DE10024788A1 - Substance for increasing an immune response during vaccinations or for increasing the production of antibodies, comprises solid lipid particles of a specific size and charge - Google Patents

Abstract

A substance (I) that increases the immune response to an immunization of human or animals, or that increases the amount of antibodies produced and which comprises solid lipid particles at room temperature, is new. A new substance (I) that increases the immune response to an immunization of human or animals, or that increases the amount of antibodies produced comprises solid lipid particles at room temperature. The particles comprise an optimal dose, size, charge and surface activity (stabilizing tenside layer) according to the need of the individual subject. (I) is simply mixed with the antigen solution.

Description

1. Background of the Invention

Antigens are applied to animals and humans to create antibodies testify. The goals are, for example, the immunization of humans or animals to protect against disease or the production of anti bodies that are subsequently isolated and processed into products. The The most common route of application for antigens is parenteral administration; alternative ways are e.g. B. oral, nasal and topical application.

A common problem is that often the strength of the immune response to that administered antigen is insufficient for the intended purpose. Sometimes the problem can be solved by administering the Dose of the antigen is greatly increased. A serious problem, however, is that antigens are often very expensive and the dose increase corresponds to one appropriate increase in the cost of the vaccine. These costs result in a heavy burden on the health system, for many population shifts - especially in the developing world - the vaccine becomes too expensive and desirable mass vaccinations can not be carried out who the.

An alternative approach is to administer antigens along with an adjuvant that enhances antigen activity and so on leads to a higher antibody titer. The adjuvant principle was first  lig described in 1948 by Jules Freund [Freund, J. J. Immunology 1948, 60, 383-398] and by two emulsions based on mineral oil rea lized. Freund's Incomplete Adjuvant - Freund's Incomplete Adjuvant - FIA) is a mixture of mineral oil with mannide monooleate (montanide, Arlacel). To use, mix it with the antigen solution and inji adorns the formed emulsion. The resulting amplification of the Immune response is so great that FIA is still a "gold standard" is attracted when new adjuvants are developed and tested. One evaluates their efficiency and quality by comparison with FIA, whereby however, many newly developed adjuvants are well below efficiency stay from FIA (e.g. at 0.2, FIA = 1.0).

The addition of killed mycobacteria to FIA (e.g. M. tuberculosis) continued to increase the immune response. This mix is called friend complete adjuvant (Freund's Complete Adjuvant - FCA). The Freund's oil emulsion, however, produces inflammatory and ulcerating Tumors (granulomas) at the injection site, some of which break open and go into spreading abscesses. According to today's standard is therefore the use of Freund's oil emulsion on humans outside of any question. It is sometimes used in animals. Often the general condition of the animals is so badly affected that it is too un economic failures is coming. The one from Freund to complete the Adjuvant suggested mycobacteria M. tuberculosis or M. buty ricum are also poorly tolerated. They also call granulomas and Fever and abscesses at the injection site [Brown, E.A. Rev Aller gy 1969 23 (5): 389-400].

There is therefore the task of finding a more tolerable adjuvant, ins especially from the point of view of inexpensive vaccines  make desirable global vaccinations against certain Er diseases that are financially real only with an inexpensive vaccine and the increasing importance of immunological products tion techniques and products. Even with increasing awareness of the need for protection of animals and the corresponding pressure of the Ge Legislation must be more tolerable (i.e. biocompatible) equally efficient tes, but at the same time inexpensive adjuvant can be found. Additional Lich it must be physically stable so that an admixture before the In jection can be eliminated and fluctuations in efficiency - z. B. by different dispersion - to be eliminated.

The most frequently used adjuvant today with approval for Human use is a fine suspension of aluminum hydroxide, the roots of which are even more archaic than those of the emulsion of Freund [Glenny, A. T. et al. J. Pathol. 1926, 29 31-40]. It is based on the Assumption that antigens are better recognized by the immune system if they are adsorbed on the surface of the aluminum hydroxide particles. However, it is disadvantageous that aluminum hydroxide is less efficient than Freund's adjuvant and it can also cause granulomas.

Starting from Freund's emulsion, emulsions have recently been added wrote, which consist of more compatible materials such. B. Squalane and Sqalen, [Sanchez-Pestador, L. et al. J. Immunol. 1988 141, 1720-1727, Masihi KN, Lunge, W., Bremer W., Ribi, E. Int. J. Immuno pharmacol. 1986 8 (3), 339-45, Hunter R.L., Bennett, B. Scand J Immunol. 1986 23 (3), 287-300, Allison AC, et al. Semin Immunol. 1990 2 (5), 369-74]. One of these systems is MF59 (European Patent Application 0399843A2).  

Based on the suspensions according to Glenny, particles from below various polymers to various inorganic and organic particles (e.g. carbon) are used [O'Hagan, D.T., Jeffrey, H., Roberts M.J., McGee, J.P., Davies, S.S. Vaccine. 1991 9 (10), 68-71, Glenny, A.T. et al. J. Pathol. 1926, 29 31-40]. Disadvantages of these systems are the cytotoxicity of some polymers (e.g. formaldehyde formation in Po lyalkylcyanoacrylates), missing or too slow degradation rate in the organism (e.g. polystyrene or polymethacrylate derivatives), insufficient appropriate biocompatibility (e.g. capsule formation with PLA particles, pH Displacement up to pH2) and inadequate approval by the Registration authorities (e.g. soot particles).

An alternative way to use particulate adjuvants such as Oil drops or solid particles is the use of macromolecular Adjuvants. It has been shown that the My cobacteria are replaced by building blocks from their capsule substance who have greater tolerance to the body. Especially be were written and synthesized MDP (N-acetylmuramyl-L-alanyl-D- isoglutamine) [Adam, A., Lederer, E. Med. Res. Rev. 1984 4, 111-152] Thr- MDP (threonyl analog of MDP) [Byars NE, et al. Vaccine. 1987 5 (3), 223-8] and the GMDP (N- acetylglucosaminyl-N-acetylmuramyl dipeptide) [Grubhofer, N. Immunolo gy Letters 1995 44, 19-24]. GMDP is a homologue of the MDP, for GMDP an increased immunostimulating effect has since been replicated pointed (patent specification DE 196 11 235 C1).

Other approaches in developing an adjuvant were the use of molecular adjuvants in high concentration so that the Solubility was exceeded and a suspension was obtained (e.g.  DDA (dimethyldioctadecylammonium bromide) [Grubhofer, N. Immunology Letters 1995 44, 19-24] and to increase the humoral response the Mi creation of macromolecular adjuvants such as solid glycopeptides substance particles (e.g. GMDP with colloidal particles) [Grubhofer, N. Im Munology Letters 1995 44, 19-24].

