JPH0749376B2 - Immunogenic and pharmaceutical compositions - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Industrial Field The present invention relates to immunogenic compositions capable of inducing selective antibody production. In particular, the invention relates to immunogenic compositions capable of inducing antibody production selectively against both synthetic and natural peptides, which peptides need not be conjugated to a heterologous protein carrier (eg BSA or KLH). Neither, nor does it need to be injected with additional adjuvant.
The invention also relates to pharmaceutical compositions using the immunogenic compositions.

[Conventional technology]

Development of effective vaccines is extremely important for disease control and prevention. The use of synthetic peptides as immunogens is one promising entry point for the production of safe and effective vaccines (Lerner, RA, Green, N, Alexander). , Etch (Alaxander, H.), Liu, F. T. (Liu, F.)
T.), Stocliffe, Sutcliffe, FG and Scenic, Shinnick, TM, Proceedings of National Academy of
Science of USA (Proc.Natl.Acad.Sc
i.USA) (1981), 78, 3403-3407; Bertnager, Bhatnager, PK, Papas, E., Blum, HEE, Blum.HE,
Milichi, DR (Milich, DR), Nitekki,
Dee (Nitecki, D.), Karelus, M.J.
ls, MJ) and Bias, G.N. (Vyas, GN), Proceedings of National Academy of Sciences of USA (1982) 79, 44
Pages 00-4404; Nuruth, A.R.
AR), Kent, S.B.H. (Kent, SBH)
And Strick, N., Journal of General Birology (J.Gen.Verol.) (1984)
Volume 65, Pages 1009-1014; Driesman, GR, Spraw, J.T.
ow, JT), Frenchic, Frenchick,
PJ) and Kennedy, R.
C.), Advances of Experimental Mendicin and Biology (Adv.Exp.Med.Bio)
L.) (1985) 185, 129-137; Sanabara, Thanavala, YM, Brown, Brown, SE, Howard, C. Howard, C.
R.), Reutt, Roitt, IM and Steward, Steward, MW, Journal of Experimental Medicine (J.Exp.Me).
d.) (1986) 164, 227-236; Muller, Muller, GM, Shapira, M.
M.) and Arnon, R., Proceedings of National Academy of Sciences of USA (1982), 79, 569-573; Kennedy, R.C., Henkel, R·
Dee (Henkel, RD), Pauletti, Dee (Paule
tti, D.), Alan, Allan, JS, Lee, Lee, TH, Essex, Essex, M. and Driesman, G.R., Science (1986) 231 vol., Pp. 1556-1559;
Bittle, JL, Biten,
Houghten, RA, Alexander, Etch, Scenic, Tm, Stcliffe, J.A.
Gee, Learner, AR, Rowlands, Dee
J (Rowlands, DJ) and Brown, F (Brcwn,
F), Nature (1982), Volume 298, pages 30-33; and Di Marchi, R., Brooke, G., Gale, C. , Cracknell, V., Dale, Doel, T. and Mowat, N., Science (1986) 232, 639-641).

[Problems to be Solved by the Invention]

However, synthetic peptide vaccines have been hampered in their development by the often undesired side effects of both the carrier and the adjuvant they require (Larner, AR, Green, N., Alexander, Etch. , Liu, F.T., St. Cliff, J. G. and Scenic, T.M., Proceedings of National Academy of Sciences of USA (1981) 78, 3403-3407.
Page; Bertnaega, Pk, Papas, Yi, Blum, Etch Yi, Milich, Dee Earl, Nittekki, Dee, Carrels, MJ and Beers, G.N., Proceedings of National Academy of・ Science of USA (1982
79), 4400-4404; Neurath, A.R., Kent, S.B.H. and Strick, H., Journal of General Birology (1984)
65), 1009-1014; and Muller, Gee, M, Shapira, M and Arnon, Earl, Proceedings of National Academy of Sciences of USA (1982) 79, 569-. 573
page). Moreover, immunization with peptide-protein carrier complexes usually sensitizes T cells to foreign carriers. Therefore, natural infection does not generate a secondary immune response, as animal T cells are not sensitized to natural carriers of neutralizing epitopes.

Therefore, there is still a need for an effective immunogen in which the peptide does not require conjugation to a foreign protein carrier and does not require injection with an additional adjuvant.

[Means for Solving the Problems]

The first object of the present invention is to increase the efficacy of natural infection or vaccination by currently available methods, including inactivated or attenuated agents, or macromolecules of the external surface of the infectious agent or subunits thereof. It is to provide immunogenic and pharmaceutical compositions that mimic while not exhibiting any of the undesirable side effects of currently available formulations.

A second object of the present invention is to provide immunogenic and pharmaceutical compositions which avoid the problem that T cells are specifically sensitized to foreign carriers, so that the T cells do not respond during subsequent natural exposure. Is to provide.

These and other objectives are achieved by providing immunogenic compositions capable of selectively inducing or enhancing antibody production.

In a first embodiment, the present invention induces antibody production selectively against a non-immunogenic peptide consisting of a peptide-lipid covalently bound substance consisting of a non-immunogenic amphipathic peptide conjugated to a lipid. An immunogenic composition capable of enhancing or potentiating is provided. In a preferred embodiment, the peptide-lipid covalent agent is associated with a mixture of one or more lipids and one or more sterols.

In a second embodiment, the invention comprises a hybrid peptide covalently bound to a lipid, said hybrid peptide comprising: (a) a non-immunogenic hydrophilic peptide, (b) a non-immunogenic neutral peptide, (c ) A non-immunogenic amphipathic peptide, and (d) a peptide selected from the group consisting of hybrid peptides consisting of any combination of two or more of said peptides (a), (b) and (c). Of the ability to selectively induce or enhance antibody production against one or more non-immunogenic peptides consisting of a peptide-lipid covalent agent consisting of non-immunogenic amphipathic peptides conjugated to Certain immunogenic compositions are provided. In a preferred embodiment, the peptide-lipid covalent agent is associated with a mixture of one or more lipids and one or more sterols.

In a third embodiment, the present invention provides a first peptide-lipid covalent substance (wherein the first peptide-lipid covalent substance is (1) a non-immunogenic amphipathic peptide conjugated to a lipid, And (2) a hybrid peptide conjugated to a lipid (wherein the hybrid peptide is (a) a non-immunogenic hydrophilic peptide, (b) a non-immunogenic neutral peptide, and (c) a non-immunogenic amphiphilic aqueous solution. Peptides, and (d)
The peptides (2) (a), (2) (b) and (2)
Consists of a non-immunogenic amphipathic peptide conjugated to a peptide selected from the group consisting of hybrid peptides consisting of any combination of two or more of (c). ) Consisting of one member selected from the group consisting of. ) And one or more additional peptide-lipid covalent substances (wherein said peptide-lipid covalent substance is (1) lipid) associated with a mixture of one or more lipids and one or more sterols. Non-immunogenic hydrophilic peptide conjugated to lipid, (2) non-immunogenic neutral peptide conjugated to lipid, (3) non-immunogenic amphipathic peptide conjugated to lipid And (4) a mixed peptide conjugated to a lipid (wherein the mixed peptide is (a) a non-immunogenic hydrophilic peptide, (b) a non-immunogenic neutral peptide, and (c) a non-immunogenic amphiphile. Peptide, and (d) the peptide (4)
(A) Non-immunogenic amphipathic conjugate to a peptide selected from the group consisting of hybrid peptides consisting of any combination of two or more of (4) (b) and (4) (c) , Hydrophilic or neutral peptides. ) Consisting of one or more members selected from the group consisting of. And an immunogenic composition capable of inducing or enhancing antibody production selectively against one or more non-immunogenic peptides consisting of

The present invention also provides a pharmaceutical composition which selectively induces or enhances antibody production, which comprises one or more of the above immunogenic compositions and a mixture of pharmaceutically acceptable carriers, diluents or excipients. provide.

