FI86597C - Process for the preparation of immune complexes - Google Patents

Description

86597

This invention relates to a novel process for the preparation of an immune complex, the so-called isoma.

It is known that killed viruses, such as influenza virus, have a good antigenic effect. However, they are pyrogenic even after extensive purification. By isolating components that are important for the induction of protective immunity, pyrogenicity is avoided, but immunogenicity is often not strong enough. Therefore, appropriate vaccines must be added to such vaccines containing isolated antigens (subunits) to enhance the immune response. On the other hand, effective excipients, when used as hitherto, cause negative side effects. Thus, adjuvanted vaccines are not superior to whole virus-based vaccines in terms of pyrogenic activity.

To increase immunogenicity, detergent gentle membrane proteins have been produced that are attached or bound to the surface of 1 i (K) som i en (KP application 7940083.0). U.S. Patent 4,148,876 describes detergent-free membrane proteins in liposomes. U.S. Patent No. 4,196,191 mentions the incorporation of excipients into such detergent-free unilamellar liposome products (without describing its effect). U.S. Patent 4,117,113 describes negatively charged liposomes containing a viral antigen. In such liposomes containing influenza hemagglutinide and neuraminidase, the incorporation of an excipient, mycobacterial cell walls, elicits a better immune response. Improved immune responses have also been achieved when adjuvants such as killed Mycobacterium tuberculosis, Bordetella pertussis, and saponins have been introduced into such negatively charged liposomes containing diphtheria toxoid (U.S. Patent 4,053,585). However, all of the above 2,86597 lipid-containing membrane protein products are unstable after prolonged storage, freeze-drying, or careless handling, e.g., high temperature.

5 Detergent-free and lipid-free protein micelles have also been produced as vaccines. Semellic Forest Virus (SMF) membrane protein micelles have been shown to stimulate the formation of circulating anti-SFC antibodies and provide protection against murine SFV infection in mice. On the other hand, such parainfluenza-3 virus membrane protein micelles did not induce antibody production in sheep and did not protect them from the pneumococcal dose of PI-3 virus unless an oil excipient was mixed with the micelles. oil auxiliaries Stiii Vei I generally with side effects in the form of local reactions at the site of inoculation (EP-A-37 931).

EP-A-0 109 942 describes an immunogenic protein or peptide complex containing 20 glycosides and a process for its preparation. According to said method, whole viruses, mycoplasmas, bacteria, parasites or animal cells can be used as starting material, but also purified peptides or proteins or proteins or peptides synthesized or produced by recombinant DNA methods.

These complexes are different in morphological structure when viewed under an electron microscope than the corresponding protein micelles produced without the addition of glycosides. The micelles have a dense central core with 30 radial peaks, while the complex according to EP 0 109 942 has an open spherical structure consisting of circular subunits or parts of a spherical structure. The shape also differs from the shape of the liposomes. The complexes and their moieties generally have a lower sedi-35 anti ov.ik io than the corresponding II, the sedimentation constant of imi <1 11> i 1 I ä ji:; uu π> nip i than that of the corresponding monomeric protein or 3,86597 peptide. form and a higher sedimentation constant than liposomes.

Complexes according to EP 0 109 942 produced by the addition of glycosides have better immunogenic activity than the corresponding protein micelles produced without the addition of a glycoside or by complexes between the protein molecule and the solubilizing agent (monomeric forms) or Lipi dira. i.e., virosomes, bound to protein molecules, and produce fewer side effects than those that result from mixing protein micelles with glycosides or other excipients. Thus, the membrane protein dose can be reduced to one tenth or less.

It has now been found that when proteins or peptides alone are used as starting material, these tend to form aggregates, i.e. micelles. This can be avoided by adding lipids in the preparation of the complex.

Also, when using bacteria as a starting material or purifying whole cells, too little lipid might be present to form micelles. This can be avoided by adding lipids.

It has also been found that this new method can be used to prepare complexes containing antigens other than peptides or proteins and containing antigenic determinants.

Thus, the present invention relates to a method of preparing an immune complex comprising antigens or antigenic determinants having hydrophobic regions, comprising mixing viruses, mycoplasmas, parasites, animal cells, antigens or antigenic determinants having hydrophobic regions with one or more solubilizing agents. to form complexes between the antigens or antigenic determinants and the solubilizing agent, after which the antigens or antigenic determinants are separated from the solubilizing agent in the presence of a glycosidic solution 4 86597 or separated from the solubilizing agent and , at a concentration of at least 5 critical micellular concentrations to form a protein complex which is isolated and purified and for which the method is characterized by the addition of lipids before isolation and purification of the complex.

In order to promote dissolution and to keep the amphipathic proteins or proteins containing hydrophobic regions naturally free or free by chemical treatment or heat (e.g. 70 ° C) or other molecules containing hydrophobic regions as dispersed monomers in aqueous solution, hydrogenated and / or polar lipids and / or polar presence may be necessary. In fact, lipids are used in nature for this purpose in both animal and plant cells. Therefore, any lipid-lipid mixture present in the membranes of animal or plant cells is suitable. Some lipids make lipid membranes stiffer, e.g. cholesterol, some more fluid, e.g. phosphatidylcholine. It is also possible to produce synthetic lipids with these required properties and there are almost no restrictions on the formulation of new lipids that can be used for these purposes.

In terms of lipid size and properties, the limitations are determined by the solubility of the system used. For example, aqueous solutions of rigid lipids (cholesterol) are stabilized by the addition of less rigid lipids (phosphatidylcholine). Polar organic solvents participate in the stabilization of a protein with hydrophobic properties present in solution as a protein monomer. In conclusion, it is not possible to limit the size of the lipid in terms of its function, because, for example, the length of the aliphatic chain, which determines the hydrophobic properties of 5,86597, can be counterbalanced by a suitable polar group.

Sugars also have a stabilizing effect that helps keep proteins containing free hydrophobic regions or other molecules with similar properties dispersed in solution as monomers. As sugars, monosaccharides such as trioses, tetroses, pentoses, hexoses, heptoses and similar ketoses, non-reducing oligosaccharides such as sucrose, trehalose and raffinose, reducing oligosaccharides such as maltose, cellobiose, cellobiose, polysaccharides. They can be added in an amount of 5 to 50% of the aqueous solution.

As water-miscible polar organic solvents, alcohols such as mono- and polyhydric alcohols having up to 10 carbon atoms such as ethanol, glycols such as ethylene glycol, ethers, diethyl ether and ketones such as acetone can be used. Lipids may be fats or fat-like substances such as triglycerides or triglyceride mixtures containing fatty acids having up to 50 carbon atoms, such as saturated fatty acids having 4 to 30 carbon atoms, for example butyric acid, caproic acid, caprylic acid, capric acid , lauric acid, myristic acid, palmitic acid, stearic acid, arachidonic acid, behenic acid or lignoseric acid or unsaturated fatty acids having up to 30 carbon atoms, such as hexadecenoic acid, oleic acid, linoleic acid, 30 linoleic acid hydroxy fatty acids such as 9,10-dihydroxystearic acid, unsaturated hydroxy fatty acids such as castor oil, branched fatty acids; glycerol ethers, waxes, i.e. esters of higher fatty acids and monohydric alcohols; phospholipids such as derivatives of glycerol phosphates such as 86597 derivatives of phosphatidic acids, i.e. lecithin, cephalin, inositol phosphatides, sphingosine derivatives having 14 to 20 carbon atoms; glycolipids, isprenoids, sulfolipids, carotenoids, steroids such as cholesterol, cholestenol 5 caprostanol, phytosterols, e.g. esters 10 or mixtures thereof.