So far, however, no solution has been found which is the same effective efficacy like Freund's adjuvant and at the same time meets other requirements: The new emulsions will not always tolerated without reaction, the synthetic macromolecules like So far, glycopeptides have not been superior to mycobacteria shows, in addition, they are far too expensive. Solid particles have Zulas solution problems. Many adjuvants show too high toxicity and thus too low biocompatibility. There are also problems with the physi stability (avoidance of aggregation or coalescence). So must the FIA emulsion is freshly made and injected, long-term location tion is not possible. Sufficient physical storage stability is always but the essential requirement for a marketable product - especially especially for pharmaceuticals.

The goals are thus the production of a new adjuvant with:

• 1. sufficient physical stability,
• 2. low cytotoxicity and sufficient biocompatibility,
• 3. comparable effectiveness to FIA,
• 4. inexpensive manufacture.

1. Description of the invention

Until now, GMDP had to be used to increase the immunostimulating effect Mixture with colloidally dispersed solid lipid particles in a particle size <200 nm can be used (USA Patent Gerbu, processing number of U.S. Patent Office is 08 / 816,787). The structure of the particles was described in Unter searches for this invention systematically modified (e.g. used Surfactants, stabilizers) to find lipid particles that the immunostimu could further increase the effects of GMDP. More surprising We have found that lipid particles alone are just as efficient are like the combination of lipid particles and GMDP (Example 9). Consequently can be dispensed with the expensive GMDP in the invention and a comparably high humoral immune stimulation by a cost-effective total lipid particles can be achieved alone.

The strength of the immunostimulating effect depends on the Surface properties of the particles (surfactants used, particleella dung) and on the particle size. If the lipid part is negatively charged kels, the effectiveness is about 1/3 of FIA when generating a positively charged lipid particle by adding EQ1 is the effect efficiency comparable to FIA (no significant difference, t-test) (Example 9). About the composition of the lipid particles used, the desired effectiveness can be set specifically. This avoids Overreaction of the organism.

From previous adjuvant research on inorganic and polymer Suspensions are known to have a particle size for each antigen maximum effectiveness, there is also a species dependence  knows. So the most suitable particle size of aluminum hydroxide suspensions for a vaccine empirically determined, polymer Particles as an adjuvant for immunization had a different one Effect as a function of their size [Kreuter J, et al. Vaccine. 1986, 4, 125-9]. The same thing happened despite the very different matrix material Surprisingly found for the lipid particles, so that the effects Strengthen antigen-specific and species by changing the particle size can be modulated specifically.

The lipid particle dispersions of the stable biocompatible adjuvant (SBA) consist of water or aqueous liquids or non-aqueous rigen, e.g. B. oily liquids dispersed lipid particles, these can be stabilized by surfactants or polymers, but not necessarily sen. With a sufficiently high viscosity of the outer phase or fineness a physically stable dispersion of the particles is obtained. Same thing also applies to low-viscosity outer phases when the particles are out carry a sufficiently high charge in the same direction and thus sediments formed can be easily redispersed.

The SBA is produced by dispersion or precipitation, being described in textbooks of pharmacy and process engineering generally known methods are used. When dispersing one divides coarsely dispersed lipids by mechanical methods. The lipids can be in the solid state (e.g. mortar mill) or are in the liquid state (e.g. emulsification of molten Lipids by stirrer). To produce the SBA dispersion, the Li pide first crushed and then in the outer (e.g. aqueous) Phase are dispersed or alternatively zer directly in the outer phase be shrunk. For the production of SBA dispersions by dispersion  tion can z. B. u. a. are used: piston-gap homogenizers (e.g. APV homogenizers, French Press), jet stream homogenizers (e.g. Microfluidizer), rotor-stator stirrer (e.g. Ultraturax, Silverson-Homo generators), static mixers on a micro-scale and macro-scale (e.g. Mixer from Sulzer), gas jet mill, rotor-stator colloid mill and mortar grinder (Example 15).

SBA dispersions show sufficient long-term physical stability lity. Determination of particle size with photon correlation spectroscopy pie (PCS) and laser diffractometry (LD) showed no or negligible ba res particle growth over periods of 1-3 years (Example 3). The The stability of SBA dispersions is that of Freunds incomplete Adjuvant (FIA) far superior (Example 1); also towards newer Adju vans emulsions show a higher stability (example 2).

stability

SBA dispersions represent a simple system In addition to glycopeptides, the auxiliaries used are chemically simple structured and robust. Use of heat in sterilization processes does not lead to chemical decomposition, the dispersion remains physical stable and particle aggregation does not occur. An example of the Sterilisa SBA dispersions by autoclaving (121 ° C, 15 minutes, 2 bar) shows example 4. FIA shows phase separation under the same conditions tion.

Many vaccines are stored at a low temperature (4-6 ° C). At optimized SBA dispersions is also storage at room temperature and even higher temperatures possible without physical Destabilization occurs (Example 11). This simplifies handling of products based on SBA, especially for hotter climates  (e.g. use as adjuvant for mass vaccinations in third country countries World).

Biocompatibility

Low is essential for a widely used adjuvant Toxicity and good biocompatibility. In a sheep study, after Application of SBA dispersion no abnormalities at the application site observed (Example 6). The good tolerance is explained with the in vi Tro in cell cultures observed extremely low toxicity of lipids. in the Compared to the German regulatory authorities and the FDA Polymers approved for parenteral administration show one around at least approx. factor 20 higher viability with high particle concentration tion (Example 5). The good biocompatibility is attributed to that body proteins in general are limited to par adsorb the particle surface - in contrast to other particles (example 7). In addition, there are no proteins detected on the surface that promote intolerance reactions.

For a wide use of an adjuvant, it offers cost reasons to make an adjuvant preparation that is used before using the Antigen solution is added. Ideally, the adjuvant in its properties be made so that it is under a number different antigens can be added. To reduce the In the mixture should be in physiological saline or otherwise isotonized solution. Physiological Saline leads to a due to the reduction in zeta potential Destabilization in bispersions and subsequent aggregation [Lucks, J. S. et al., Int. J. Pharm 1990 58, 229-235]. The SBA adjuvant should after admixing to the antigen in isotonic solution for a sufficiently long time be physically stable. Example 8 shows that the lipid particles according to Zumi  stable to physiological saline even over 6 hours are; measurable aggregation does not occur.

In addition to the particle size, surface properties such as La targeted and thus the SBA adjuvant is effective can be produced specifically for the target. Positive and negative charges can by adding appropriately charged surfactants or stabilizers be generated. About the concentration of the additive, the strength of the Charge can be set. The ideal addition are charged substances zen that like cetylpyridinium chloride as a preservative for the pa renteral application are permitted (example 12).