The alphabet nomenclature used herein for the description of peptide sequences is the standard amino acid alphabet shown in the table below.

For purposes of this invention, the following terms are defined below.

Peptide: A series of amino acids polymerized head to tail through a peptide bond between one carboxylic acid group and the next amino acid group. Peptides can be naturally occurring, recombinantly produced, synthetically produced, or produced by chemical or enzymatic cleavage of the large polypeptides or proteins thus obtained, or from smaller units. It can be produced by chemical or enzymatic peptide construction, and the peptide has non-amino acid moieties such as sugars, or undergoes various chemical transformations such as phosphorylation, methylation, sulfation or acetylation. These transformations can result from production in microorganisms or abrupt transformations using chemical or enzymatic synthetic techniques.

Non-Immunogenic Peptide: A peptide as defined above that does not stimulate an immune response in an animal when administered to the animal without being conjugated to or mixed with additional substances (a peptide may immunize one animal Is non-immunogenic to animals of different allotypes, though it is. Further, as used herein, the term "non-immunogenic peptide" is immunogenic, but its immunogenicity includes peptides which are enhanced by the formation of an "immunogenic composition" according to the invention. You can

Amphipathic peptide: A peptide as defined above containing distinct hydrophobic and hydrophilic regions. Examples of amphipathic peptides include (1) a peptide containing a range of predominantly hydrophobic amino acids adjacent to it followed by a range of predominantly hydrophilic amino acids, or (2) one side consisting primarily of hydrophobic amino acids, and The side of is α consisting mainly of hydrophilic amino acids
A peptide that can assume a helix structure in solution. It can be said that the second type of amphipathicity of the peptide is α-helix periodicity.

The hydrophobicity or hydrophilicity of a region is clarified by computer analysis using a program that considers the hydrophobicity or hydrophilicity of amino acid side chain residues.

Examples of such programs that can be applied to the measurement of the presence of hydrophobic or hydrophilic ranges are Hop and Woods (Hopp, TP) and Woods, Wood.
s, KR), Proceedings of National Academy of Sciences of USA (19
1981) 78, 3824-3827, and Hop, Tee Pee, Woods, K. R., Molecular Immunology (Mol.Immunol.) (1983), 20: 483-489.
Page) or Kite and Doo-liter (Kyte, J.) and Doo-liter, Dof (Doo)
little, RF), Journal of Molecular Biology (J. Mol. Biol.) (1982) 157, 105-131.
Page) but other similar programs can also be used.

A computer that can measure the amphipathic properties of peptides when they form an α-helix structure in solution
The program was also developed. In this case, the apparent hydrophilic region is associated with one side of the α-helix peptide, while the apparent hydrophobic region is associated with the other side of the peptide. Examples of such programs are Delisi and Berzofskv (Proceedings
It is provided by the National Academy of Sciences of USA (1985) 82, 7048).

Also, for the purposes of this invention, hydrophobic, hydrophilic or neutral regions are defined by computer analysis in the primary sequence or in relation to the α-helix periodicity described above.

One physical test of amphipathicity is the measurement of its ability to disrupt the integrity of phospholipid membranes. This property can be tested by measuring the peptide's ability to promote the release of carboxyfluorescein from preformed liposome internal aqueous regions, as described below. However, not all amphipathic peptides necessarily perturb the integrity of phospholipid membranes, so for the purposes of this invention perturbation of phospholipid membranes is not an absolute requirement for the definition of amphipathic peptides. Absent.

Hydrophobic peptide: In addition to being hydrophobic in the Hop-and-Woods or Kite-and-Doulit type analysis for the adjacent range as described above, or the Delicy and Verzovsky type analysis for the α-helix periodicity. A peptide as defined.

Hydrophilic peptide: In addition to being hydrophilic in Hop-and-Woods type or Kite-and-Douliter type analysis for the adjacent range as described above, or Delicy and Verzovsky type analysis for α-helix periodicity A peptide as defined.

Neutral peptide: Neutral in the Hop-and-Woods or Kite-and-Doulit type analysis for contiguous regions as described above, or the Delicy and Verzovsky type analysis for α-helix periodicity A peptide as defined.

The present invention envisions at least three general embodiments of immunogenic peptides.

The first embodiment provides an immunogenic composition capable of selectively inducing and enhancing antibody production against non-immunogenic amphipathic peptides. The second and third embodiments are the ability to selectively induce and enhance antibody production against non-immunogenic amphipathic peptides as well as non-immunogenic hydrophilic and / or non-immunogenic neutral peptides. An immunogenic composition is provided.

The composition according to the first embodiment comprises a peptide-lipid covalent agent, which comprises a non-immunogenic amphipathic peptide conjugated to a lipid. In a preferred embodiment, the peptide-lipid complex is associated with a mixture of one or more lipids and one or more sterols.

Peptide-lipid covalent agents are known in many known ways, for example cross-linking to glysphingolipids (Heath, TD, et al., Biokimica Biophysica Actor (BBA) (1981) 640, 66. ~ Page 81) or by N- (p-aminophenylstearylamide) (Snyder, SL)
And Bannier, Wannie, Vannier, WE, Biokimika Biophysica Actor (1984) 772, 288
~ 294) or the method using N-hydroxysuccinimide ester of palmitic acid (Huan
g, A.), Journal of Biological Chemistry (JBC) (1980), 255, 8015-8018)
Is produced by conjugating an appropriate lipid to an amphipathic peptide by conjugation.

Covalent attachment of the peptide to the lipid is preferably by cross-linking, methods known in the art, such as N-succinimidyl-4- (p-maleimidophenyl) butyrate (Martin, FJ). And Papahadjopoulos, D.,
Journal of Biological Chemistry (19
(1982) 257, pp. 286-288), N-hydroxysuccinimidyl-3- (2-pyridyldithio) propionate (Barbet, J. Out, J.
Supra Struct & Cell Biochem (J.Supr
a.Struct. and Cell Biochem.) (1981) 16 volumes, 243--2
P. 58), m-maleimidobenzoyl-N-hydroxysuccin indo ester (Hashim, Washi
oto, Y.), Journal of Immunological Methods (1983), 62, 155-162), citraconylation (Jansons, VK) and Mallet. Mallett,
PL), Analytical Biochemistry (Anal.B
iochem.) (1981) 111, 54-59).

As the peptide, any amphipathic peptide that fits the above definition can be used. Of course, if the immunogenic composition is used as a vaccine, the peptide must not be toxic or otherwise harmful to the health of the animal.

As further mentioned above, amphipathicity can be tested by measuring the ability of the peptide to promote the release of carboxyfluorescein from the internal aqueous region of preformed liposomes (Szoka). And Papaha Jopulos, Proceedings of National
Academy of Sciences of USA (1978) Vol. 75, pp. 4194-4198, and Weinstein, J. Outside Weinstein, JN, Science (1977) 195, 489-491), which is not a mandatory test for the purposes of the present invention.

In particular, carboxyfluorescein (CF) is a known method (Zoca and Papahajopoulos, Proceedings of National Academy of Sciences).
Vol. 75, pp. 4194-4198, of U.S.A. (1978), self-extinguishing concentration (20
0mM, Weinstein, outside JN, Science (1
977) Volume 195, pages 489-491).
CF is Ralston et al. (Biochim.Biophys.Acta) (1981) 649
Vol., Pages 133-137). The ability of peptides to perturb the integrity of liposome bilayers can be determined by measuring the emission at 520 nm 30 minutes after peptide addition to liposome suspensions. Generally, an increase in fluorescence of greater than 5% of the total possible increase is amphipathic. The overall possible increase in fluorescence is due to the 0.1% Triton (Trito
n) It can be determined by measuring the emission at 520 nm before and after dissolution with X-100.