These and other useful lipids are described in Lipid Biochemistry an introduction, ed. MI. Gurr and A.I. James, Chapman and Hall, London, New York, University Press Cambridge 1980, which is hereby incorporated by reference.

Preferably cholesterol, phosphatidyl

R

choline, liposomes or intralipid (Oleum soya fractionate 200 g, Lechitinum fractionate vitello door 12 g, glycerol 22.5 g, make up to 1 liter with water).

Lipids could be added at any stage of the process, preferably before the addition of the glycoside, but lipids could also be added after the glycoside. In the absence of lipids, antigens or antigenic determinants tend to form micelles. The addition of lipids breaks the micelles so that a complex can form.

Lipids are added such that the molar ratio of lipids to antigens or antigenic determinants is at least 0.1 / 1, preferably at least 1/1.

If a molar ratio higher than 100/1 is used, the complex becomes sticky and difficult to handle. If a lower molar ratio is used, mi-miles may occur and less complex formation. However, at a ratio of 0.1 / 1, some complex is still formed. The molar ratio of lipids to antigens or antigenic determinants is thus 0.1/1 to 100/1.

7 86597 Currently available analytical methods do not indicate whether lipids are included or not. The complexes prepared by lipid addition according to the new method are similar in morphological structure when viewed under an electron microscope (EM, see figure) and have the same sedimentation constant as complexes prepared without lipid addition (see above).

However, unless the complexes prepared by the addition of lipids are subsequently purified, their contours are cloudy in EM and it is assumed that the lipids aggregate on their surfaces. When the complexes are purified, their outlines in EM are quite clear and it cannot be shown at this time that they contain Lipi-15s.

Macromolecular compounds can be used as antigens. In addition to proteins, polypeptides, glycoproteins, and lipoproteins, polysaccharides, oligosaccharides, polynucleotides, or mixtures thereof may also be used.

Antigenic determinants suitable for the preparation of complexes by the method of the invention include, for example, small polypeptides, small oligosaccharides, oligonucleotides, glycoprotein fragments, and hapten or mixtures thereof.

By small polypeptides is meant herein polypeptides that contain at least three and at most about 40 amino acids. In general, the antigenic determinant does not contain more than 4-10 amino acids. However, sometimes a somewhat higher number of amino acids is required to ensure the specific structure of the antigenic determinant and / or the immune response that occurs after immunization.

By small oligosaccharides is meant a linear or branched sugar one kkkel juju, each containing at least four and at most about 20 sugar units, preferably 6-10 sugar units. These 86597 oligosaccharides can be prepared synthetically or by chemical or enzymatic cleavage of naturally occurring polysaccharides or glycoproteins.

By oligonucleotides is meant compounds consisting of at least one and at most about 18 deoxyribonucleotides or ribonucleotides, obtained synthetically or by enzymatic or chemical cleavage of polynucleotides such as RNA and DNA. These oligonucleotides may optionally be double-stranded.

By hapten is meant small molecules that are not immunogenic per se, but become immunogenic when bound to a carrier molecule. Examples are steroids and prostaglandins.

Antigens or antigenic determinants must have hydrophobic groups or at least one reactive group that can form a bond between the antigen or antigenic determinant and hydrophobic compounds, for example, compounds listed on page 23, chapters 20-20, page 25, first chapter.

Molecules that are not immunogenic per se can, if they contain hydrophobic groups, be complexed into isomers with larger groups that are immunogenic and act as carriers in the isomic complex. The complex may thus contain mixtures of haptens and antigens.

Proteins or peptides having hydrophobic regions that bind to hydrophobic bases ('is') of the glycosides may be amphipathic proteins or peptides having hydrophilic and hydrophobic groups derived from the following sources or encapsulated in capsules. membrane proteins or membrane peptides of viruses, bacteria, mycoplasma, parasites or animal cells, or are such proteins or peptides, molecules prepared by the hybrid DNA technique or synthetically prepared, B) hydrophilic proteins or peptides made therefrom, groups. These proteins or peptides may be derived from viruses, bacteria, mycoplasmas, parasites, animal cells or may be synthesized or obtained by hybrid DNA technology; . These proteins may be derived from the above-mentioned microorganisms or cells, or may be obtained by hybrid DNA technology or synthesized. Preparation of the complex a) The preparation of membrane peptides or membrane proteins derived from whole cells or viruses basically comprises three steps: purification and isolation of animal cells or microorganisms or fragments thereof; dissolving the hydrophobic proteins and removing the solubilizing agent while transferring the desired antigen to the complex by means of a glycoside in immunogenic form (immune complex).

Purification and isolation 25 Viruses, mycoplasmas, bacteria, parasites and animal cells are concentrated and purified in a known manner / for references see "The Tools of Biochemistry", T.G. Cooper, John Wiley & Sons (1977) New York, which is incorporated herein by reference, for example, by gel filtration or by gradient centrifugation through a sugar gradient or by gradient centrifugation through a percoll or by hollow fiber dialysis. For bacteria, it may be necessary or more advantageous to first dissolve and cleave cell walls / reference pathways, cf. Cota-Robles and Stein, CRC Handbook of Microbiology Part II (1973) pp. 833-844, which is incorporated herein by reference7, e.g., by sonication or treatment with a French press.

Dissolution 5 The purified animal cells or microorganisms or parts thereof are then mixed with a non-ionic, ionic or amphoteric ion-containing detergent which is used in excess. Typical examples of suitable nonionic detergents are polyglycol esters and poly-10 glycol ethers with aliphatic or aralylate acids and alcohols. Examples of these are alkyl polyoxyethylene ethers of the general formula ^ n ^ 2n + 1 (OCl ^ Ci ^) χΟΗ, abbreviated <2ηΕχ; alkylphenyl polyoxyethylene ethers containing between the alkyl group of the phenyl ring and the polyoxyethylene chain, abbreviated as C «SE, e.g., Triton X-100 = tert-CQ ^ EQ, (poly-o X o 7.0 ethylene oxide octylphenol ether), acyl polyethylene-ethyl acyl polyoxyethylene sorbitan esters, abbreviated C-sorbitan-E, e.g. Tween 20, Tween 80,

Π X

20 N-D-alkyl ligosides, e.g. β-D-octyl glucoside. The glycosides listed below, in particular saponin, may also be used. However, these are weak doters and should be used in combination with other detergents. Typical examples of suitable gallic acid iodine-25 reagents are, for example, deoxycholate and cholate. Even conjugated detergents such as taurodeoxycholate, glycodeoxycholate and glycocholate can be used. Possible amphoteric detergents include lysolecithin as well as synthetic lysophos-30 folipids. Mixtures of the above detergents can also be used.

Dissolution can be achieved with alcohols, organic solvents or small amphipathic molecules such as heptane-1,2,3-triol, hok-35 sunni-1,2,3-triol, acetic acid, water-soluble peptides and proteins or their mixtures or detergents.

The solvent is used in excess of the amount of lipid and hydrophobic proteins. Suitable cells and microorganisms as well as the detergent are mixed in a weight ratio of 1: 3 to 1:10.

The cells or microorganisms and the solubilizing agent are mixed in a buffered, possibly saline solution. The molarity of the saline solution is 0.02 to 0.5 M, preferably 0.05 to 0.25 M. 0.1 to 0.2 M is preferred. The detergent should act for about an hour at room temperature.

Sodium chloride is preferred as the salt, but other salts may be considered, especially salts with alkali ions, alkaline earth ions and ammonium ions, as well as strong acids and organic acids such as acetic acid, trichloroacetic acid, formic acid and oxalic acid. Suitable buffers are all substances which buffer between pH 6.5 and 9. When cholates and deoxycholates are used, pH 8 to 9 is preferred and when nonionic detergents are used, pH 7.4 is preferred. When organic acids are used to dissolve proteins, the buffer can be omitted.