A variety of different lipids can be used to make SBA Dispersions are used. These are both chemically uniform Lipids as well as their mixtures. The lipids are characterized by that in the end product SBA dispersion in the crystalline state (e.g. β-, βi modification) or in the liquid-crystalline state (α modification) lie or in their mixture. When lipid mixtures are used also liquid lipids (e.g. oils, lipophilic hydrocarbons, lipophilic orga liquids such as oleyl alcohol) the solid lipids (e.g. glycerides, lipophilic hydrocarbons such as hard paraffin) are added (so-called "lipid blends").

Find z. B. following lipids as a dispersed phase and can be as individual components or as a mixture: natural che or synthetic triglycerides or mixtures thereof, monogly cerides and diglycerides, alone or mixtures thereof or with e.g. B. Triglycerides, self-emulsifying modified lipids, natural and synthetic waxes, fatty alcohols, including their esters and ethers  as well as in the form of lipid peptides, or any mixtures thereof ben. Synthetic monoglycerides, diglycerides and Triglycerides as individual substances or as a mixture (e.g. hard fat), Imwitor 900, triglycerides (e.g. glycerol trilaurate, glycerol myristate, Gly cerolpalmitate, glycerol stearate and glycerol behenate) and waxes such as B. Cetyl palmitate and white wax (DAB). Also hydrocarbons, such as B. Hard paraffin.

The proportion of the inner or lipid phase based on the total formulation tion is 0.1% to 80% (m / m) and is preferably in the range of 1% up to 40% (m / m). Should the addition of dispersion stabilizing Additi ven may be necessary or desired, e.g. B. emulsifiers to stable dispersers To be able to produce sion, these can be in the form of pure sub punch or be incorporated in the form of mixtures to the particles to stabilize.

The amount of such additives, which can be added in relation to the total weight of the aqueous dispersion, is in the range from 0.01% to 30% and preferably in the range from 0.5% to 20%. To stabilize the SBA dispersions or for their targeted surface modification, the surfactants, stabilizers and polymers can be used, which are generally known from the production of dispersions. Examples include:

• 1. sterically stabilizing substances such as poloxamers and poloxamines. (Polyoxyethylene-polyoxypropylene block copolymers), ethoxylated Sorbi Tan fatty acid esters, especially polysorbates (e.g. Polysorbate 80 or Tween 80®), ethoxylated mono- and diglycerides, ethoxylated lipids, ethoxylated Fatty alcohols or fatty acids, and esters and ethers of sugars or of  Sugar alcohols with fatty acids or fatty alcohols (e.g. sucrose mono stearate, sucrose distearate, sucrose cocoat, sucrose stearate, sucrose Tue palmitate, sucrose palmitate, sucrose laurate, sucrose octanoate, sucrose Oleate).
• 2. charged ionic stabilizers such as diacetyl phosphates, phosphates dylglycerol, lecithins of various origins (e.g. egg lecithin or So jalecithin), chemically modified lecithins (e.g. hydrogenated lecithins), ge just like phospholipids and sphingolipids, mixture of lecithins with Phospholipids, sterols (e.g. cholesterol and cholesterol derivatives, ge just like stigmaster) and also saturated and unsaturated fat acids, sodium cholate, sodium glycocholate, sodium taurocholate, natri umdeoxycholate or their mixtures, amino acids or anti Flocculants such as B. sodium citrate, sodium pyrophosphate, sodium sor bat [Lucks, J.S. et al. Int. J. Pharm., 1990, 58, 229-235]. Zwitterionic Surfactants such as B. (3 - [(3-cholamidopropyl) dimethylammonio] -2-hydroxy-1- propanesulfonate) [CHAPSO], (3 - [(3-cholamidopropyl) dimethylammonio] - 1-propanesulfonate) [CHAPS] and N-dodecyl-N, N-dimethyl-3-ammonio- 1-propanesulfonate. Cationic surfactants, especially as preservatives medium-sized compounds, such as. B. Benzyldimethylhexadecylammo nium chloride, methylbenzethonium chloride, benzalkonium chloride, cetylpy ridinium chloride.
• 3. viscosity-increasing substances such. B. cellulose ether and cellulo se esters (e.g. methyl cellulose, hydroxyethyl cellulose, hydroxypropylcel lulose, sodium carboxymethyl cellulose), polyvinyl derivatives and Po lyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl acetate, alginates, polyacrylates (e.g. Carbopol), xanthans and pectins.

The loaded stabilizers are, if necessary or desired, in front preferably with 0.01% to 20% (m / m) and in particular in an amount of 0.05% up to 10% contained in the SBA dispersion. Viscosity increasing Substances are in a similar ratio if necessary or desired nis incorporated in the formulation, preferably in an amount of 0.01-20% and in particular in an amount of 0.1% to 10% (m / m) and preferably in the range between 0.5% and 5%.

Can be used as the outer phase (dispersion medium, continuous phase) Water, aqueous solutions or liquids miscible with water, see above such as glycerin or polyethylene glycol and oily liquids such as miglyols (medium chain triglycerides - MCT) and other oils (castor, peanut, Soybean, cottonseed, rapeseed, linseed, olive, sunflower, Di stel oil can be used.

Surfactant-free SBA are produced by dispersing the lipid phase in an aqueous solution containing one or more viscosity increasing sub punching contains, either alone or in combination with other sub punch, as well as sugar, sugar alcohols, especially glucose, mannose, Trehalose, mannitol, sorbitol and others. Furthermore it is possible a combination of the viscosity-increasing substances or the combination this with sugar or sugar alcohols, or in another combination nation with charge stabilizers or anti-flocculants.

SBA dispersions can be used as an adjuvant to many different anti genes for vaccination against various diseases. Examples include:
Glycoproteins such as e.g. B. Gonococcal Protein I, Brucella abortus antigen, Tetanus toxoid, Diphteria toxoid, Listeria monocytogenes,
Virus antigens such as Semliki Forest Virus, Enceohalomyocarditis Virus, Porcine rarovirus, Pseudorabiesvirus, Newcastle desease Virus, Bovine vi ral diarrhea, HIV, Influenza, Cytomegalievirus, Herpes Simplex, Hepatitis-C, Measles,
Parasites such as B. Malaria, Eimeria Spp.

In summary, it can be said that the SBA dispersions provide an adjuvant that:

• 1. has sufficient physical stability to function as a product and to be made specifically as a medicine
• 2. has low toxicity and good biocompatibility, in particular if biodegradable lipids such as glycerides are used,
• 3. a comparable effect to Freund's incomplete adjuvant (FIA) owns and
• 4. inexpensive from low-priced excipients with inexpensive Manufacturing process can be produced.

SBA dispersions can find wide application to deal with toxicologically acceptable excipients to reduce the antigen dose and thus the costs adorn, because by adding SBA at low antigen dose the same immunostimulating effect is achieved.

Antigens with previously insufficient antigenicity for a vaccine can by adding immune response stimulating SBA to an effici vaccine to be transferred.  

