica Acta. 733: 172-179.

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van Rooij, E.M.A., H. L. Glansbeek, L.A.T. Hilgers, E.G te Lintelo, Y.E de Visser, W.J.A Boersma, B.L. Haagmans and A.T.J. Bianchi, 2002, Protective Antiviral Immune Responses to Pseudorabies Virus Induced by DNA Vaccination Using Dimethyldioctadecylammonium Bromide as an Adjuvant, Journal of Virology, 10540- 10545

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Yamagami, H., T. Matsumoto, N. Fujiwara, K. Kaneda, I. Yano and K. Kobayashi. 2001. Trehalose 6,6'-dimycolate (cord factor) of Mycobacterium tuberculosis induces foreign-body- and hypersensitivity-type granulomas in mice. Infect. Immun. 69:810- 815.

Claims

1. An adjuvant comprising a quaternary amine with a halogen counter ion of the formula NR¹R²R³R⁴-hal, wherein R¹ and R² independently each is a short chain alkyl group containing 1 to 3 carbon atoms, preferable methyl groups, R³ and R⁴ independently each is an alken, alkadien or alkatrien containing from 12 to 20 carbon atoms, preferable from 14 to 18 carbon atoms, and hal is a halogen atom, with the proviso that the adjuvant does not contain nucleic acid as adjuvant or carrier.

2. An adjuvant of claim 1 , for use together with an antigen e.g. a peptide, a polypeptide, a fusion polypeptide or a protein.

3. An adjuvant of claim 1 or 2, wherein the quaternary amine with a halogen counter ion is dimethyl dioctadecenyl ammonium-bromide or -chloride (DODAC-Br or DODAC-CI).

4. An adjuvant of claim 2 or 3 additionally comprising a quaternary amine of the formula NR¹R²R³R⁴-hal, wherein R¹ and R² groups may e.g. be methyl, ethyl, propyl and isopropyl and wherein at least one of the radicals R³ or R⁴ 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.

5. An adjuvant of claim 1-4, comprising a second adjuvant component.

6. An adjuvant according to claim 5, where the second component is selected from the group of Toll-Like Receptor (TLR) ligands derived from microbes, comprising trehalose dimycolate and trehalose dibehenate 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.

7. An adjuvant according to any of the preceding claims comprising: a) an aqueous composition comprising a first adjuvant component which is a quaternary amine with a halogen counter ion of the formula NR¹R²R³R⁴-hal, wherein R¹ and R² independently each is a short chain alkyl group containing 1 to 3 carbon atoms, preferably methyl groups, R³ and R⁴ independently each is an alken containing 12 to 20 carbon atoms and hal is a halogen atom, and b) an aqueous composition comprising a lipophilic second adjuvant component.

8. An adjuvant of claim 7, wherein the quaternary amine with a halogen counter ion is dimethyl dioctadecenyl ammonium-bromide or -chloride (DODAC-Br or DODAC- CI).

9. An adjuvant of claim 7-8, where the second component is selected from the group of Toll-Like Receptor (TLR) ligands derived from microbes, comprising trehalose dimycolate and trehalose dibehenate 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 therof.

10. An adjuvant of claim 7-9, wherein the composition (a) comprises about 2.5 mg DODAC-Br or DODAC-CI per ml of composition (a) and the composition (b) comprises about 1 mg MPL-A and about 2 μl triethylamine per ml of composition (b).

11.An adjuvant of any of the preceding claims, wherein the aqueous compositions (a) and (b) are combined into one single aqueous composition.

12. An adjuvant of any of the preceding claims, which comprises DODAC-Br or DODAC-CI and MPL-A in 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.

13. An adjuvant of any of the preceding claims, which comprises DODAC-Br or DODAC-CI and TDB in a ratio of from 20:1 to 2:1 by weight,

14. An adjuvant of any of the preceding claims for use of intradermally, subcutaneous or mucosal delivery of an antigenic substance.

15. An immunization kit comprising an adjuvant comprising a quaternary amine with a halogen counter ion of the formula NR¹R²R³R⁴-hal, wherein R¹ and R² independently each is a short chain alkyl group containing 1 to 3 carbon atoms, preferable methyl groups, R³ and R⁴ independently each is an alken, alkadienes or alkatrienes containing from 12 to 20 carbon atoms, preferable from 14 to 18 carbon atoms, and hal is a halogen atom, with the proviso that the adjuvant does not contain any nucleic acid adjuvant or carrier components, and an antigenic substance.

16. The immunization kit of claim 15, wherein the quaternary amine with a halogen counter ion is dimethyl dioctadecyl ammonium bromide or chloride (DODAC-Br or DODAC-CI).

17. The immunization kit of claim 15-16, wherein a second lipophilic adjuvant component is selected from the group of Toll-Like Receptor (TLR) ligands derived from microbes, comprising trehalose dimycolate and trehalose dibehenate 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.

18. The immunization kit of claim 15-17, wherein the antigenic substance is derived from a cell homogenate or fractions thereof.

19. The immunization kit of claim 15-18, wherein the antigenic substance is a culture filtrate (CF).

20The immunization kit of claim 19, wherein the antigenic substance is a culture filtrate (CF) stemming from the cultivation of a micro-organism.

21. The immunization kit of claim 18-20, wherein the antigenic substance is derived from a culture of metabolizing Mycobacterium tuberculosis.

22. The immunization kit of claim 15-21 wherein the antigenic substance is an antigenic fragment, e.g. a substantially pure molecular species.

23. The immunization kit of claim 22, wherein the antigenic molecular species is of synthetical or recombinant origin.

24. The immunization kit of claim 21 -3, wherein the antigenic molecular species comprises one or more antigens from the ESAT-6 gene family from Mycobacterium tuberculosis, such as ESAT6, Ag85A, Ag85B, TB10.4, ORF2c, Rv1036 or Rv0285.

25. The immunization kit of any of the preceding claims for intradermally, intramuscular, subcutaneous or mucosal delivery of an antigenic substance.

26. A method comprising the regulation of main phase transition state temperature by regulating the proportion between the quaternary amines mentioned in claim 1 and 4.

27. The method of claim 26, comprising a second adjuvant component.

28. A method according to claim 26-27 where the phase transition temperature of DODAC is regulated by adding up to 95 mol% DDA.

29. A method according to claim 28 where the ratio of DODAC to DDA is between 19:1 and 1 :19 by weight.