PREPARATION OF TMUNOGOON COMPLEXES Once the cells and microorganisms have been dissolved, a mixture of solubilizing agent and fragments of the cell or microorganism (hereinafter referred to herein as fragments) is formed. Among the fragments are charged, monomeric antigenic proteins, in hydrophobic regions, in complex with a solubilizing agent. The immunogen complex (primary isom) is prepared by separating charged monomeric antigen proteins from a solubilizer in the presence or direct transfer of one or more glycosides, wherein the glycosides must have hydrophobic or hydrophilic regions and be at least at a critical micelle concentration.

12 86597

The residue of the fragments is removed before the complex according to the invention is prepared, while it is prepared or afterwards, preferably before.

The carrier complex can be prepared either by removing the solubilizing agent, e.g., by dialysis, gel filtration, or chromatography, from a mixture of solubilized, monomeric antigenic proteins, glycoside, and optionally other fragments, or by separating the charged, monomeric antigenic proteins from said mixture, e.g. by electrophoresis. An essential feature is that the monomeric antigen proteins are separated from the solubilizing agent in the simultaneous presence of glycosides or, after separation, they are transferred directly to the glycoside, the micellar form of which must be present. When monomeric antigen proteins are separated from a solubilizing agent so that they can be in direct contact with a glycoside, a specific complex is formed in accordance with the present invention. It is assumed that the micelle form 20 of the glycoside is the basis for the formation of the complex and this is formed by the attraction between the hydrophobic regions on the surface of the glycoside micelles and the hydrophobic regions on the surface of the membrane proteins. The amount of glycoside in the complex varies depending on the glycoside used and the complexed membrane proteins and is from 0.5 to 50% by weight, in particular from 0.5 to 25% by weight, preferably from 0.5 to 15%, often from 0.5 to 15% by weight. 10 and in particular 2-8% by weight. However, if the charged monomeric antigen proteins are separated from the solubilizer in the absence of glycoside 30, protein micelles of the type prepared according to EPC Application No. 81102213.6 are formed.

It is convenient to remove other fragments by gradient centrifugation because the sedimentation constants of the components in question decrease in the following order: cell fragment, protein complex with solubilizer 86597 or glycoside, monomeric proteins, and solubilizer. Thus, other fragments can be removed by gradient centrifugation from a mixture of solvent, monomeric proteins, and other fragments before the glycoside is added and the solvent removed, e.g., by dialysis, gel filtration, chromatography, or the monomeric proteins are removed from the solvent, e.g., by electrophoresis. , by chromatography or gradient centrifugation. In the latter method, it is also possible to remove other fragments by the same gradient centrifugation, just as the complex is formed. It is also possible to separate the other cellular components after the complex has formed as mentioned above, e.g. by centrifugation, α-finite chromatography-15 or gel filtration. Substances that are not associated with the structure of the isomers but are bound to them by hydrophobic forces can be removed by centrifugation through a detergent zone, e.g., with a sugar gradient or a detergent-20 tiger gradient containing a sugar solution.

The glycoside can be any glycoside only in the hydrophilic and hydrophobic regions. Preferably, the saponins described in R. Tcshesche and Wulf, Chemie and Biologie der Saponine in Fortschritte 25 der Chemie Organischer Naturstoffe, W. Herz, II. Grisebach, G.W. Kirby, Vol. 30 (1973) in particular strongly polar saponins, primarily polar triterpene saponins such as polar, acidic bisdesmosides, e.g. saponin extract obtained from Quillajabark Araloside A, Chikosetsusaponen IV, Calendula glycoside C, V, Achyranthes-Saponin B, Calendula Glycoside A, Araloside B, Araloside C, Putranjia-Saponin III, Bersamasaponoside, Putranjia-Saponin IV, 35 Trichoside A, Trichoside B , Saponaside A,

Aescine from Trichoside C, Gypsoside, Nutanoside, Dianthoside i4 86597 C, Saponaside D, preferably from Aesculus hippocasta-num (T. Patt and W. Winkler: Das therapeutisch wirksame Prinzip der Rosskas-Tanipp) , Arzneimittelforschung 10 5 (4), 273-275 (1960) or sapoalbine from Gypsofila struthium (R. Vochten, P. Joos and R. Ruyssen: Physico-chemical properties of sapoalbin and their relation to foam stability, J. Pharm Belg. 42, 213-226 (1968), in particular the saponin extract from Quilla and the saponaria Molina, en-10 essentially a DQ extract prepared according to K. Dalsgaard: Sapon Adjuvants, Bull. Off. Ont. Epiz. 77 (7-8). ), 1289-1295 (1972) and Quil A, prepared according to K. Dalsgaard: Adjuvants III of Saponin, Archiv fur die Gesamte Virusforschung 44, 243-254 (1974) Mixtures of glycosides are also used. at least 1-3 times their critical micelle form concentration (CMC), preferably at least 7 to 12 times this amount. It is contemplated that in this case the glycoside may be bound to or adhered to the monomeric forms of the membrane proteins. Quil A with a critical micelle formation concentration of 0.03% by weight is preferred. The amount of Quil A should then be at least 0.02% by weight, in particular 0.05 to 0.5% by weight, preferably 0.2% by weight. The above citations for glycosides 25 are incorporated herein by reference.

Separation of charged monomeric antigen proteins from the solubilizing agent can be accomplished by centrifugation, dialysis electrophoresis, and various chromatographic methods.

centrifuging

A mixture of fragmented cells or microorganisms and a solvent prepared as described above is subjected to gradient centrifugation and deposited, e.g., on a surface of a sugar or saline solution containing a solvent and having a gradient, 86597 containing a glycoside such as sugar gradients. -tin or glycerol gradient or metric amide or a heavy metal, e.g. CsCl (i.e. relatively inert substances with a suitable density, viscosity to act as a gradient material), for example at the concentrations given below for the sugar gradient.

The gradient system is centrifuged at at least 100 000 g (depending on time and gradient, according to practical conditions). As the sugar, mono-10 saccharides such as glucose, lactose, maltose, disaccharides such as sucrose, but also trioses, tetroses and glycerin can be used. Sucrose is preferred. The concentration of sucrose in the gardener is suitably at least 5, preferably 15 to 25% by weight as the initial concentration (at the top of the gradient) and the final concentration is at least 20, preferably 45 to 60% by weight (at the bottom of the gradient). For example, the gradient may consist of an upper layer with 5 to 25% by weight of sugar and a lower layer with a sugar content of 20 to 60% by weight. However, the gradient may also have several layers, whereby the concentration differences of the private layers are reduced accordingly. The sugar gradient contains a glycoside or a mixture of glycosides. The glycoside should be 1 to 3, especially at least 5, preferably at least 25 to 12 times the CMC Quil A, at least 0.02, especially at least 0.05 to 0.5, preferably at least 0.2% by weight. %. In this glycoside-containing gradient, the solubilizing agent is separated and the complex between the solubilizing agent and the protein is converted to a protein-glycoside complex.

At the top of the sugar gradient is a layer of sugar or heavy saline containing a solvent or mixtures of solvents? lipids remain in this layer. The concentration of the solvent in this layer 35 is less than or equal to that of the mixture of microorganisms or cells and the solvent used 16 86597 and is suitably 0.25 to 3% by weight, preferably 0.75 to 1.5% by weight, 1% by weight -% is preferred. The sugar or salt concentration may be the same or less than the concentration in the upper layer of the lower glycoside-containing gradient, preferably 5-25% by weight, especially 15% by weight of sugar.