Due to the inexpensive manufacture of existing comparable Efficiency to FIA, SBA dispersions are suitable as an adjuvant for vaccinations in the veterinary sector, where for profitability reasons only very low-priced Vaccines can be used.

Adjuvants used to date have focused on the increase the humoral immune response. Given the effectiveness of SBA Dispersions it is no longer necessary to add another adjuvant to the SBA Add lipid particles or add adjuvants such as GMDP no further increase in humoral immune response. Obviously the immune system at its maximum response capacity, additional This adjuvant can no longer bring an additional effect. So brin additions to SBA for the humoral answer no advantage, additions like in the Gerbu patent (patent specification DE 196 11 235 C1) ben, by the surprisingly found potency of the Er SBA described invention superfluous.

Surprisingly, however, it was found that after application of GMDP in combination with SBA dispersions at a renewed late vaccination for a significantly increased cellular immune response came as if previously only SBA dispersion was used alone as an adjuvant has been. It was thus newly found that a combination of SBA Dispersion specifically for increasing the cellular immune response in he new vaccination is suitable. There is an increased immune response in the event of a subsequent second vaccination, if an additional adjuvant such as GMDP was used in a mixture with SBA dispersion (Example 13).

For the preparation of an adjuvant with the aim of increasing the cellular It is therefore advantageous to use an SBA dispersion with an immune response  to combine another adjuvant. Possible adjuvants for a station wagon nation are: N-acetylglucosaminyl- (β1-4) -N-acetylmuramyl-L-alanyl-D- isoglutamine [GMDP], dimethyldioctadecylammonium bromide [DDA], N- acetylmuramyl-L-alanyl-D-isoglutamine [MDP], N, N di- (β-stearoylethyl) - N, N-dimethylammonium chloride [EQ1], glycopeptides, components of the Cell wall of mycobacteria, saponins, quaternary amines, such as B. Cetyl pyridinium chloride and benzalkonium chloride, zwitterionic amines such as CHAPS (3 - [(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate), Dextran sulfate, dextran, 3-odesacyl-4'-monophosphoryl lipid A [MPL®], N- Acetyl-L-alanyl-disoglutaminyl-L-alanine-82) -1,2 dipalmitoyl-sn-glycero - 3- (hydroxy-phosphoryloxy)) ethylamide, Monosodium salt [MTP-PE], Gra nulocyte-macrophage colony stimulating factor [GM-CSF], block copolymers, e.g. B. P1205, Poloxamer 401 (Pluronic L121), dimyristoyl phosphatidylcholine [DMPC], dehydroepiandrosterone-3β-01-17-one [DHEA], Dimyristoylphosphatidylglycerol [DMPG], deoxycholic acid sodium salt, Cytokines, imiquimod, DTP-GDP, saponins, 7-allyl-8-oxoguanosine, mon tanide ISA 51, Montanide ISA 720, MPL, Murametid, Murapalmitin, D- Murapalmitin, 1-monopalmitoyl-rac-glycerol, dicetyl phosphate, polyme ethyl methacrylate [PMMA], PEG sorbitan fatty acid esters such as Polysorbate 80 (TWEEN® 80), Quil A saponin, sorbitan fatty acid esters such as sorbitan trioleate (SPAN®85, Arlacel85), DTP-DPP, stearyl-tyrosine, N, N dioctadecyl-N ', N'- bis (2-hydroxyethyl) propanediamine, calcitriol.

In addition to increasing the humoral immune response through SBA Dispersions open up the possibility of climbing through the invention Combination of the cellular immune response with a second vaccination of SBA dispersions with other adjuvants.  

Instead of adding adjuvants to increase cellular The adjuvants can also incorporate immune responses into the lipid particles be cured. Incorporation is possible through storage in the solid Particle matrix, enrichment in the interface in the case of amphiphilic Adjuvants or by simple adsorption on the particle surface. The incorporation of adjuvants can occur during particle production or carried out subsequently (e.g. in the case of incorporation, example 16). For incorporation during manufacture, adjuvants are used in the ge melted lipid phase dissolved, solubilized or dispersed and the ad The lipid phase containing juvans is then further processed. In the case of on phiphile adjuvants can also be used in the external phase of SBA Dispersion can be solved and then accumulate in the particle boundary surface or by adsorption on the surface. By incorporation There is an extended release of adjuvants by the Diffusion or in the course of particle breakdown by enzymes. A delayed one Prolonged release increases the immune response.

Another attractive area of application is antibody production in the animal. The antibody yield can be clearly seen by adding the adjuvant increase.

Examples example 1 Determination of the physical stability of SBA-versus- Freund's Incomplete Adjuvant (FIA)

An aqueous SBA dispersion was made by high pressure homogenization at 95 ° C from 20% beeswax, 2% Tween 80 manufactured (PCS diameter 289 nm polydispersity index 0.101). FIA was obtained using the method described by Freund [Freund, J. J. Immunology 1948, 60, 383-398]. SBA and FIA were  stored at the temperatures of the climate zones, which at the Stabili testing of medicinal products [EMEA guideline CPMP / QWP / 159/96, January 1998)]. The storage temperatures were: 5 ° C, 25 ° C, 40 ° C. The physical stability was determined using mes Particle size determination with laser diffractometry, characterization para meters were the diameter 50% and the diameter 95% (50% or 95% of the particles are below the specified size, sensitive Pa parameters for particle aggregation). FIA showed up after a few minutes Storage - even at room temperature - significant particle growth, FIA is therefore not a storage-stable adjuvant. In contrast, the par SBA particle sizes under the 3 storage conditions over a period of time unchanged from 1 year (Table 1).

Table 1

Investigation of the stability of SBA (20% beeswax, 2% Tween 80) in comparison with Freund's incomplete adjuvant (FIA)

Example 2 Determination of the physical stability of SBA-versus- Squalene adjuvant

SBA was prepared as described in 1; it contained 10% cetyl palmitate and 1.2% miranol (PCS diameter 210 nm, PCS polydispersity index 0.189). Squalene adjuvant was made as described in European patent application 0 399 843 with registration date May 25, 1990 (adjuvant MF59). Particle size measurement solution was done with laser diffractometry; Storage was as in Example 1 performed at 3 temperatures (Table 2). In addition, a be accelerated stability test carried out, SBA and MF59 dispersions were shaken at 40 ° with a frequency of 50 Hz (Table 3). Like this SBA shows an increased rate, probably in normal storage as well as in the stress test Stability.  

Table 2

Investigation of the stability of SBA (10% cetyl palmitate, 1.2% miranol) in comparison with MF59

Table 3

Stability of MF 59 against SBA at 40 ° C and a shaking frequency of 50 Hz

Example 3 Long-term stability of SBA

SBA dispersion consisting of 20% Beeswax, 2% Tween 80 was stored at 4-6 ° C for one year. The PCS Data and laser diffractometer diameter showed little or no Change (table 4).