Examples of amphipathic peptides include the following human immunodeficiency viruses, the HIV envelope protein HIV (487-511),
HIV (469-511) and HIV (578-608) (Outside Starcich, Cell (1986) Volume 45, 637-6
Page 48). In addition, the following sequence, HIV (735-752) (D-R-P-E-G-I-E-
E-E-G-G- E-R-D-R-S-NH 2, Kennedy (Kennedy) outside, Science (1986, March) 231 Volume
1556 to 1559), HIV (340 to 368) (N-N-T-
L-K-Q-I-D-S-K-L-R-E-Q-F-G
-N-N-L-Q- S-S-G-C-NH 2) alone, or HIV with amphipathic peptides of HIV to enhance (e.g., 487-511) antibody production (299-329) ( H-R-P-N
-N-N-T-R-K-I-R-I-E-R-E-P-
E-R-A-E-K-I-E-N-M-R-Q-C-NH
Antibodies against ₂ ) recognize sequences contained in the region where HIV surface glycoproteins or neutralizing antibodies are produced. HIV (340 ~
368) and HIV (299-329) both originate from the region of the virus that produces neutralizing antibodies (Putney et al., Science (December, 1986) 234, 1392-1395).

HIV (487-511), HIV (469-511) and HIV (578-60)
8) The primary sequence is shown in FIG.

HIV (487-511) and HIV (469-511) hop and
The woods distribution is shown in FIG. 2, and the hop-and-woods distribution of HIV (578 to 608) is shown in FIG.

As the lipid conjugated to the amphipathic peptide, any lipid can be used, provided that a method for coupling the peptide to the lipid can be found. Those skilled in the art can easily find such a method, if any.

Examples of suitable lipids include phospholipids such as phosphatidylethanolamine (PE), sterols such as cholesterol, sphingolipids such as sphingomyelin, glycolipids such as myelin, and other diacyl groups. Included are lipid structures, including diacylamines.

Phospholipids are preferred and phosphatidyl ethanolamine is especially preferred.

The peptide-lipid covalently bound substance can be purified for use by removing unreacted components and surfactant by dialysis. The substance that remains in the dialysis bag after dialysis is the immunogen. No further purification is necessary.

In addition to covalently linking peptides to lipids to produce peptide-lipid covalently bound substances, naturally occurring covalently bound substances can also be used. An example of such a naturally occurring peptide-phospholipid covalent agent is a covalent agent consisting of a protein bound to phosphatidylinositol.

The above-mentioned peptide-lipid covalently binding substance can be used alone as an immunogenic composition according to the present invention. However, in a preferred embodiment, the peptide-lipid covalent agent is associated with a mixture of one or more lipids and one or more sterols.

A complex consisting of a peptide-lipid covalently bound substance in association with a mixture of one or more lipids and one or more sterols can be used for all components or peptides in a non-toxic detergent at high limiting micelle concentrations. -Can be produced by solubilizing a lipid covalent agent, additional lipids and sterols. The solution is then dialyzed to remove the detergent. The product is a particulate suspension that is vesicular in nature or has an amorphous particulate structure when viewed by light microscopy.

Without intending to be limited by the following description, the nature of the peptides in the complex appears to be a determining factor in the resulting structure. That is, peptides that are hydrophilic or neutral more often appear to form vesicle structures, while peptides that are hydrophobic appear more often to form amorphous particulate aggregates.

Peptides are most often associated with each other and with additional lipids and sterols by hydrophobic interactions derived from the acyl chains of the lipid to which the peptide is attached and from other lipids and the hydrophobic region of the sterol. However, as in the membrane structure, ionic interactions and van der Barska will also be included.

Association can be tested by centrifuging the dialysis solution by ultracentrifugation at high speed (100,000 to 300,000 xg). When the peptide-lipid covalent agent associates with additional lipids and sterols to form the immunogenic composition according to the present invention, the peptide-lipid covalent agent pellets with the additional lipids and sterols.

The fate of peptide-lipid compositions can be monitored, for example, by radioactive labeling, fluorescent labeling, spectroscopy, thin layer chromatography or high pressure liquid chromatography.

More particularly, there are at least two methods of producing a complex consisting of a peptide-lipid covalent agent associated with a mixture of one or more lipids and one or more sterols.

In one method, the derivatized lipid (for peptide-lipid covalent agent formation) is dissolved in a detergent together with a mixture of one or more underivatized lipids and one or more sterols. The pre-made peptide is then added to the solution and reacted with the derivatized lipid to form the peptide-lipid covalent bond. After the reaction is complete, the solution is dialyzed into an appropriate aqueous buffer solution to remove the detergent, thereby forming a particulate suspension containing the immunogenic complex.

The derivatized lipids used in the above method are prepared by conventional methods as described for producing peptide-lipid covalently bound materials by themselves. For example, when the cross-linking agent is used for forming a peptide-lipid covalent substance, the lipid is derivatized by binding the cross-linking agent to the lipid. If necessary, the derivatized lipid is then extracted from the reaction mixture by conventional methods.

The peptide is activated by the method used to attach it to lipids. For example, if a cross-linking agent is used to attach the peptide to the lipid, the peptide is reduced with dithiothreitol by conventional methods. In this way, the activated peptide is purified by conventional methods such as affinity chromatography or HPLC (high pressure liquid chromatography).

The surfactant in which the derivatized lipid produced in this way is dissolved together with one or more underivatized lipids and one or more sterols is a non-toxic surfactant of high critical micelle concentration. Can be "High"
The limit micelle concentration means that the detergent can be removed by dialysis. Triton X-100 or C ₁₂ E ₈ (N-dodecyl octaethylene monoether (N-dodecyloctaethyren
Other detergents with "low" critical micelle concentrations, such as e monoether)) are also suitable, but removal is achieved by other methods than dialysis, such as adsorption to SM-2 beads. . Suitable surfactants with high limiting micelle concentrations can be readily determined by one of ordinary skill in the art, examples of which include octyl-β-D-glucoside and octyl-β-D-thioglucopyranoside.

Octyl-β-D-glucoside is preferred.

In preparation for solubilizing the lipids and sterols in a detergent, they are first dissolved in an organic solvent that solubilizes but does not oxidize or in any case destroy the integrity of the phospholipid. Suitable organic solvents include diethyl ether, chloroform, benzene and acetone.

Derivatized lipids, non-derivatized lipids and sterols are used in a molar ratio of about 2: 3: 5, but a wide range of ratios can be used and about 10 mg of organic solvent (lipids and sterols). Although it dissolves at a concentration of / ml (solvent), a range of concentration ratios can be used.

The sample is then dried in a stream of nitrogen or non-oxidizing gas such as argon.

The dried sample is resuspended at a concentration of about 4 mg (lipids and sterols) / 1 ml (solvent) in an aqueous buffer solution such as an aqueous phosphate buffer solution or a citrate buffer solution having a pH of 4.5 to 6.5, and the sample is stored at room temperature. And sonicate to form a particulate suspension of vesicles.

A surfactant is then added to this sample and the sample is again sonicated at room temperature to dissolve all components.

The amount of surfactant added is sufficient to solubilize the components of the mixture. A suitable amount is about 10: 1 by weight based on the total weight of lipid and sterol. One of ordinary skill in the art can readily determine other suitable amounts of surfactant.

To form the peptide-lipid covalent agent, the activated peptide prepared as described above is added to the detergent mixture in an amount necessary for optimal binding of the peptide to the derivatized lipid, For example, an amount of about 1: 2 moles of peptide is added per mole of derivatized lipid. The pH and other reaction conditions are adjusted according to the particular reaction to be carried out and so that the peptide and the derivatized lipid react appropriately. Reaction conditions are easily determined by one of ordinary skill in the art. A suitable reaction time is generally overnight.