After centrifugation at at least 100,000 g for at least 16 hours, preferably 20 hours at 20 ° C, the proteinaceous fractions are pooled and dialyzed against buffer (0.5-0001 M), preferably 0.005 M Tris. HCl, 0.01 M NaCl, pH 7.4 or 0.02 M in ammonium acetate buffer, pH 7.0 and concentrated, e.g. as TG Cooper describes in The Tools of Biochemistry, published by John Wiley & sons (Nes York 1974), which is incorporated herein by reference, for example, by lyophilization, vacuum dialysis, or ultrafiltration. During centrifugation, all the ingredients.1 have just been released, whereby the solubilizing agent is released from the complex formed by the protein and the solubilizing agent and the raonomeric proteins are transferred to the glycoside and form a complex with it. Subsequent dialysis removes the sugar.

The complex can be further purified, e.g. glycosid-free, by gradient centrifugation, e.g.

Using a sugar gradient containing 26 to 60% by weight of sugar, preferably 10 to 40% by weight of sucrose. If the top of the gradient contains a detergent, the free glycoside will bind even more.

Dialysis Method 30 After purification of the cells or microorganisms as described above and after mixing with the solvent in the weight ratio described above, the mixture of cells and solvent in the buffer described above may alternatively be mixed directly with Quil A with at least 1-3, preferably 7-12 times the amount of 0.05 to 2% by weight of 17,85597% glycoside, preferably 0.2% by weight of glycoside and diluted against a buffer such as 0.5 to 0.001 M, preferably 0.005 M Tris-HCl, 0.01 M NaCl, pH 7.4; especially 0.2 M ammonium acetate buffer, pH 7.0 r>. The mixture of cell solubilizer and cells may contain lipids and ethanol, which are particularly useful for immunogenic proteins purified by affinity chromatography or for synthetically prepared peptides. Dialysis separates the solubilizing agent in the presence of a glycoside. The membrane-protein complex formed can then be isolated by precipitation gradient centrifugation, for example, as described on page 9 in the fourth whole paragraph, however, the glycoside moiety separates and is released from the other fragments and the free glycoside.

The mixture of cells and microorganisms and the solubilizing air in the buffer is also subjected to a gradient distillation and, for example, layered with a sugar gradient of 5 to 60% by weight in the above-mentioned buffer, preferably 10 to 12% by weight. to a precipitate-precipitate and centrifuge at 250,000 g for at least 20 minutes, preferably 30 minutes at 250,000 g. In this case, the other fragments are separated from the complex between the solubilizing agent and the protein.

The upper protein-containing solution, called the peak fraction, is extracted and the glycoside is added in an amount of at least 1-3, preferably 7-12 times the CMC Quil A, 0.05 to 0.5% by weight, preferably 0.2 as% by weight and is dialyzed against 0.5 to 0.001 M buffer, in particular 0.005 M Tris-HCl, 0.01 MIIC1, pl. 7.4; preferably against 0.2 M ammonium acetate. The solvent is removed in the presence of the glycoside. Further purification can be performed by sedimentation gradient centrifugation (see page 16, second full paragraph). Further purification can be performed by centrifugation through a sugar gradient containing 5 to 60% by weight of sugar, preferably 20 to 50 or 10 to 40% by weight of 18,86597 sugars, and optionally containing sugar in the top layer together with the detergent.

Elektro foree s imene rack

Alternatively, a fragmented solution of fragmented microorganisms or cells and a solvent or protein-containing supernatant (other fragments and free solvent removed) obtained by gradient centrifugation of this mixture at e.g. 5-60% by weight, preferably 20% by weight. - 50% by weight or 10 to 40% by weight of a sugar gradient in buffer, separated by electrophoresis from the solubilizing agent and transferred to a solution containing at least 1-3, preferably at least 7-12 times the Quil A of CMC: n, 0.05 to 0.5% by weight of glycosides, preferably 0.2% by weight of glycoside. The charged, monomeric antigen proteins are then separated from the solubilizing agent. For separation by electrophoresis, it is suitable that the solvent-buffer solution contains an addition of salt which can interfere with the electrophoresis and provide a very high temperature. It is possible to use, for example, zone electrophoresis with or without a carrier. Common substances that can be used as carriers are, for example, polyacrylamide, agar, silica gel, starch, cellulose, polyvinyl chloride, ion exchanger, celite.

Isolation and concentration of the complexes are performed as described on page 10, lines 23-26. Further purification is performed by gradient centrifugation (see page 17, second paragraph).

If hydrophobic mem-30 crane proteins with different charges or weights are present in the starting material, it is possible to separate them by electrolysis or centrifugation methods and prepare separate complexes therefrom. Using these conditions, it is possible to separate and enrich the complexes formed by different membrane proteins.

86597 19

Chromatographic methods

The dissolved proteins, optionally after purification for cell fragments, can be separated from the solubilizing agent by chromatographic methods, for example by gel filtration, hydrophobic chromatography or affinity chromatography, e.g. ion exchange chromatography, so that the antigenic structure is dissolved cellulose, agarose, dextran,] () acrylamide and glass. Various Ugandans are coupled to a matrix structure, which then acquires specific properties that are exploited during separation. The antigenic structure is usually adsorbed at the same time as the solvent used passes through the matrix without adsorption. Deadsorption of the antigen then follows. Prior to or during the deadsorption step, the solubilizing agent, salt and buffering agent may be exchanged so that the solubilizing agent is displaced by the glycoside and a complex is formed. The solubilizing agent can be supplemented with lipid-20 and alcohol, which promotes the formation of isomers.

Ligand molecules charged in ion exchange chromatography, such as dea (yAK I i uin i noe 1 yy 1 i ä (DEAK), are coupled to the matrix and used as cation exchangers. Carboxyl methyl (CM) or phosphate groups (P) are coupled to the matrix and an anion exchange is used. These molecules are separated by taking advantage of the differences in total charge between the antigenic structures and the solubilizing agent, usually the solubilizing agent is uncharged and the protein charged, eluting with a salt gradient such as a K or NaCl gradient or a suitable buffer, e.g. a phosphate buffer in the presence of solubilizing agent. (for concentration and examples, see section Dissolution above), by adjusting the pH, the solubilizing agent can be supplemented with lipid and alcohol, which promotes the formation of isomers, by eluting the protein, 86597 20 ion exchange solubilizing agent or forming a com plexiglass if a glycoside is added to the eluent instead of a solubilizing agent. Next, the salts are removed by dialysis.

Gel filtration uses a solubilizing agent that is lower in molecular weight than the antigenic structures and comes out in the following fractions.

By immunoaffinity chromatography, the antibodies can be irreversibly bound to the above matrix, after which the unique specificity and affinity of the antibodies is exploited to purify the desired antigenic structure. The solvent has no affinity for antibodies. Elution is performed with slight denaturation, for example by lowering the pH to about 4 and in the presence of a solubilizing agent or glycoside.

Lectin chromatography uses lectins, a group of proteins that can reversibly react with specific sugar groups, making it possible to bind, for example, glycoproteins to them. Lectin 20 is coupled as a ligand, for example to Sepharose (Pharmacia, Uppsala) or is commercially available, pre-coupled to a suitable matrix. Detergents (solubilizers) have no affinity for immobilized lectin. The adsorbed antigen structure is usually deadsorbed by the addition of a low molecular weight sugar, optionally methylated, which has an affinity for the lectin in question, for example by adding mannose, methyl mannoside, glucose, methyl glycoside and N-acetylglucosamine in a buffered saline, buffered amine,

In covalent chromatography, an antigenic structure having a thiol group attached by a covalent bond is bound to a matrix. The thio-35 group in the antigen is selectively linked to an activated thiol group attached to a suitable matrix by thiodisulfin-86597 dio exchange. This bond is reversible, and after removal of the solvent by washing, the thiol-bearing antigenic structure can be eluted by reduction of the disulfide bond with mercaptoethanol or dithiothreitol in the presence of a solvent or glycoside.