Table 4

Long-term stability of SBA: SBA dispersion consisting of 20% beeswax, 2% Tween 80 was stored at 4-6 ° C for one year

Example 4 Heat stability of SBA during autoclaving (heat, pressure) ver sus FIA and MF59

The SBA dispersion was composed of 18% Hard paraffin, 4% Tween 80 / Span 85 (7/3) and water. Sterilization of 20 mL each was given in injection vials according to the standard conditions European Pharmacopoeia (121 ° C, 2 bar, 15 minutes). Parti The size was determined using PCS and laser diffractometry (LD 95%) (P. I .: polydispersity index, measure of the width of the particle distribution, MW: Average of 3 measurements, staff: standard deviation, P.I. Polydis persistence index) (Table 5). The FIA emulsion showed after autoclaving  Phase separation, MF59 significant particle growth. SBA is physi calically stable and can be sterilized by autoclaving.

Table 5 Heat stability of SBA during autoclaving (heat, pressure) versus FIA and MF59

The SBA dispersion was composed of 18% hard paraffin, 4% Tween 89 / Span 85, 7/3 and water. Sterilization of each because 20 mL in injection vials at 121 ° C, 2 bar, 15 minutes. Parti The size was determined using PCS and laser diffractometry.

Example 5 Physiological tolerance

To estimate the contract The cytotoxicity of SBA in cell cultures was determined (human granulocytes, HL60 cells). To quantify toxicity the viability of the cells was checked using the MTT test [Mosmann, T., J. Immu nol. Meth. 1993, 65, 55-63]. The SBA dispersion was together composed of 10% cetyl palmitate, 0.5% poloxamer 188 and water. The Cell count per well was 200,000 in human granulocytes and 200,000 in HL60 cells. Incubation was for 12 hours. The SBA was the via 80% in the granulocytes and 85% in the HL60 cells. At Nanoparticles made of PLA were only 5% viable, for nanoparticles from PLA / GA it dropped to 0%. The compatibility of SBA is in the Cell cultures are at least 20 times better than those approved by the FDA polymers for parenteral administration.

Example 6 Compatibility after parenteral administration

Was used aqueous SBA dispersion with the composition 5% hard paraffin, 5% Tween 80 / Span 85 (7/3) and water. Parenteral injection was carried out in Sheep (n = 30), injection site was the side chest wall, the injection volume was 5 mL spread over 4 injection sites. The sheep showed none Abnormalities, neither at the injection site nor in their behavior.

Example 7 Biocompatibility - interaction with body proteins

The SBA Dispersion was composed of 10 Compritol, 2.5% Poloxamer 407 and water. Production was carried out with high pressure homogenization. The par particles were incubated with human plasma for 5 minutes, then separated from the plasma and adsorbed on the particle surface Body proteins with two-dimensional polyacrylamide gel electrophoresis [Blunk, T et al. Electrophoresis 14, 1382-1387 (1993)]. With ver  comparable particle surfaces adsorbed on SBA with 96.41 cpm (counts per minute) a very low pro compared to emulsions Quantity of stone (comparison values: 472 cpm on emulsion, 390 cpm on polysty roll particles [Harnisch, S. et al. Elektrophoresis 1998, 19, 349-354, Blunk, T., Electrophoresis 1993, 14, 1382-1387]. Complement factors, the one Promote intolerance were not detected on the SBA surface animals.

Example 8 Stability in phosphate buffered physiological saline solution (PBS)

SBA composed of 20% hard paraffin, 5% Tween 80 / Span85 (7/3) and water were mixed with PBS (2 mL SBA + 2 mL Saline solution). The physical stability in the physiological table salt solution was determined as a function of time using laser diffractometry. There was no increase in particle size over 6 hours (Diameters 90% and 95%, Table 6) (staff: standard deviation).

Table 6

Stability in phosphate buffered physiological saline solution (PBS) SBA (20% hard paraffin, 5% Tween 80 / Span85 (7/3)) mixed with PBS (2 mL SBA + 2 mL saline). Determination of the physi Kalische stability in the physiological saline solution with Laserdif fractometry as a function of time.  

Example 9 Adjuvant effect compared to molecular adjuvant (GMDP - N-acetylglucosaminyl-N-acetylmuramyl dipeptide) and FIA

Sheep were vaccinated with the Mycoplasma Bovis PG 45 R9 strain. The vaccine antigen was cultivated in stand culture over 72 hours under microaerophilic conditions in Hayflick medium. Inactivation was carried out by adding 0.1% β-propiolactone. The cells were separated, washed with phosphate buffer pH 7.4 and adjusted to a content of 1 × 10 ¹⁰ CFU / mL. The sterility of the preparation was checked in accordance with the German Pharmacopoeia, Edition 10. The dry matter determination gave a content of 1 mg / mL Mycoplasma Bovis antigen. The adjuvant SBA, GMDP and FIA were mixed equally with the antigen in buffer. Injection volume was 5 mL, spread over 4 injection sites.

Composition of SBA was 4% hard paraffin, 1% EQ1 (N, N Di- (β- Stearoylethyl) -N, N-dimethylammonium chloride) and 4% Tween 80 / Span  85 (7/3). Composition of the GMDP adjuvant was 5% lipid and 0.5% surfactant. FIA was made as in Example 1.

Blood was drawn on day 0 before vaccination, on day 35 and on day 63. The antibodies were determined using ELISA. For the ELISA test a commercially available marked anti-IgG sheep from Sigma puts.

SBA showed a comparable intensity of action as the combination of lipid particles with GMDP. Furthermore, SBA was as effective as FIA ( Fig. 1). There was no significant difference in the intensity of action between the three adjuvants.

Fig. 1 Adjuvant effect compared to molecular adjuvant (GMDP - N-acetylglucosaminyl-N-acetylmuramyl dipeptide) and FIA

To composition of SBA was 4% hard paraffin, 1% EQ1 (N, N Di- (β- Stearoylethyl) -N, N-dimethylammonium chloride) and 4% Tween 80 / Span 85 (7/3). Composition of the GMDP adjuvant was 5% EQ1 and 0.5% Montanide 888. FIA according to example 1.

Example 10 Effect of SBA composition on immune titer

SBA dispersions were produced with identical lipid, but different surfactants on the surface, ie they differ in their surface properties. The formulation SBA-1 consists of 4% hard paraffin, 4% Tween 80 / Span 85 (7/3) and water, the formulation SBA-2 contains 4% hard paraffin, 1% EQ1 and 4% Tween 80 / Span 85 (7 / 3) and water. The efficiency to increase the immune titer was tested analogously to example 9; FIA served as a comparison. Depending on the surface properties, there are different levels of immune response for the two SBA dispersions ( Fig. 2). The strength of the desired immune response can thus be adjusted by varying the surface properties (surfactants, stabilizers, charge, etc.).