Dialysis is performed by conventional methods, with time intervals at which essentially all of the surfactant is removed and sufficient buffer exchange.

As a dialysis buffer, any aqueous buffer solution that does not oxidize lipids or destroy the integrity of the immunogenic composition can be used.

The product is a microparticle suspension of an immunogenic composition consisting of a peptide-lipid covalent agent associated with a mixture of one or more lipids and one or more sterols.

According to a second method of producing an immunogenic composition consisting of a peptide-lipid associated with a mixture of one or more lipids and one or more sterols, according to a second method, previously prepared as described in the first method. Suspension comprising vesicles consisting of a derivatized lipid prepared by making the activated peptide prepared as described in the first method and a mixture of one or more lipids and one or more sterols. It is added to the liquid and the peptide is allowed to react with the derivatized lipid in the vesicles
Form a covalent lipid binding material. Immunogenic compositions in which there is already a peptide-lipid covalent agent can also be used as vesicles. After the reaction is complete, all components are solubilized in detergent and the solution is dialyzed into an appropriate aqueous buffer solution to remove detergent, as in the first method, thereby removing the immunogenic composition. A fine particle suspension of matter is formed.

In the second method, the solution containing the unilamellar vesicles is made in a manner similar to that of the first method to make a microvesicular suspension. The peptide is allowed to react before the addition of detergent. Surfactant is added at the end of the overnight coupling reaction. All other steps, times, concentrations, temperatures, pH etc. are the same as described in the first method.

More particularly, the amount of active peptide required to optimize the coupling of peptide and derivatized lipid to the mixture to make a peptide-lipid covalent agent, e.g. about 1: 2 moles per mole of derivatized lipid. Amount of peptide is added. The pH and other reaction conditions are controlled by the particular reaction being performed. Reaction conditions are easily determined by one of ordinary skill in the art. A suitable reaction time is usually overnight.

The surfactant used to solubilize the components in the reaction mixture is the same as described above for the first method.

The sample is made by solubilizing the surfactant by adding to the reaction mixture an amount sufficient to solubilize the components in the mixture. A ratio of about 10 mg surfactant to 1 mg total lipid and sterol is generally suitable. The sample is then sonicated to dissolve all components.

Once solubilized in detergent, dialysis is performed as described above for the first method.

The lipids and peptides used in this embodiment of the invention include:
Any lipid and any amphipathic peptide as described above can be used to make an immunogenic complex consisting only of an amphipathic peptide conjugated to a lipid.

Phospholipids are preferred and phosphatidyl ethanolamine is especially preferred.

The lipid in the lipid and sterol mixture can be any lipid provided it is not immunogenic, ie the animal does not show an immune response either in the lipid alone or in a complex. Also, the lipid must not interfere with the animal's ability to mount an immune response to the peptide.

One of ordinary skill in the art can readily determine whether a lipid is immunogenic by assaying the antibody reactive with the lipid by standard assays.

In addition, the lipid component of the lipid and sterol mixture is all 1
It may consist of one type of lipid or a mixture of two or more lipids, and the lipid may be the same as or different from that present in the protein-lipid covalent material.

Examples of lipids useful as the lipid component of a lipid and sterol mixture include phosphatidylserine (PS), phosphatidylcholine (PC), sphingomyelin (SP), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylglycerol (PG). , Phosphatidic acid (PA), and cardiolipin.

Phospholipids are preferred.

As a sterol, any sterol does not solubilize lipid aggregates and is not immunogenic, that is, it does not induce an immune response against itself and does not interfere with the animal's ability to respond to the peptide. It can be used as a condition, for example, to have anti-inflammatory properties.

Mixtures of more than one sterol can be used, but it is preferred to use only one sterol.

As used in the case of lipid and sterol mixtures, sterol refers to many types of steroids containing an alcoholic hydroxyl group at the C ₃ carbon and a branched aliphatic chain of 8 carbons or more at the C ₁₇ carbon. They exist at the C ₃ carbon as free alcohols or long chain fatty acid esters of hydroxyl groups.

Examples of sterols that can be used in the mixture include cholesterol and lanosterol.

A particularly preferred sterol is cholesterol.

The immunogenic compositions in the second and third embodiments are not only for non-immunogenic amphipathic peptides, but also for non-immunogenic hydrophilic and / or non-immunogenic neutral peptides. It has the ability to selectively induce or enhance antibody production.

One of these two immunogenic compositions consists of a peptide-lipid covalently bound substance consisting of a hybrid peptide conjugated to a lipid, said hybrid peptide being (a) a non-immunogenic hydrophilic peptide, (b) ) Non-immunogenic neutral peptide, (c)
One peptide selected from the group consisting of non-immunogenic amphipathic peptides, and (d) a hybrid peptide consisting of any combination of two or more of the above peptides (a), (b) and (c). It consists of a non-immunogenic amphipathic peptide conjugated to. In a preferred embodiment, the peptide-lipid covalent agent is associated with a mixture of one or more lipids and one or more sterols.

Peptide-lipid complex alone or with one or more lipids
An immunogenic composition associated with a mixture of one or more sterols is prepared in the same manner as described above for an immunogenic composition consisting of a non-immunogenic amphiphilic peptide conjugated to a lipid. Provided that the peptide in the peptide-lipid covalent bond is (a) a non-immunogenic hydrophilic peptide, (b) a non-immunogenic neutral peptide, (c) a non-immunogenic amphipathic peptide, And (d) a non-immunogenic amphiphile conjugated to one of the hybrid peptides consisting of any combination of two or more of peptides (a), (b) and (c) as defined above. It consists of a hybrid peptide consisting of a sex peptide.

The peptides forming both hybrid peptides are covalently linked together, preferably end-to-end.

Further, since the bond between amino groups and the bond between carboxyl groups and carboxyl groups are usually unstable in an aqueous environment, it is preferable that the end-to-end bonds be amino-terminal and carboxyl-terminal.

As hydrophilic or neutral peptide, any hydrophilic or neutral peptide which meets the above definition can be used. If the immunogenic composition is used as a vaccine, the peptide must not be toxic or otherwise harmful to the health of the animal.

Examples of hydrophilic peptides include HIV (469-485), HIV (500-5
11) and HIV (647-659).

The primary sequences of these peptides are shown in Figure 1.

HIV (469-485) and HIV (500-511) hop and
The woods distribution is shown in FIG. 2, and the hop-and-woods distribution of HIV (647-659) is shown in FIG.

An example of a neutral peptide is (NANP) _n .

The primary sequence of (NANP) ₃ is shown in FIG. 1, and the hop-and-woods distribution is shown in FIG.

The amphipathic peptides used in the hybrid peptides are the same as described above for the first embodiment of the immunogenic composition.

In particular, the amphipathic peptide T ₂ (HIVenv (121-124), H-
E-D-I-I- S-L-W-N-Q-S-L-K) and _{T 1 (HIVenv (428-443),} L-Q-I-I-N-M-
W-Q-E-V- L-A-M-Y-A-NH 2) ( sheath (Cease) outside, Proceedings of National Academy of Sciences of USA (1987) 84 Vol. , 4249-4253) is for HIV (eg H
It is useful for binding hydrophilic or neutral peptides from IV (299-329)) to induce an immune response to these non-immunogenic peptides.

A hybrid peptide is any one of the known techniques of peptide synthesis reactions.
One can be made either manually or on an automated peptide synthesizer. Alternatively, the hybrid peptide can be produced by standard techniques of genetic engineering. Moreover, hybrid peptides can be produced by enzymatic assembly from peptide fragments or by cleavage from larger hybrid polypeptides or proteins.