Hydrophobic Chromatography This technique utilizes the interaction between the immobilized hydrophobic ligand of the octyl or phenyl type and the hydrophobic surfaces of the antigenic structure. Alternatively, this technique may be a method in which the solubilizing agent is bound from the mixture to the ligand at the same time as the non-adsorbed antigenic structure can be recovered for continuous processing according to Example 4 (dialysis method). In other conditions, the anti-15 antigenic structure may bind to the ligand, and since the solubilizing agent has no affinity for the ligand, the procedure is followed according to the dialysis method. At high ionic strength, the immobilization is carried out, for example, using ammonium sulphate and the elution is carried out at low ionic strength -20 Ia with water or ethylene glycol.

The solubility of chromatographically purified or isolated proteins or other immunogens containing hydrophobic domains is improved when the solubilizing agent is supplemented with lipid and / or polar organic solvents which are miscible with water and also when sugars are added.

The complexes may thus contain bacterial membrane proteins, in which case it is preferred that the cell walls be disrupted first before the cell material is treated by the method described above. Examples of bacteria from which hydrophobic proteins can be produced are e.g. Escherichia, Staphylococci, Haemaophilus, e.g. H. influenzae, Bordetella, e.g. B. Pertussis, Vibrio, e.g.

V. chlolerae, Salmonella, e.g. S. typhi, S. paratyphi, 35 preferably an adhesion factor in Coli, e.g. pili K 88, and a porin protein, e.g.

Especially in the case of bacteria which contain relatively little lipids and in respect of which the lipoproteins and outer membrane proteins are strongly hydrophobic, the addition of lipids, sugars or polar organic solvents is suitable.

Examples of useful viruses with a capsule are Orthomyxoviridae, such as influenza A, B, C, Paramyxoviridae, especially measles virus, mumps virus, parainfluenza 1, 2, 3 and 4 viruses, smallpox virus and Rhabdoviridae, especially catarrhal virus, Rabies virus, and bovine leukemia virus, human immunodeficiency virus (HIV), Herpesviridae, especially Pseudora-bies, Herpes simplex I and II, Cytomegalovirus, Cotrona-viridae, Togaviridae, such as EEE, WEE, VEE (Eastern, Western and Venezuelan) equine encephalopathy , yellow fever virus, in particular bovine diarrhea virus and European swine fever virus Arenaviridae, Poxviridae, Bunyaviridae, in particular Huntan virus, Iridoviridae, in particular African swine fever virus and among unclassified viruses human hepatitis B virus and Marburg-Kbola virus.

Examples of parasites which can be used according to the present invention are Protozoa, such as Toxoplasma, for example Toxoplasma gondii, Plasmodium, for example Plasmodium vivax, malariae, falciparium, Teileria parvum, ovale and Filaroidae, mie-30 plum Parafilaria and Onchocerca, , different types of anaplasmas, Schistosoma, such as Schistosoma hematobium, mansoni, japonicum, and Trypanosoma, for example Trypanosoma gambiense, brusei or congolesi.

b) It is also possible to start with hydrophobic proteins that are not membrane-derived or non-hydrophobic proteins or peptides. Non-hydrophobic proteins or peptides can be rendered hydrophobic by attaching hydrophobic groups to them or rendering inaccessible hydrophobic regions accessible by denaturation. Non-hydrophobic groups may be derived from enveloped or non-enveloped viruses, bacteria, mycoplasmas, and parasites. Examples of non-capsular viruses with non-hydrophobic proteins are Picornaviridae (which are also assumed to have hydrophobic proteins), for example foot-and-mouth disease virus, poliovirus, Adenoviridae,

Parvoviridae, for example feline plague virus and porcine parvovirus, Reoviridae, for example Rotavirus. Examples of mycoplasmas are M. pneumoniae, mycoides, bovis, suis, hyorinos, orale, salivarium, hominis and fermentans.

15 These proteins can be obtained purified as described in a) Purification and Isolation.

Hydrophobic groups that can be attached to non-hydrophobic proteins are straight, branched, saturated, or unsaturated aliphatic chains, preferably having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms or hydrophobic amino acids or peptides or other hydrophobic structures, such as steroids. The length of the hydrophobic structure 25 is adapted to the size and nature of the protein. By way of example, a peptide of 10 to 15 amino acids (foot-and-mouth disease virus) is suitably provided with two tyrosines at the amino or carboxy terminus. A protein with a Mole-30 rabbit monkey of 70,000 daltons needs about 20 hydrophobic amino acids. The experiment is done empirically. Thus, peptides of 1 to 20 amino acids, preferably 1, 2, 3, 4, 5 amino acids, selected in particular from Trp, Ile, Phe, Pro, Tyr, Leu: n, 35 Var, especially Tyr; cholesterol derivatives such as cholinic acid, ursodeoxycholinic acid.

These hydrophobic groups must be attached to a functional group that may be attached to a non-hydrophobic protein, such as a carboxyl, amino, disulfide, hydroxyl, sulfhydryl, and carbonyl group, for example, an aldehyde group.

The hydrophobic group that can be attached is preferably selected from methane, ethane, propane, butane, hexane, heptane, octane and Cys, Asp, Glu, Lys, preferably octanal and Tyr.Tyr.Tyr-Cys, -Asp or -Glu. carboxyl, aldehyde, amino, hydroxyl and disulfide derivatives of peptides containing Hydrophobic groups containing a linking group must be dissolved in water, for example, with the above-mentioned solvents or detergents, or with hydrochloric acid, acetic acid containing 67% by volume of acetic acid, lye solution or ammonia, depending on the substance to be dissolved. The pH is then adjusted to neutral without precipitation; in this case, it must be ensured that the pH is not in the range that would denature the protein to which the hydrophobic group is to be attached. Lipid can improve solubility.

The hydrophobic molecule is added to the non-hydrophobic protein in molar ratios of 10: 1 to 0.1: 1, preferably 1: 1.

Hydrophobic groups with a carboxyl group as a coupling molecule can be coupled to a protein by means of water-soluble carbodiimides or mixed anhydrides. In the first case, the carboxyl group is activated at pH 5 with carbodiimide and mixed with a protein dissolved in a buffer having a pH of 8 and a high phosphate content. In the latter case, the carboxy compound is reacted with isobutyl chloroformate in the presence of triethylamine in dioxane or acetonitrile and the resulting anhydride is added to the protein at pH 8-9. It is also possible to convert the carboxyl group with hydrazine to a hydrazide which, together with aldehydes 35 and ketones in the protein, periodate 86597 25 account oxidized sugar units give hydrazone bonds.

Amino groups can be converted with nitric acid, at low temperature, to diazonium salts, which form azo bonds with Tyr, 5 His and Lys.

Hydroxyl groups can be converted to hemisuccinate derivatives by succinic anhydride, which can be coupled like carboxyl groups.

Aldehyde groups can react with amino groups located in the protein to form a Schiff's base.

Numerous coupling groups and methods are described in Journal of Immunological Methods, 59 (1983) 129-143, 289-299, Methods of Enzymology 93 280-283, and Analytical Biochemistry, 116 (1981) 402-407, which are incorporated herein by reference.

The proteins and peptides thus prepared, which have received hydrophobic groups, are then complexed with a glycoside as described in a), but at this stage the purification steps used to remove cell fragments are omitted.

c) It is also possible to make hydrophobic domains and encapsulating hydrophilic proteins accessible by denaturing the protein. This can be done, for example, at a low pH, at a pH of about 2.5, or in a well-alkaline solution at a pH of about 9.5, 3-M urea, or at a high temperature, e.g., above 70 ° C. Examples of such proteins are immunoglobulins IgG, IgM, IgA, etc. or other globular proteins, albumin, proteins from non-encapsulated viruses such as poliovirus proteins, envelope proteins from a virus that reveals the hydrophobic region of a protein, as is often the case with a retroviral envelope protein. The immunoglobulin may be, for example, an anti-idiotopic antibody.