Fig. 2 Effect of SBA composition on immune titer

Formu SBA-1 consists of 4% hard paraffin, 4% Tween 80 / Span 85 (7/3) and water, formulation SBA-2 contains 4% hard paraffin, 1% EQ1 and 4% Tween 80 / Span 85 (7/3) and water.

Example 11 Storage stability of SBA-2

The SBA dispersion SBA-2 from Example 11 was stored at different temperatures and the physical stability determined by measuring the particle size with PCS. Particle growth did not occur ( Fig. 3).

Fig. 3 Storage stability of SBA-2

The SBA dispersion SBA-2 from Example 11 was stored at different temperatures and the phy Physical stability determined by measuring the particle size with PCS.

Example 12 Surface modification of SBA dispersions

To Modifi Cation of the surface charge were SBA dispersions with interfac active positive stabilizers (EQ1 - Distearoylethyldia monium chloride, cetylpyridinium chloride) and negatively charged Stabilisa gates (sodium lauryl sulfate, (SDS)). The composition of the SBA dispersions were: SBA-EQ1 (20% cetyl palmitate, 4% Tween 80 / Span 85 (7/3), 1% EQ1), SBA-CPC (18% lipid, 10% surfactant, 0.1% cetylpyridi nium chloride) and SBA-SDS (20% cetyl palmitate, 1% SDS 80. As a measure of the charge, the zeta potential was measured in millivolts (mV)  (Electrophoresis measurement), Zetasizer4 (Malvern Instruments, UK). Um electrophoretic mobility was converted into zeta potential with the Helmholtz-Smoluchowski equation. The zeta potentials were +40 mV, +28 mV and -35 mV for the 3 SBA dispersions.

Example 13 Increased cellular immune response in chickens when refreshed Vaccination vaccine containing the first immunization with GMDP Adjuvant (SBA) had been treated

SBA (5% EQ1, 0.5% Montanide 888 was mixed 1: 1 with the antigen (rabbit IgG) and injected subcutaneously. SBA contained 5 µg GMDP, and the immunization schedule was as follows: first immunization and two booster shots per day 14 and day 28. The antibody determination took place on day 42, and the IgY titer was measured in the egg yolk. To test for a strengthened cellular immune response, a new antigen contact took place on day 100 (renewed vaccination) and on day 120 the antibody determination. The results show that in the case of the first immunization (day 14 and 28) with SBA containing GMDP and with renewed antigen contact, a significantly increased antibody production takes place. Columns 2, 3 and 4, Fig. 4.

Fig. 4 Antibody production in chickens after basic Immunization and renewed antigen contact on day 100

The last antibody determination of the basic immunization took place on day 42, that of the booster vaccination (day 100) on day 120. The composition of the vaccines of Examples 1-5 is as follows:

• 1. Day 42: antigen in PBS, day 100: antigen in PBS (n / a: no adjuvant, Antigen in PBS).
• Day 42: Antigen in SBA with GMDP, Day 100: Antigen in SBA.
• 3. Day 42: Antigen in SBA with GMDP, Day 100: Antigen in FCA (FCA = Freund's complete adjuvant).
• 4. Day 42: Antigen in SBA with GMDP, Day 100: Antigen in PBS.
• 5. Day 42: antigen in FCA, day 100: antigen in PBS.

Example 14

The adsorption of GMDP on SBA particles was determined using PCS examined. GMDP was mixed with SBA (4% hard paraffin, 4% tween 80 / Span 85 (7/3) and left for 30 minutes at room temperature, to enable adsorption of the GMDP on the particles. The end concentration was 1.435 mg / ml. The increase in size is 3.9 nm. (PCS diameter without GMDP: 99.4 nm, standard deviation: 0.764, Diameter with GMDP: 103.3 nm, standard deviation: 0.755).

Example 15 Modification of particle size

The SBA dispersion with EQ1 from Example 12 (SBA-EQ1) was produced using various manufacturing processes in order to vary the particle size. The particle size was measured using laser diffractometry (laser diffractometer LS 230, Coulter Electronics, Germany, measuring range: 40 nm-2000 µm). The diameter 50% of the particles is given as a characterization parameter. The following manufacturing methods were used:

• a) High pressure homogenization:
  The lipid was melted, added to the aqueous surfactant solution, dispersed with a stirrer and the crude emulsion obtained was homogenized at 80 ° C. using a high-pressure homogenizer (Micron LAB 40, APV Gaulin Homogeniser GmbH, Germany). Homogenization parameters were 500 bar pressure, 3 homogenization cycles. The particle diameter 50% was 0.15 µm.
• b) The raw emulsion was prepared as described under a) and homogenized with a microfluidizer (device type 110-Y, Microfluidix Inc., USA).
  Homogenization parameters were 700 bar, 10 minutes circulation time. The average particle diameter was 0.452 µm.
• c) Rotor-stator dispersion:
  The crude emulsion was prepared as described under a) and then dispersed with an Ultraturrax (type T25, Jahnke and Kunkel, Staufen, Germany) at a speed of 10,000 revolutions per minute for 1 minute and 10 minutes, dispersion temperature 80 ° C. The particle diameter were 7.5 and 1.2 µm
• d) Static mixer:
  Lipid and aqueous surfactant solution from a) were heated to 80 ° C. and mixed in a static mixer (Sulzer, Germany). The particle size was 15.8 µm.
• e) Gas jet mill:
  Beenic acid triglyceride was air-jet milled (Jetmill, Mosokawa Alpine AG) and then dispersed with stirring in the aqueous surfactant solution at room temperature. The particle diameter 50% was 37.03 µm.
• f) Mortar grinder:
  The coarsely powdered lipid was milled in a mortar mill with the addition of liquid nitrogen for 3 minutes and 15 minutes (Retsch Mörsermühle, Reetsch, Germany). The lipid was dispersed in water as in e). The mean particle size was 40 µm.

Example 16 Molecular adjuvant to increase cellular Im munantwort, incorporated in SBA

GMDP was in Span 85- (W / O) Emul gator dissolved and added cetyl palmitate. The mixture was at 70 ° C melted and after re-cooling in the mortar mill ground addition of liquid nitrogen. The ground lipid GMDP mixture was dis in a 2.5 percent Tween-80 solution pergiert and predispersed with the Ultraturrax for 1 minute at 8000 rpm. This dispersion was at 4 ° C by means of high pressure homogenization in 3 cycles homogenized at 1000 bar. The PCS diameter is 260 nm with a polydispersity index of 0.430.

Example 17 Molecular adjuvant to increase cellular Im munesponse, incorporated into the interface

Saponins are generally known knows to increase the cellular immune response. The manufacture of the Particles were made using a rotor stator analogous to example 15. Die Zu The composition of the SBA dispersion is 5% cetyl palmitate, 0.5% saponin (Quil-A-Saponin) and water. The saponin was in the aqueous phase dissolved, heated to 80 ° and added the melted lipid. Forth position with an Ultraturrax, stirring at 10000 RPM for 5 mi grooves. The diameter determined with the laser diffractometer 50% was 2.28 µm.  