Further, by embodying the present invention, it is possible to obtain an immunogenic composition in which any number and kind of peptides can be bound to produce multiple antibodies. Of course, regarding the length of the final peptide, if the peptide is too long or too short, there are not only structural problems due to peptide folding, but also there are technical limitations on the length when making synthetic peptides. There are practical limits. This limit can be easily determined by those skilled in the art.

The constitutive peptide thus produced is as described above for the first embodiment of the immunogenic composition without regard for which of the peptides forming the hybrid peptide the conjugate bond with the lipid is formed. The immunogenic peptide-lipid composition can be made by coupling to lipids in the same manner as described.

Examples of hybrid peptides include (a) HIV (469-511) (non-immunogenic hydrophilic peptide HIV
(469-485) and non-immunogenic amphipathic peptide HIV (487
-511). ) And (b) HIV (487-511) (non-immunogenic hydrophilic peptide HIV
(500-511) and non-immunogenic amphipathic peptide HIV (487
-499). ).

When HIV (469-511) forms part of the immunogenic composition according to the invention, the antibody is directed against the non-immunogenic hydrophilic peptide HIV (496-485 (as well as peptide HIV (487
-511), (469-511) and (500-511)).

When HIV (487-511) forms part of the immunogenic composition according to the invention, the antibody is directed against the non-immunogenic hydrophilic peptide HIV (500-511) (as well as the peptide HIV (48
7-511) and (469-511)).

The present invention capable of selectively inducing or enhancing antibody production not only against non-immunogenic amphipathic peptides but also against non-immunogenic hydrophilic peptides and / or non-immunogenic neutral peptides The other of the two compositions according to claim 1, wherein the peptide is associated with a first peptide-lipid covalent substance, which consists at least in part of an amphipathic peptide, and a mixture of one or more lipids and one or more sterols. Consisting of one or more additional peptide-lipid covalently bound substances.

The first peptide-lipid covalent agent comprises one of the immunogenic covalent agents described above having an amphipathic peptide associated with it.

That is, the first peptide-lipid covalently bound substance is a peptide-lipid covalently bound substance as described above consisting of a non-immunogenic amphipathic peptide conjugated to a lipid, or a hybrid peptide conjugated to a lipid. Can be a covalent peptide-lipid covalent binding agent as described above, wherein the hybrid peptide is (a) a non-immunogenic hydrophilic peptide, (b)
A non-immunogenic neutral peptide, (c) a non-immunogenic amphipathic peptide, and (d) a peptide (a) as defined above,
Consists of a non-immunogenic amphipathic peptide conjugated to one of the hybrid peptides consisting of any two or more combinations of (b) and (c).

One or more additional peptide-lipid covalent binders are the following covalent donors (1) non-immunogenic hydrophilic peptides conjugated to lipids, (2) non-immunogens conjugated to lipids. One or more of a neutral neutral peptide, (3) a non-immunogenic amphipathic peptide conjugated to a lipid, and (4) a conjugated peptide conjugated to a lipid, in which case said hybrid peptide Is (a) a non-immunogenic hydrophilic peptide,
(B) a non-immunogenic neutral peptide, (c) a non-immunogenic amphipathic peptide, and (d) any two or more of the peptides (a), (b) and (c) defined above. It consists of a non-immunogenic amphipathic, hydrophilic or neutral peptide that is conjugated to a peptide selected from the hybrid peptides consisting of the above combinations.

That is, the one or more additional peptide-lipid covalent agents can be any as described above, but can further include a peptide-lipid covalent agent without an amphipathic peptide. .

This embodiment of the invention makes it possible to raise antibodies against these peptide-lipid covalent substances which are not themselves immunogenic, ie any covalent substance which does not comprise the amphiphilic peptide.

Thus, for example, one peptide peptide is a parents aqueous peptide, such as T ₂ or T ₁ of the the HIV - lipid covalently binding agent and peptide other peptides are hydrophilic peptides as HIV (299-329) - An immunogenic composition comprising a mixture of one or more lipids and one or more sterols in association with a lipid covalently bound substance is an amphipathic peptide T ₂
Or not only for T ₁ , but also for the hydrophilic peptide HIV (299-
329) also induces antibodies. If other peptide-lipid covalent agents are included, one or more antibodies included in the additional complex will be elicited.

This immunogenic composition according to the third embodiment of the invention comprises 2
Other immunogenic compositions are described above, except that one or more peptide-lipid covalently bound substances are associated with a mixture of one or more lipids and one or more sterols. Can be made in the same way. This result can be achieved, for example, by reacting one or more peptides with a simultaneously derivatized lipid by any of the two above-mentioned methods.

Hydrophilicity used for peptide-lipid covalently bound substance consisting of non-immunological hydrophilic peptide conjugated to lipid, non-immunogenic neutral peptide conjugated to lipid, and hybrid peptide conjugated to lipid Alternatively, the neutral peptide is similar to that described above for the hybrid peptide, and any peptide-lipid covalent agent that uses the amphipathic peptide is the amphipathic peptide that is the first of the immunogenic compositions. It is the same as described above for the instantiation.

Hydrophilic and neutral peptides are covalently attached to lipids in the same manner as described above for amphipathic peptide-lipid complexes.

Further, according to the present invention, any two or more of the above immunogenic compositions can be mixed and combined to obtain an immunogenic composition. This method can also be used to produce antibodies to one or more peptides.

The immunogenic composition according to the present invention is useful as an immunogen. Practical applications include clinical and non-clinical use. Examples of non-clinical uses include immunization to raise monoclonal antibodies and production of antibodies against defined specific epitopes used in diagnostics.

Examples of clinical uses include the induction of systemic or mucosal antibodies to sperm for use in vaccine production and contraception.

〔Example〕

Although the present invention will be described in connection with particular embodiments, it is not limited thereto.

Unless stated otherwise, all percentages, ratios, etc., are by weight.

Example 1 Production of an immunogenic composition capable of selectively inducing antibody production against a non-immunogenic amphipathic peptide-Preparation of the first peptide-lipid covalent agent (I) Crosslinker phosphatidyl The coupling with ethanolamine was performed by Martin and Papahajopoulos (Journal of Biological Chemistry (1982) 257.
Vol., Pages 286-288).

(A) Reagent (1) Succinimidyl-4- (p-maleimidophenyl) butyrate (SMPB) (2) Phosphatidylethanolamine (PE) (3) Anhydrous methanol (4) Triethylamine (B) Method (1) Reagent (eg 2 ml) Of methanol, 5 μ of triethylamine, and 17 ml of SMPB were added to 25 mg of dry PE.) Under a nitrogen gas atmosphere at room temperature for 2 hours.

(2) Methanol was removed from the reaction product in a nitrogen stream.

(3) 2 ml CHCl ₃ was added and the mixture was diluted with 1% NaCl in water.
It was extracted twice by centrifuging in 2 ml at 1500 rpm for 5 minutes. All operations in this step were performed under nitrogen gas.

(4) Then, the chromatograph on silica gel
(A) The column was washed with 50 ml CHCl ₃ and (b) 5 ml CHCl 3
The sample in ₃ was loaded onto the column, then 40: 1, 3: 1 CHCl ₃ : MeOH.
It was carried out by sequentially applying 10 ml each of a 0: 1, 25: 1, 20: 1, 15: 1 mixture, then 40 ml of a 10: 1 mixture, while collecting 5 ml fractions.

(5) Then, silica gel column fractionation (solvent, CHCl ₃ : Me
OH: H ₂ O, 65: 25: 4) thin layer chromatography. The coupled cross-linker and phospholipid (MPB-PE) eluted with a 10: 1 fraction of early CHCl ₃ : MeOH showed a single spot co-migrating with the PE standard. The spots of derivatized PE were ninhydrin negative and iodine positive. PE standard was ninhydrin positive. The silica gel column fraction containing MPB-PE was combined and stored at -20 ° C under nitrogen gas.