35 Proteins can be prepared as pure proteins as described in b) and then complexed to a 26 8 6 5 9 7 complex with a glycoside as described in c).

Starting from purified or synthetic proteins or peptides according to b) or c), they tend to aggregate in the form of micelles in isom and 5. Therefore, the addition of one or more lipids, especially cholesterol, increases the formation of the primary complex. Sometimes it is useful to add a polar solvent, e.g. ethanol. Lipids are added to the protein and peptide while solubilizing agents are adjusted. The amount is not decisive. The molar ratio of lipid to peptide should be at least 1: 1. In this case, one of the four methods mentioned above can be used. When radioactive lipids are used, no radioactivity can be detected in the primary, immune complex. Hydrophilic peptides / polypeptides may be covalently bound to fatty acids contained in liposomes consisting of, for example, cholesterol, phosphatidylcholine, and fatty acids in a ratio of 1: 7: 2. The peptide / polypeptide is extracted from the liposomes with detergent 20 and separated from the excess lipid by centrifugation in a sucrose gradient (10-30% sucrose) containing the detergent.

Ischomes can be prepared as described above, preferably by centrifugation or dialysis.

25 When using the centrifugation method, can be used

For dissolving the Triton X-100 liposome complex. When using the dialysis method, it should be possible to dialyze off the detergent (e.g. octyl glycoside).

The immunogen complex of the invention can be used for specific immune stimulation in humans and animals. They can thus be used for immunoregulation and as diagnostic agents and vaccines against diseases caused by bacteria, viruses, mycoplasmas and parasites, as well as for the production of antibodies for research purposes.

27 86S97

Mixtures of amphipathic proteins derived from various bacteria or viruses can also be added to prepare vaccines for a variety of purposes.

The complexes can be used in scientific compositions of human or veterinary medicine containing the isomers according to the invention, optionally together with customary additives and excipients, preferably in the form of a buffer solution of the isom, e.g. as a TN solution. They can also be used as analytical agents and carriers for substances whose immunogenicity is to be enhanced.

The invention is illustrated in more detail by the following examples, which do not limit the scope of the invention.

Example 1 By performing mild denaturation at low pH, the hydrophobic regions of the bovine serum albumin molecule (BSA 17) are made available for ischemia formation.

USA (2nd my) dissolved in 0.15 M citrate-20 buffer (pH 2.5) was incubated for two hours with a solution containing 2% N-decanoyl-N-methylglucamine and an equal molar amount of lipid ( cholesterol / phosphatidylcholine 1: 1). The lipid stock solution contained 10 mg lipid / ml in distilled water and 20% N-decanoyl-N-methyl-glucamine. Quil A was added to a concentration of 0.1%. The mixture was thoroughly dialyzed against phosphate buffer (pH 7.2) first for six hours at low temperature and then for two days at 4 ° C. Electron microscopy confirmed the formation of isomers.

To purify the preparation from iscomb-unbound material, it was centrifuged through a 20% sucrose solution containing 200 μl of a 10% sucrose solution containing 0.1% Triton X-100. A Kontron Tst 54 rotor was used and centrifuged for 10 to 35 hours at 50,000 rpm at 20 ° C. BSA-containing isoms were recovered from a centrifuge tube 28,86597 by dissolving the pellet in 500 μl of phosphate buffer (pH 7.2). 5-10% of BSA was recovered from Isko-Meina.

The immunogenicity of BSA isomes was tested by performing 5 immunization experiments in mice. A group of eight mice was immunized once with 1 μg in BSA isomers. Another group of five mice was immunized once with 0.1 μg of BSA strokes. The corresponding groups of eight mice were immunized with BSA that did not bind to Iskomme. Mice were bled after 10 months and serum was collected. Serum antibodies against BSA were measured by ELISA. The eight mice immunized with 1 μg of isomes had a mean serum value of 1/450 (range 1/100 to 1/500), while the mice immunized with 1 μl or 0.1 ug of unbound BSA did not respond to immunization with 1 or 0.1 ug of BSA. Three of the mice immunized with 0.1 μg of BSA isomers did not respond to BSA with low serum antibody levels, while two of the mice responded.

Example 2 Mild denaturation of BSA releases the hydrophobic regions of the molecule. This can be done at basic pH. To control the reaction in the direction of the formation of isomers, an alcohol, for example ethanol, was included.

BSA (2 mg) dissolved in 0.5 M carbonate buffer (pH 9.6) containing 30% ethanol (ethylene glycol can also be used) was incubated for six hours. N-decanoyl-N-methylglucamine was added to a concentration of 2% and equal molar amounts of lipid (cholesterol / phosphatidyl-lycoline, see Example 1). Quil A was added to a concentration of 0.1%. The mixture was incubated for 30-60 minutes and then thoroughly dialyzed against phosphate buffer (pH 7.2) first overnight at room temperature and then at 4 ° C. The isomes were purified from unbound material by centrifugation as described in Example 1. Electron microscopy confirmed, 86597 29, the presence of isomic complexes. In addition, these isomes sedimented on a sucrose gradient from 10 to 50% sucrose at the same rate as IgM, i.e., the sedimentation constant was 19S. The yield of BSA in the isomes was over 50%.

Example 3 2 mg of glucagon was dissolved in 5.5 M carbonate buffer (pH 9.6) containing 30% ethanol. This glucagon solution was treated in exactly the same manner as bovine serum albumin (BSA) in Example 2. More than 50% of glucose-10 gon was recovered in isomers. The formation of isomers was confirmed by electron microscopy and the sedimentation of the complexes in a sucrose gradient of 10 -> 50% was determined using BSA (4S), thyroglobulin (19S) and IgM (19S) as standards.

Example 4 2 mg of angiotensin was dissolved in 0.5 M carbonate buffer (pH 9.6) containing ethanol. This angiotensin solution was treated in the same manner as bovine serum albumin (BSA) in Example 2. More than 50% of angiotensin-20 was recovered in isomers. The formation of isomers was confirmed by electron microscopy and the sedimentation of the complexes in a sucrose gradient of 10 -> 50% was determined using BSA 84S), thyroglobulin (19S) and IgM (19S) as standards.

25 Example 5

The Epstein Barr herpesvirus envelope protein gp 340 was treated with ischos.

gp 340 (200 μg) dissolved in 0.15 M phosphate buffer (pH 7.2) was incubated in a mixture containing 70 μg lipid (cholesterol / phosphatidylcholine 1: 1, see Example 1) and 2 % N-decanoyl-N-methylglucamine, two hours at room temperature. Quil A was added to a concentration of 0.1%. The mixture was thoroughly dialyzed against phosphate buffer (pH 7.2) first for six hours at room temperature and then for two days at 4 ° C. Electron microscopy confirmed that isomers 86597 were formed, gp 340 isomers were purified from unbound material by centrifugation through sucrose as described in Example 1.

Example 6 5 gp 340 isomers were prepared in various ways and a variant is described below.

300 μg of gp 340 in 2 μl of phosphate buffer (pH 7.2) was added to a tube dried on the wall with 70 μg of cholesterol. Triton X-100 was added to a concentration of 2% and the mixture was kept at room temperature for two hours.