Example 18 Production of SBA in the presence of an amphiphilic adju vant

The amphiphilic surfactant CHAPS is described in the literature as Means to increase the immune response. The particles consist of 5% Cetyl palmitate and 0.5% CHAPS. The particles were produced ana log Example 20. The diameter 50%, using laser diffractometry true, is 1.897 µm.

Example 19 Comparison of SBA versus pure molecular adjuvant

SBA dispersion No. 2 from Example 10 (SBA-2) was tested in sheep (conditions as in Example 9) against molecular adjuvant, ie pure GMDP (N-acetylglucosaminyl-N-acetylmuramyl dipeptide). The concentration of GMDP (0.1 mg / ml) was analogous to Example 9. The in vivo testing was carried out as described in Example 9. SBA 2 shows a higher active intensity than pure GMDP ( Fig. 5).

Composition of SBA 2

4% hard paraffin, 1% EQ1 (N, N Di- (β- Stearoylethyl) -N, N-dimethylammonium chloride) and 4% Tween 80 / Span 85 (7/3).

Example 20 Effect of the charge (surface property) on the Im munantwort (sheep study analogous to example 9)

There is no difference in the potency between the positively charged particles SBA 4 and SBA 2. EQ1 from SBA 2 can be replaced without loss of efficacy by the cetylpyridinium chloride, which has been investigated toxicologically and approved as a pharmaceutical preservative (SBA 4). Compared to the negatively charged particles of the formulation SBA 5, a stronger effect of the positively charged particle formulations is observed ( Fig. 6).

The SBA formulations have the following composition: SBA 4:
4% hard paraffin, 4% Tween 80 / Span 85 (7/3), 0.5% cetylpyridinium chloride. SBA 5: 4% hard paraffin, sodium deoxycholate 0.2%, sodium cholate 0.2%, sodium oleate 1%, lipoid E80 2%. SBA 2: 4% hard paraffin, 1% EQ1 and 4% Tween 80 / Span 85 (7/3). The surface charge (zeta potential) was determined in conductivity water with a conductivity of 50 µS / cm: SBA 4: +41.2 mV, SBA 2: +40.5 mV, SBA 5: -36.4 mV. The sizes (PCS diameter and polydispersity index (PI)) were: SBA 4: 103 nm (PI 0.110), SBA 5: 107 nm (PI 0.115), SBA 2: 101 nm (PI 0.101).

Example 21 Species independence of the effect (chickens)

The antigen from Example 9 was mixed with SBA 1 and SBA 2 in a ratio of 1: 1 and injected with 0.5 ml per chicken. The antibody titers were determined from the chicken eggs. An ELISA test was used for quantification. In contrast to the description in Example 9, a labeled anti-IgG chicken was used in the ELISA test. The first immunization took place on day 0. A booster with the same preparations took place on day 31. Analogous to the results in example 10, the formulation SBA 2 had a stronger effect ( FIG. 7).

Composition SBA 1: 4% hard paraffin and 4% Tween 80 / Span 85 (7/3), PCS diameter: 107 nm (P.I. 0.112) and SBA 2: 4% hard paraf fin, 1% EQ1 and 4% Tween 80 / Span 85 (7/3), PCS diameter: 101 nm (P.I. 0.101).  

Example 22 Influence of the lipid matrix on the adjuvant effect

In the formulation SBA 2, the non-biodegradable hard paraffin was exchanged for the biodegradable glycerol tribehenate (SBA 3). The intensity of the effect does not differ; Hard paraffin can be replaced by glycerol tribehenate ( Fig. 8).

Composition SBA 2: 4% hard paraffin, 1% EQ1 and 4% tween 80 / Span 85 (7/3), PCS diameter: 101 nm (P.I. 0.101) SBA 3: 4% Gly ceroltribehenat, 1% EQ1 and 4% Tween 80 / Span 85 (7/3), PCS-Durch knife: 105 nm (P.I. 0.112).

Example 23

The formulations SBA 1 and SBA 2 were tested in comparison to aluminum hydroxide (carried out analogously to Example 9). The effect of SBA 1 and SBA 2 is equal to the effect of aluminum hydroxide (control: antigen in PBS). As in Example 9, SBA 2 is more effective than SBA 1 ( Fig. 9).

Composition SBA 2: 4% hard paraffin, 1% EQ1 and 4% tween 80 / Span 85 (7/3), PCS diameter: 101 nm (P.I. 0.101) SBA 1: 4% hard paraffin, 4% Tween 80 / Span 85 (7/3), PCS diameter: 107 nm (P.I. 0.112).  

List of pictures

Fig. 1: Adjuvant effect compared to molecular adjuvant (GMDP - N-acetylglucosaminyl-N-acetylmuramyl dipeptide) and FIA.

Fig. 2: Effect of SBA composition on immune titer.

Fig. 3: Storage stability of SBA 2.

Fig. 4: Antibody production in chickens after basic immunization and renewed antigen contact on day 100, the last antibody determination of the basic immunization took place on day 42, that of the booster vaccination (day 100) on day 120.

Fig. 5: Adjuvant effect of SBA compared to molecular adjuvant GMDP (example 19).

Fig. 6: Effect of different particle loads (example 20).

Fig. 7: Adjuvant effect in chickens (example 21).

Fig. 8: Influence of the lipid matrix on the adjuvant effect (example 22).

Fig. 9: Comparison of the effect of SBA 1 and SBA 2 with aluminum hydroxide (Example 23).

Claims (22)