(6) Phospholipids were quantified by the method of Bartlett (Journal of Biological Chemistry (1959) 234, 466 pages). The average yield was 50%.

(II) Reduction of peptide by Roche method (A) reaction (1) Solid phase method (Yarney and Marrifield), “The Peptide: Analysis, Synthesis, Biology” , Synth
Academic Press (Academic Pre)
ss) (New York), Vol. 2, p. 1-284), the amphipathic peptides HIV (487-511), HIV
(469-511) or HIV (578-608) was dissolved in 1 M acetic acid (HAc) (eg 2 mg peptide in 1 ml HAc).

(2) Dithiothreitol (DTT) was added to a concentration of 100 mM.

(3) The sample was degassed, filled with nitrogen, and then kept at 37 ° for 3 hours in a tube with a screw stopper.

(B) Affinity chromatography on Bonddelute column (3cc column is from Analytichem Internationa)
l) Obtained from C18, part 607203. ) (1) Load the column with 2 volumes of 100% MeOH, then 10 volumes with 1 MH
Washed with Ac.

(2) The peptide was loaded on a bond rate column.

(3) Column is 5 volumes of HAc, then 5 volumes of 0.02%
It was washed with trifluoroacetic acid (TFA).

(4) Sample is 5 times the volume of 60% acetonitrile, 0.02% TF
Elute with A.

(5) The eluted sample was freeze-dried overnight and stored at -20 ° C. An essentially complete recovery was made.

(III) Preparation of immunogenic composition consisting of peptide-lipid covalent substance associated with mixture of lipid and sterol (cholesterol) (A) lipid; MPB-PE, sphingomyelin (SP), phosphatidylcholine (PC), Phosphatidylserine (P
S). Sterol; cholesterol (Ch).

(B) Method (1) MPB-PE: SP: PC: of lipid and cholesterol (Ch)
It was dissolved in ether so that the PS: Ch molar ratio was 2: 1: 1: 1: 5 (10 mg / ml).

(2) The sample was dried in a nitrogen stream.

(3) Dry sample in buffer (20 mM citric acid, 35 mM disodium phosphate, 108 mM NaCl, 1 mM EDTA, pH 4.5).
Resuspended to give mg. lipid and cholesterol / ml.

(4) The sample was sonicated to form a fine particle suspension.

(5) Octyl-β-D-glucoside (10 mg / mg lipid and sterol) was added to the sonicated sample.

(6) The sample was sonicated again to dissolve all lipids.

(7) The sample was stored under nitrogen gas in the dark.

(IV) Coupling of peptide with lipid mixture containing derivatized phosphatidylethanolamine (MPB-PE) (A) Method (1) Pre-prepared reduced peptide from step II above at MPB-at pH 4.5. Added to the lipid and sterol mixture from step III above containing PE (MPB-PE molarity was twice the peptide molarity).

(2) The pH was adjusted to 6.5 with 1 N NaOH.

(3) MPB-PE and the peptide in the mixture were reacted under nitrogen gas at room temperature overnight.

(4) The mixture was dialyzed at 4 ° C. and pH 7.2 against phosphate buffered saline with several changes.

(5) An immunogenic composition consisting of a protein-lipid complex associated with a mixture of lipid and cholesterol was recovered from the dialysis bag and stored in a refrigerator (4 ° C).

Example 2 Preparation of an immunogenic composition capable of selectively inducing antibody production against a non-immunogenic hydrophilic peptide, capable of selectively inducing a non-immunogenic neutral or hydrophilic peptide Immunogenic compositions include (1) eg Barney, G. and Marifield, Earl. "The Peptides, Analysis, Synthesis, Biology" by B. (Marrifield, RB) (edited by E. Gross and J. Meinhofer) (Academic)
Press (New York), Volume 2, pp. 1-284, by standard methods as described in assembling neutral or hydrophilic peptides adjacent to an amphipathic peptide to form a hybrid peptide, Reduced and then conjugated conjugate of the reduced hybrid peptide to the derivatized lipid present in solution with other lipids and corresterol as described in Example 1 and dialyzed as described in Example 1. To form an association of the mixed peptide-lipid covalent substance with the mixture of lipid and cholesterol, or (2) carry out the mixture of the reduced non-immunogenic neutral or hydrophilic peptide and the reduced amphiphilic peptide Covalently bound to a derivatized lipid present in solution with other lipids and cholesterol as described in Example 1 and dialyzed as described in Example 1 One of the two peptide-lipid covalent substances was prepared by two methods of forming an association between a lipid and a sterol.

Using method 1, HIV peptides (487-511), (469-51)
An immunogenic composition was prepared using 1) and (578-608).

Example 3 Antibody Production of the Immunogenic Composition of Example 1 Six to eight week old female same-line mice were prepared with the immunogenic composition of Example 1 (and prepared as in Example 1). However, the peptide is (NANP) ₃ , IL-2 (1-1
2) -trans (the primary amino acid sequence is shown in Table 1), HIVenv (469-485), HIVenv (500-511) and
HIV env (578-608), all of which were hydrophilic or neutral) were immunized intraperitoneally. Each used an equivalent amount of 30 μg of peptide on days 0 and 14.
On day 28, mice were immunized with an amount equivalent to 7.5 μg peptide of the conjugate. Mice were bled from the retro-orbital cistern on days 0, 14, 28 and 42. After collection, blood was allowed to clot and serum was separated by centrifugation. Antibodies were measured by an enzyme-linked immunosorbent assay (ELISA) described below for serum.

ELISA-Direct binding test (A) Plate coating (1) Antigen (immunized or face-to-face test) diluted to approximately 0.5-1 μg / ml in bicarbonate-carbonated saline (pH 9.6) Peptide was also used).

(2) As a control, the tank was coated with bovine serum albumin (BSA) or gelatin diluted to 10 times the antigen concentration.

(A) 100 μl BSA in bicarbonate-carbonate buffer was added to each tank.

(B) Plates were dried overnight at 37 ° C.

(C) Distilled water or PBS / Tween (3x)
It was washed 5 times.

(B) Blocking reaction (1) 1% in pH 8.2 borate buffered saline (pH 8.2)
1% in gelatin or PBS (without Tween added) (pH 7.0)
200μ of BSA was added to each tank.

(A) A flat plate was covered and kept at 37 ° C. for 1 hour.

(B) After keeping the temperature constant, the bath was washed 5 times with 1xPBS / Tween.

(C) Binding reaction Tween-containing PBS (0.5M NaCl) (pH 6.5) containing antibody-containing serum
Diluted with 1% BSA in medium (starting from 1: 100 and serial 2-fold dilution).

(A) A 100 μ sample was added to each tank, and the plate was covered.

(B) The sample was kept at room temperature on a shaker for 2 hours.

(C) The plate was washed 5 times with PBS / Tween.

(D) Detection reaction of mouse antibody (1) Goat anti-mouse immunoglobulin G (IgG) and immunoglobulin M (IgM) -HrP (Boehringer Mannheim (BOEH.MANN.), Cat. # Purified by affinity chromatography 605-26) in PBS / Tween (pH
6) diluted 1: 1000 with 1% BSA in.

(A) 100 μ of solution was added to each bath, the plate was covered and protected from light.

(B) The solution was kept at room temperature on a shaker for 2 hours.

(C) The plate was washed 5 times with PBS / Tween.

(E) Substrate reaction (1) O-phenylenediamine / di-salt machine (Sigma (SI
GMA), # P-1526) was mixed with 0.1 M citrate buffer at pH 4.5 to give a final concentration of 0.4 mg / ml. 4 μ of 30% H ₂ O ₂ was added to each 10 ml of buffer.