The mixture, adjusted to a volume of 300, was layered on a gradient with a top of 200 of a 15% sucrose solution containing 1% Triton X-100 and a bottom of 12 ml of a 20% sucrose solution as described above. in phosphate buffer containing 0.1% Quil A. Centrifuged in a Beckman SW 40 rotor at 40,000 rpm for 16 hours at 20 ° C. The gradient was taken from the bottom of the plate in 500 μl aliquots and fractions containing gp 340 were identified by ELISA using rabbit gp 340 antiserum. Electromicroscopy revealed the formation of isomes.

Example 7 gp 70 from feline leukemia virus purified from F 422 tissue culture fluid by immunosorbent assay was denatured at low pH as described in Example 1 for bovine serum albumin (BSA). The gp 70 isoms were also formed by the procedure of Example 1. The formation of strokes was confirmed by electron microscopy. About 10% of gp 70 out of 30 was integrated into isomers.

Example 8

The feline leukemia virus gp70 purified according to Example 7 was slightly denatured as described in Example 2 for bovine serum albumin (BSA). 35 gp 70 was also formed according to the procedure of Example 2 at basic pH in the presence of ethanol.

31 86597

The formation of strokes was confirmed by electron microscopy. More than 50% of gp 70 was integrated into isomers.

Example 9 HIV gp120 purified from the culture medium of HIV-infected H9 cells by the im-5 immunosorbent method was treated at low pH as described in Example 1 for bovine serum albumin. The same procedure was also used to generate gp120 isomes. The formation of strokes was confirmed by electron microscopy.

Example 10 HIV gp120 prepared from HIV-infected H9 cells by the immunosorbent time method was treated as described for bovine serum albumin (BSA) at alkaline pH and in the presence of ethanol to reveal the hydrophobic regions of the molecule. The procedure described in Example 2 in connection with BSA was used to form the isomes. The formation of strokes was confirmed by electron microscopy.

Example 11 A peptide having the amino acid sequence 144-159 of the foot-and-mouth disease virus (FMDV) VP1 was synthesized in five variants.

I. (Tyr) 3-FMD

II. N-palmitic acid-FMD

25 III. N-capric-FMD

IV. N-capric acid (FMD) ^

V. Lys-capric acid-FMD

1 mg of peptide was mixed with an equal molar amount of lipid (cholesterol / phosphatidylcholine 1: 1, see

Example 1), in the presence of N-decanoyl-N-methylglucamine at a concentration of 2% and a final concentration of Quil A of 0.1%. The mixture was incubated for 30-60 minutes at room temperature and dialyzed against phosphate buffer (pH 7.2). The permeability limit of the dialysis membrane was 35 mol / kg. Electron microscopy showed the formation of isomers.

86597 32

Example 12

A peptide with the amino acid sequence (palm) 2 Lys Glu Glu Asn or His Asp Ala of the Plasmodium falciparum antigen Pf 155 (malaria) was synthesized with palmitite-5 nic acid attached at the N-terminus.

1 mg of this peptide was mixed with an equal molar amount of lipid (cholesterol / phosphatidylcholine 1: 1, see Example 1) in the presence of N-decanoyl-N-methylglucamine at a concentration of 2% and a final concentration of Quil A of 0.1%. . The mixture was incubated for 30-60 minutes at room temperature and dialyzed against phosphate buffer (pH 7.2). The permeability limit of the dialysis membrane was 1,000 molecular weight. Electron microscopy showed a typical isomira pattern.

15 Example 13

A peptide having the amino acid sequence Ala Glu Glu Asn Asn Glu Glu Asn Glu Glu Val Glu Glu Asn Val was synthesized from the Plasmodium falciparum antigen Pf 155 (malaria). Every third amino acid is hydrophobic and the structure of the peptide 20 is (^ .- helix.

1 mg of peptide was mixed with an equal molar amount of lipid (cholesterol / phosphatidylcholine 1: 1, see Example 1) in the presence of N-decanoyl-N-methylglucamine at a concentration of 2% and a final concentration of Quil A of 0.1%. The mixture was incubated for 30-60 minutes at room temperature and dialyzed against phosphate buffer (pH 7.2). The permeation limit of the dialysis membrane was 1,000 molecular weight. Electron microscopy showed a typical isomic structure.

Example 14 2 mg of Hepatitis B (HBs) surface protein introduced by yeast recombinant DNA methods and produced by yeast particle was solubilized with N-decanoyl-N-methylglucamine at a concentration of 2%. Equal molar amounts of lipid (cholesterol / phosphatidylcholine-35: 1: 1, see Example 1) and Quil A were added to a concentration of 0.1%. The mixture was incubated at room temperature for two hours.

33 86597

The mixture was then thoroughly dialyzed against phosphate buffer (pH 7.2) first for 4-6 hours at room temperature and then at 4 ° C. Electron microscopy showed typical piglet formation.

Example 15 Bovine viral diarrhea virus (BVDV) (3 mg) dissolved in TN was made soluble by adding Triton X-100 to a concentration of 1%. The solubilized virus was applied to a lectin column consisting of lens lectin immobilized on Sepharose 4B (Pharmacia, Uppsala). The column was equilibrated with TN *, and after the viral material was applied to the column, this was rinsed with a volume of five columns of TN containing 0.1% by volume of Triton X-100 and then 10 columns with a volume of T-15 of Tn. Viral envelope proteins were deadsorbed with elution drop buffer containing 0.1 mol / l methyl <* - D-mannoside and 0.5% by weight of octyl -? - D-glucoside dissolved in TN and added to the columns. Fractions containing viral envelope proteins were collected, Quil A 20 was added to a concentration of 0.1% by weight and 120 ug of lipid (cholesterol / phosphatidylcholine 1: 1, see Example 1). The mixture was incubated for 30-60 minutes at room temperature and dialyzed against phosphate buffer (pH 7.2).

* (0.05 mol / l Tris, 0.1 mol / l NaCl, pH 7.2) The above example was repeated, but intralipid * was used instead of the lipid mixture described above. The formation of isomes was confirmed by electron microscopy.

* Vitrun, Stockholm 30 Example 16

The smooth and coarse variant (3 mg) of the 2308 porin protein of Brucella abortus strain were very pure of contaminating proteins but contained LPS contamination. The porin protein was kindly supplied by 35 A.J. Winter, Department of Veterinary Microbiology,

Cornell University, Ithaca, USA. Porin proteins 34,86597 were dissolved in 1 ml of phosphate buffer containing 20% sucrose and 350 μg lipid (cholesterol / phosphatidylcholine 1: 1, see Example 1) while maintaining this mixture at 4 ° C overnight. Zvitter-5 gent was added at 0.05%, N-decanoyl-N-methylglucamine at 0.1% and Quil A at 0.1%. The mixture was thoroughly dialyzed against phosphate buffer (pH 7.2) for two days, the first four hours at room temperature. Electron microscopy-identified isomers were purified from non-isome-unbound material by centrifugation as described in Example 1.

Example 17

The cytomegalovirus, a herpesvirus, nucleo-15 protein - kindly supplied as a crude extract by Dr. B. Warren, National Institute of Health, Stockholm - was denatured at low pH as described in Example 1. Ischomes were prepared by the procedure of Example 1. Electron microscopy showed that 20 isomes were formed.

Balb / c mice were immunized twice (25 μg, 10 μg) with isomers prepared from malaria peptides described in Example 13 (A) and a mixture of peptides described in Examples 12 and 13 (AB).

25 Table 2

Immune- Amount 1. immunization Amount 2. immunization gene after_after _

(peptide peptide A peptide B peptide A peptide B

30 dl) A 1: 270 l: 2M0 1: 1380 1: U00 A 1: 300 1: 220 1: 1590 1: 660 A 1: 630 1: U80> 1: 2100 1: 800 AB 1: U10 1: 250 1: 2089 1: 500 86597 35

Example 18

The mixture of cytomegalovirus glycoprotein and nucleoprotein was kindly provided as a crude extract by Dr. B. Wahren, National Institute of Health, Stockholm.