1. Means to increase the immune response in vaccinations of humans and animals and to increase the yield of antibodies in immunology and antibody production, characterized in that it is solid at room temperature (= 20 ° C) lipid particles, which in an optimal dose characteristic for the respective subject to be immunized, as well as optimal particle size, particle charge and surface properties (stabilizing surfactant layer) are applied, whereby they are simply mixed into the solution of the antigen. 2. Composition according to claim 1, which are particles in the range of 10-1000 nm. 3. Composition according to claim 1, wherein it is particles in the range of 1-10 microns acts. 4. Composition according to claim 1, which are particles in the range of 10-200 µm, especially around particles in the range of 10-100 µm acts. 5. Composition according to claim 1, which are particles in the range of 200-1000 µm, especially around particles in the range of 200-500 µm acts. 6. Composition according to claims 1-5, characterized in that the lipids used to produce the lipid particles at room temperature are solid, for example ethyl stearate, octadecane, DDA, natural  che or synthetic triglycerides or mixtures thereof, Mo noglycerides and diglycerides, alone or mixtures thereof or with z. B. triglycerides, self-emulsifying modified lipids, natural Liche and synthetic waxes, fatty alcohols, including their Esters and ethers, and in the form of lipid peptides, or any Mixtures of the same. Synthetic mono are particularly suitable glyceride, diglyceride and triglyceride as individual substances or as a mixture (e.g. hard fat), Imwitor 900, triglycerides (e.g. Gly ceroltrilaurate, glycerol myristate, glycerol palmitate, glycerol stearate and Glycerol behenate) and waxes such as. B. cetyl palmitate and white Wax (DAB), which is used individually or in mixtures can, also hydrocarbons such. B. Hard paraffin. 7. Means according to claim 6, characterized in that the for Production of the lipid particles used solid lipids liquid Li pide such as liquid gycerides (miglyols), peanut oil, castor oil, soybean oil, Cottonseed oil, rapeseed oil, linseed oil, olive oil, sunflower oil, Safflower oil is added, with the particles made from it are solid at room temperature (20 ° C). 8. Composition according to claims 1-7, characterized in that it are positively charged. 9. Composition according to claim 8, characterized in that for the production the positive charge in the production of the lipid particles the sub punch benzyldimethylhexadecylammonium chloride, methylbenze thonium chloride, benzalkonium chloride, cetylpyridinium chloride, N, N-di- (β-stearoylethyl) -N, N-dimethylammonium chloride, dimethyldiocta  decylammonium bromide individually or in a mixture with one another be set. 10. Composition according to claims 1-7, characterized in that it are negatively charged. 11. A composition according to claim 10, characterized in that for generating the negative charge in the production of the lipid particles following compounds: diacetylphosphates, phosphatidylglycerol, Lecithins of different origins (e.g. egg lecithin or soy lecite hin), chemically modified lecithins (e.g. hydrogenated lecithins), ge just like phospholipids and sphingolipids, a mixture of Lecithi with phospholipids, sterols (e.g. cholesterol and cholesterol Derivatives, just like stigmasterol) and also saturated and un saturated fatty acids, sodium cholate, sodium glycocholate, sodium taurocholate, sodium deoxycholate, individually or in a mixture with one another other be added. 12. Composition according to claims 1-7, characterized in that it are uncharged or only slightly charged. 13. Composition according to claim 12, characterized in that for their Er generation in the production of lipid particles uncharged substances such as polyethylene glycol sorbitan fatty acid esters (especially the Tween Series such as Tween 80), polyoxyethylene-polyoxypropylene copolymers (in particular the Poloxamer series and the Poloxamine series), ethoxylated mono- and diglycerides, ethoxylated lipids, ethoxylated Fatty alcohols or fatty acids, and esters and ethers of sugars or of sugar alcohols with fatty acids or fatty alcohols (e.g. Saccha  rose monostearate) added individually or in a mixture with one another become. 14. Composition according to claims 1-13, characterized in that ih one or more adjuvants are added. 15. Composition according to claim 14, characterized in that it is mo lecular adjuvants such as N-acetylglucosaminyl- (β1-4) -N- acetylmuramyl-L-alanyl-D-isoglutamine [GMDP], dimethyldioctadecy lammonium bromide [DDA], N-acetylmuramyl-L-alanyl-D-isoglutamine [MDP], N, N di- (β-stearoylethyl) -N, N-dimethylammonium chloride [EQ1], glycopeptides, components of the cell wall of mycobacteria, Saponins, quaternary amines such as e.g. B. cetylpyridinium chloride and Benzalkonium chloride, zwitterionic amines such as CHAPS (3 - [(3- cholamidopropyl) dimethylammonio] -1-propanesulfonate), dextran sulfate, dextran, 3-odesacyl-4'-monophosphoryl lipid A [MPL®], N- Acetyl-L-alanyl-disoglutaminyl-L-alanine-82) -1,2 dipalmitoyl-sn-glycero-3- (hydroxy-phosphoryloxy)) ethylamide, monosodium salt [MTP-PE], granulocyte-macrophage colonies stimulating fac tor [GM-CSF], block copolymers, e.g. B. P1205, Poloxamer 401 (Pluronic L121), dimyristoylphosphatidylcholine [DMPC], dehydroepiandro sterone-3β-01-17-one [DHEA], dimyristoylphosphatidylglycerol [DMPG], deoxycholic acid sodium salt, cytokines, imiquimod, DTP- GDP, saponins, 7-allyl-8-oxoguanosine, Montanide ISA 51, Montani de ISA 720, MPL, Murametid, Murapalmitin, D-Murapalmitin, 1- Monopalmitoyl-rac-glycerol, dicetyl phosphate, polymethyl methacrylate [PMMA], PEG sorbitan fatty acid esters such as polysorbate 80 (TWEEN® 80), Quil A saponin, sorbitan fatty acid esters such as sorbitan trioleate  (SPAN®85, Arlacel85), DTP-DPP, stearyl-tyrosine, N, N dioctadecyl- Contains N ', N'-bis (2hydroxyethyl) propanediamine, calcitriol. 16. Composition according to claim 14, characterized in that it is par ticular adjuvants such as B. aluminum hydroxide, polymer particles, Liposomes. 17. A composition according to claim 14, characterized in that the Efficacy of adjuvant enhancing substances from the group of divalent transition metal ions, quaternary amines, Dex tran, dextran sulfate, vitamin E derivatives such as vitamin E phosphate and Vitamin E hemisuccinate, and isoprinosine have been added. 18. Agent according to claims 1-17 prepared in that the Li pidparticles by crushing lipids in the solid aggregate were manufactured, z. B. mortar mill, gas jet mill, elec trosputtering, high pressure homogenization, microfluidization. 19. Composition according to claims 1-17, characterized in that the Li pidparticles by crushing lipids in the molten zu was under dispersion in an external phase (e.g. high-speed Stirrers, static mixers on a micro and macro scale, Rotor-stator mills, colloid mills, high pressure homogenization, Mi crofluidization, spray processes such as spray drying and electro spray process) are produced, the outer phase being liquid (Water, organic liquids, oils or their mixtures) or can be gaseous (air, nitrogen, noble gas).   20. Composition according to claims 1-19, characterized in that the Lipid particles and possibly the additional adjuvant in an outer Phase are dispersed, e.g. B. aqueous liquids such as water, isoto African sugar solutions and isotonic sodium chloride solution, non-aqueous liquids such as PEG 400 or 600, organic rivers liquids such as oils (miglyols, peanut oil, castor oil, soybean oil, cotton seed oil, rapeseed oil, linseed oil, olive oil, sunflower oil, safflower oil and other oils or their mixtures. 21. Composition according to claim 20, characterized in that the au higher phase viscosity-increasing substances were added, e.g. B. 22. Composition according to claims 1-19, characterized in that the Lipid particles and possibly the additional adjuvant in a dry Form, e.g. B. as a lyophilisate, spray-dried product, solid Dispersion, pellet or tablet.