(A) 100 μ of solution was added to each bath to protect the plate from light.

(B) The solution was reacted at room temperature on a shaker for 15 minutes.

(C) The progress of antibody binding was determined by measuring the optical density at 488 nm.

ELISA solution bicarbonate - carbonate (B-C) substantially mixed equal portions until buffer _{-10x 0.5MNaHCO 3 0.5MNa 2 CO 3 pH9.6} .

Bicarbonate-carbonate saline 25 ml 10xB-C buffer 7.5 ml 5M NaCl 1.8 ml 1M NaN ₃ Make up to 250 ml with distilled water and filter (pore size 0.20μ).

1% BSA 20 ml 0.5 M NaPO ₄ buffer in PBS (pH 7.0), pH 7.0 15 ml 5 M NaCl 0.5 ml 1 M MgCl ₂ 1.0 ml 0.5 M CaCl ₂ 5.0 g Bovine serumalbumin,
Fraction V, Reagent Grade Make up to 500 ml with distilled water and filter (pore size 0.45μ). 4 ° C
Save to.

0.5MNaPO ₄ buffer _{(1) Na 2 HPO 4 ·} 7H 2 O-67gm / 500ml (2) NaH 2 PO 4 · H 2 O-34.5gm / 500ml (1) and (2) until the desired pH Mix. pH 6.5
To achieve this, (1) and (2) are mixed in almost equal amounts. pH
To make 7.0, mix 500 ml (1) and 200 ml (2).

1% BSA in PBS / Tween pH 6.5 20 ml 0.5 M NaPO ₄ buffer, pH 6.5 15 ml 5 M NaCl 0.5 ml 1 M MgCl ₂ 1.25 ml 20% Tween-20 1.0 ml 0.5 M CaCl ₂ 5.0 g bovine serum Albumin, Fraction V, reagent grade Make up to 500 ml with distilled water and filter (pore size 0.45μ). 4 ° C
Save to.

1% BSA in PBS with Tween (0.5 M NaCl) (pH 6.5) 20 ml 0.5 M NaPO ₄ buffer, pH 6.5 50 ml 5 M NaCl 0.5 ml 1 M MgCl ₂ 1.25 ml 20% Tween-20 1.0 ml 0.5 M CaCl ₂ 5.0 g of bovine serum albumin, Fraction V, reagent grade distilled water to 500 ml and filter (pore size 0.45μ). 4 ° C
Save to.

0.1M citrate buffer, pH 4.5 trisodium citrate _{7.35g (Na 3 C 6 H 5} O 7 · 2H 2 O) 5.25g of Citric acid monohydrate 500 ml, and pH 4.5.

2.5 MH ₂ SO ₄ and 50 mM Na ₂ S ₂ O ₅ (sodium metabisulfite) 69.4 ml concentrated H ₂ SO ₄ 4.75 gm sodium metabisulfite Make 500 ml with distilled water and filter (pore size 0.45μ). 4 ° C
Save with.

1% gelatin in BBS (pH 8.2) 10 gm gelatin 6.18 gm H ₃ BO ₃ 9.54 gm Na ₂ B ₄ O ₇ · 10H ₂ O 4.38 gm NaCl Distilled water up to 500 ml and filtered (0.45μ pore size) To do. Store at room temperature.

Tween 20 (0.15%) in PBS (1M NaCl) (pH 6.5) 2
% BSA 100 ml 5 M NaCl 20 ml 0.5 M NaPO ₄ buffer, pH 6.5 0.5 ml 1 M MgCl ₂ 1.0 ml 0.5 M CaCl ₂ 3.75 ml 20% Tween-20 distilled water to 500 ml and filter (0.45 μ pore size). 4 ° C
Save to.

The results for sera obtained 42 days after the first immunization are shown in Table 1 below.

Table 1 Antibody titers of mice immunized with various peptide-lipid conjugates Peptide Log ₁₀ AT ^* hydrophilic or neutral (NANP) ₃ 0.0 (6) ^** IL-2 (1-12) trans 1.2 ± 1.3 (6) HIVenv. (469-485) 0.0 (5) HIVenv. (500-511) 0.0 (5) HIVenv. (647-659) 0.0 (5) Amphipathic HIVenv. (469-511) 4.4 ± 0.5 ( 5) HIV env. (487-511) 4.3 ± 0.8 (4) HIV env. (578-608) 4.4 ± 0.4 (5) Note ^* AT represents the antibody titer against the immunized peptide.

Note ^** Numbers in parentheses indicate the number of mice per group.

The results in the above table clearly show that covalent binding of amphipathic peptides to phospholipids and association with additional phospholipids and cholesterol induces a significant immune response when injected into mice. In contrast, neutral and hydrophilic peptides covalently bind phospholipids, resulting in no complex immune response in mice upon conjugation with additional phospholipids and cholesterol.

It is clear from the data presented in Table 4 of Example 5 that the amphipathic peptides are not immunogenic in their own right.

Example 4 Antibody Production by the Immunogenic Composition of Example 2 Mice were prepared as in Example 1 and the immunogenic composition prepared in Example 2, except that HIV peptides (469-485) and (5) were used.
00-511) was used for immunization with the complex. Antibody titer was determined as in Example 3.

The results are shown in Table 2 below.

The results in the above table are non-immunogenic peptides (469-486, 500-51
When 1) was synthesized adjacent to an amphipathic peptide, it was shown that mice were able to produce antibodies against neutral and hydrophilic regions. Therefore, these regions became immunogenic by binding to larger amphipathic regions, covalently binding phospholipids and associating with additional phospholipids and cholesterol.

As a method of adding an amphipathic structure, purified viral glycoprotein (S. Goul-fogerite (S. Goul
d-fogerite) and J. Mannino, Analytical Biochemistry (Anal.Biochem.)
(1985) 148, 15-26) by mixing with dissolved peptide-lipids, phospholipids and simple lipids, some of them are naturally bound to non-immunogenic peptide-phospholipid covalent substances. The conjugate was added at 1/200 of the concentration present in and the mixture was dialyzed overnight at room temperature against phosphate buffered saline pH 7.2 with 3 changes. The results are shown in Table 3 below.

As shown in Table 3, the addition of amphipathic molecules in the form of viral glycoproteins leads to antibody production on the two above non-immunogenic peptides. In each case, the increase in antibody titer is greater than 2 as its log value.

Therefore, neutral or hydrophilic peptides are immunogenic when complexed with phospholipids and added to a preparation containing an amphipathic structure and additional phospholipids and cholesterol.

Example 5 Importance of Coupling Non-Immunogenic Peptides to Lipids to Create an Immune Response Table 4 shows the importance of coupling amphipathic peptides to lipids to create immunogenic structures. ing.

The results in Table 4 show that the peptide is covalently bound to phospholipids as lipids and associates with phospholipids as additional lipids and cholesterol as sterols to produce immunogenic compositions. This composition is the first MP to couple
Significantly immunogenic whether B-PE is already associated with other lipids and cholesterol in the structure or is free in solution by being dissolved in octyl-β-D-glucoside. It is sex. In contrast, no immune response is observed when the peptide is mixed with preformed lipid-cholesterol structures and is not covalently materialized with phospholipids. Furthermore, 487-51 solubilized in sterile PBS
No immune response is observed when animals are injected with 1.

Example 6 Assembly of a synthetic vaccine against malaria Peptide (NANP) _n is a repetitive peptide that has great immunological importance for protection against malaria pathogens. However, (NANP) _n is a neutral peptide and is not immunogenic by itself in most animals.