This glycoprotein-nucleoprotein preparation was treated at low pH and isomes prepared as described in Example 1. Electron microscopy showed the formation of isoms. Table I shows that monkeys immunized with cytomegalovirus isomes induced potent cell-mediated immunity. It is important for the intended cytomegalovirus vaccine to develop cell-mediated immunity.

Table 1 Proliferative reactivity of peripheral blood cells of CMV-immunized monkeys to CMV antigen in solid phase 2

Monkey Immunizing CMV- Net Pulse Rate Uiiv No. Antigen Datasheet i SD IgG- 20 Dose Tu Combined Amount

Lymphocyte- Monocyte- PHA

tit tit 1 small - 4 99+ 22 9 800 + 1044 200 160 25 large - <100 <100 500 6400 2 small - <100 <100 300 400 large - 700+ 519 <100 400 24000 large monocytes. 3800 + 1700 n.d. 13000 30000 3 small - 854+ 250 <100 300 500 30 large - <100 <100 3200 24000 large monocytes. 2800+ 240 n.d. 22000 18000 4 small monocytes. 423+ 166 441 ^ 317 2000 400 large monocytes. 3516+ 3202 106+ 160 10000 18C00 36 86597 5 small monocytes. 866 · * - 709 833+ 288 10000 100 large monocytes. 30200+ 5300 660+ 280 27000 22000 6 small monocytes. <100 490+ 440 6000 160 large monocytes. 27000+ 21000 <100 52000 25000 5 7 small to isomers 36400+ 6500 n.d. 32700 2100 9 small isomers 49500+ 9000 n.d. 15300 15000

Human control sample, CMV seropositive 37300+ 6500 2600+ 513 45000 12000 10

Human control CMV ceronegative <100 <100 50000 100 2

Prior to immunization, all animals had a net pulse rate of 1,000 min for lymphocytes and CMV antigen (546 μ 58 min for CMV, net pulse rate for 100 min); monocytes and CMV (322 i 208 min net pulse rate 100 min *) or lymphocytes and monocytes in combination with CMV (709 h 425 min * CMV, net pulse-20 volume 211 h 300 min when examined ^ CMV IgG levels in all animals were 100 prior to immunization, except in monkey # 8 pre-immunized with CMV nucleocapsid antigen.

Example 19 300 μg of gp 240, Epstein Barr virus (herpesvirus) envelope protein, is added in 200 μl to a PBS tube with 70 μg of cholesterol dried on the walls. Triton X-100 is added and the mixture is kept at room temperature for two hours. A mixture of 300 [mu] l is placed on a gradient, the top of which consists of 200 [mu] l of a solution containing 15% sucrose and 1% of TX-100 and the bottom of 12 ml of a solution containing 20% of sucrose and 0.1% Quil A in PBS. The mixture was centrifuged at 40,000 rpm in a Beckman SW rotor for 16 hours at 20 ° C. The gradient was recovered from the bottom at doses of 500. Gp 340 mixed with detergent and cholesterol 37 86597 can be transferred to isomers by dialysis. In this case, Quil A was added to a final concentration of 0.1% before dialysis. In this case, it is preferable to use a detergent which can be easily dialyzed off, for example the octyl glycoside used in this case.

The mixture was then dialyzed for 48 hours at 4-6 ° C against PBS (TN buffer has also been used). The formation of isomers was confirmed by electron microscopy. For example, the advantages of using different viral envelope proteins purified by different methods, i.e. the method chosen, are obvious.

Example 20

The three sequences of the FeLV gp 70 hybrid DNA product were conjugated to liposome-incorporated stearylamine by a two-step glutaraldehyde method. 10 mg (10 mg / ml) Liposome and activated in PBS (pH 7) with glutaraldehyde (final concentration 1.25%) at room temperature overnight. Excess glutardialdehyde was removed by either dialy-20 ringing or gel filtration.

3 mg of activated liposomes were mixed with 1 mg of each FeLV fp 70 polypeptide, the volume was adjusted to 1 ml, and the pH was raised by adding 100 μl of 1 M NaCO 3 solution (pH 9.6). The mixture was incubated overnight and purified from unbound polypeptide by gel filtration (e.g., S-300).

The polypeptide fatty acid was extracted with 2% N-decanoyl-N-methylglucamine (MEGA-10) and separated from the excess lipid by centrifugation with a sucrose gradient (5 -> 30% sucrose) containing 0.3% MEGA-10. .

The polypeptide was taken up, Quil A was added to a final concentration of 1% and the mixture was thoroughly dialyzed against PBS, first for 4-6 hours at room temperature and then at 4 ° C.

38 86597

Example 21 3 mg of bovine IgG was incubated for six hours in 0.5 M carbonate buffer (pH 0.6) containing 30% ethanol. N-decanoyl-N-methylglucamine was added at a concentration of 5% and at the same time equal molar amounts of lipid (cholesterol / phosphatidylcholine 1: 1, see Example 1). Quil Aita was added to a concentration of 0.1%. The mixture was incubated for 30-60 minutes and then thoroughly dialyzed against phosphate buffer (pH 7.2) first overnight at room temperature and then at 4 ° C. The isomers were purified from iscomb-unbound material by centrifugation as described in Example 1. Electron microscopy showed the presence of isoms. In addition, these isomes sedimented in a sucrose gradient of 10 -> 50 50% at the same rate as IgM, i.e., the sedimentation constant was 19S.

Example 22

Two peptides with the amino acid sequences of the Plasmodium falciparum antigen Pf 155 (malaria) (described in Examples 12 and 13) were synthesized. Both peptides were used to generate peptide-protein isomers. Equal molar amounts of peptide, membrane protein and lipid (cholesterol / phosphatidylcholine 1: 1, see Example 1) were mixed with N-decanoyl-N-methylglucamine 25 and Quil A at final concentrations of 2 and. . : 0.1%, respectively. The mixture was incubated for 30-60 minutes at room temperature and dialyzed against phosphate buffer (pH 7.2). The permeability limit of the dialysis membrane was a molecular weight of 1,000 kD. Electron microscopy showed a typical 30-stroke structure.

Claims (6)

A process for producing an immunogenic complex containing antigenic proteins or peptides with hydrophobic regions, which complex has an open spherical structure consisting of circular subunits or parts of the spherical structure, and which complex usually has a lower sedimentation constant than the corresponding micelles and higher sedimentation constant than the corresponding monomeric form of protein or peptide, from amphipathic proteins or peptides or from such containing viruses, bacteria, mycoplasma, parasites or animal cells, mixing at least one of the above components with at least one solubilizing agent, forming complexes between amphipathic antigenic proteins or peptides and the solubilizing agent, after which the amphipathic antigenic proteins or peptides are separated from the solubilizing agent, in the presence or separated from the solubilizing agent, and transferred directly to a solution containing at least e. n triterpene saponin having hydrophobic and hydrophilic regions at a concentration of at least the critical micelle concentration (CMC), characterized in that lipids are added before the complex is isolated and purified. 2. A method according to claim 1, characterized in that the lipids are miscible in the solubilizing agent. Method according to claim 1, characterized in that the lipids are selected from membrane lipids in animal or plant cells. 30 4. A process according to claim 1, characterized in that the lipids are selected from phospholipids and isoprenoids. 5. A process according to claim 4, characterized in that the lipids are phosphatidylcholine and cholesterol. «86597 6. The method of claim 1, wherein the lipids are added in a molar ratio of lipids to antigens or antigenic determinants of at least 0.1, preferably at least 1.