Recently, immunologically important amphipathic peptides have been identified from malaria pathogens (MFGood).
Outside, Science (1987) Volume 235, 1059-1062). This amphipathic peptide has the following sequence, P-S-D-
K-H-I-E-Q-Y-L-K-K-I-K-N-S
-I-S.

The vaccine is produced as follows.

Bound peptide, P-S-D-K-H-I-E-Q-
Y-L-K-K-I-K-N-S-I-S- (NANP) _n
(N = 3) was synthesized by a synthetic protein synthesizer.

The bound peptide was then reduced with dithiothreitol and derivatized phosphatidylethanolamine (P
E), lipid mixtures such as those consisting of sphingomyelin, phosphatidylserine, and phosphatidylcholine, and with cholesterol as a sterol, solubilized in a nonionic detergent such as octyl-β-D-glucoside at high threshold micelle concentrations .

The bound peptide was conjugated to PE derivatized by one of the known methods as described in Martin and Papahajopoulos, Journal of Biological Chemistry (1982) 257, 286-288. Cross-linked.

The solution was dialyzed against phosphate buffered saline (pH 7.2) to give a fine particle suspension of the immunogenic complex, which was brought to an appropriate concentration in phosphate buffered saline. Adjusted.

Example 7 Construction of a synthetic vaccine against HIV III The following non-immunogenic hydrophilic peptides of immunological importance:
HIV III: (299 to 329) HR-P-N-N-N-T-R
-K-I-R-I-R-E-P-E-R-A-E-K-
I-E-N-M- R-Q-C-NH 2 and (735~752) D-
R-P-E-G-I-E-E-E-G-G-E-R-D
-RS is known.

The following immunologically important amphipathic peptides are also derived from HIV III and have recently been published by KBCease (Proceedings of National Academy of Sciences of USA (1987, 6 Month) 84
Vol. 4, pages 4249-4253, T ₂ (HIVenv (112-12
4), H-E-D-I-I-S-L-W-H-L-Q-
S-L-K), and _{T 1 (HIVenv (428-443),} L-Q-
I-N-M-W-Q-E-V-L-A-M-X-A
-NH ₂ ), but also Putney et al. (Science (198
6 years, December) Volume 234, pp. 1392-1395) HIV (340
-368), H-N-N-T-L-K-Q-I-D-S-
K-L-R-E-Q-F-G-N-N-L-Q-S-S
-G-C-NH ₂₎ is described.

One or more vaccines are produced by the method of the invention by forming an immunogenic composition with a protein, lipid complex, wherein said protein is:
-368 alone, 340-368 bound to 487-511, 299-324 bound to 340-368, 299-324 bound to 487-511, 578
299-324 bound to -608, and 340 bound to 578-608
-368.

More preferably, an immunogenic composition comprising one or more of the above protein-lipid covalent agents is formed, eg 48
340-368 bound to 7-511 and 299-3 bound to 370-608
24 are included in the same composition.

Although the present invention has been described in detail in relation to particular embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope thereof.

〔The invention's effect〕

The results in Table 2 show that when a non-immunogenic peptide was synthesized adjacent to an amphipathic peptide, it was administered to mice and allowed the animals to produce antibodies to neutral and hydrophilic regions. Have shown that these regions become immunogenic by binding to larger amphipathic regions, covalently binding phospholipids and associating with additional phospholipids and cholesterol.

The results in Table 3 show that addition of an amphipathic peptide in the form of a viral glycoprotein to a non-immunogenic peptide-lipid covalent agent results in antibody production in the latter, thus neutral or It has been shown that complexing hydrophilic peptides with phospholipids and adding an amphipathic structure and a preparation containing additional phospholipids and cholesterol makes them immunogenic.

The above results indicate a method of selectively imparting immunogenicity to synthetic and natural peptides which are originally non-immunogenic.

[Brief description of drawings]

FIG. 1 shows the primary amino acid sequences of various peptides useful in the immunogenic compositions of the present invention. 2, 3, 4, and 5 are contours obtained from Hop and Woods analysis of peptides useful in the immunogenic compositions of the invention. Figure 2 shows amphipathic peptides,
Showing contours of HIV (469-511) and HIV (487-511) and hydrophilic peptides, HIV (469-485) and HIV (500-511),
Fig. 3 shows the outline of the amphipathic peptide, HIV (578-608), Fig. 4 shows the outline of the hydrophilic peptide, HIV (647-659), and Fig. 5 shows the neutral peptide (NANP). The contour of _n is shown. * Indicates the degree of hydrophilicity or hydrophobicity. Hydrophilicity is shown above the one-letter amino acid abbreviation, and hydrophobicity is shown below the one-letter amino acid abbreviation. The greater the number of ★, the greater the hydrophilicity or hydrophobicity.

Claims (23)

[Claims] 1. A peptide-lipid covalent substance comprising a non-immunogenic amphipathic peptide conjugated to a lipid, which induces antibody production selectively to the non-immunogenic peptide. An immunogenic composition capable of enhancing. 2. The immunogenic composition according to claim 1, wherein the peptide-lipid covalent substance is further associated with a mixture of one or more lipids and one or more sterols. 3. A peptide-lipid covalent substance comprising a conjugated peptide conjugated to a lipid, the hybrid peptide comprising (a) a non-immunogenic hydrophilic peptide and (b) a non-immunogenic intermediate. From a group consisting of a sex peptide, (c) a non-immunogenic amphipathic peptide, and (d) a peptide consisting of a combination of any two or more of said peptides (a), (b) and (c). An immunogen capable of selectively inducing or enhancing antibody production against one or more non-immunogenic peptides consisting of non-immunogenic amphipathic peptides conjugated to one selected peptide Sex composition. 4. The immunogenic composition according to claim 3, wherein the peptide-lipid covalent substance is further associated with a mixture of one or more lipids and one or more sterols. 5. A first peptide-lipid covalent bond substance and 1.
Consisting of one or more additional peptide-lipid compositions associated with a mixture of one or more lipids and one or more sterols, wherein the first peptide-lipid covalent agent is (1) a lipid A non-immunogenic amphipathic peptide conjugated to, and (2) a hybrid peptide conjugated to a lipid, which is a member selected from the group consisting of (a) a non-immunogenic peptide. A hydrophilic hydrophilic peptide, (b) a non-immunogenic neutral peptide, (c) a non-immunogenic amphipathic peptide, and said peptide (2) (a), (2) (b) and (2)
(C) a non-immunogenic amphipathic peptide conjugated to a peptide selected from the group consisting of a mixed peptide consisting of a combination of any two or more thereof, wherein said one or more additional peptides -The lipid covalent substance is conjugated to (1) a non-immunogenic hydrophilic peptide conjugated to the lipid, (2) a non-immunogenic neutral peptide conjugated to the lipid, and (3) a lipid. A non-immunogenic amphipathic peptide that is chemically bound, and (4) one or more members selected from the group consisting of a conjugated peptide that is conjugated to a lipid. A hydrophilic hydrophilic peptide, (b) a non-immunogenic neutral peptide, (c) a non-immunogenic amphipathic peptide, and (d) the peptide (4) (a), (4) (b) and ( 4) From any two or more combinations of (c) A non-immunogenic amphipathic, hydrophilic or neutral peptide conjugated to a peptide selected from the group consisting of: antibodies selectively against one or more non-immunogenic peptides An immunogenic composition capable of inducing or enhancing production. 6. The immunogenic composition according to claim 3, wherein the mixed peptide comprises a non-immunogenic hydrophilic peptide conjugated to a non-immunogenic amphipathic peptide. 7. The immunogenic composition according to claim 3, wherein the mixed peptide comprises a non-immunogenic neutral peptide conjugated to a non-immunogenic amphipathic peptide. 8. The immunogenic composition according to claim 4, wherein the mixed peptide comprises a non-immunogenic hydrophilic peptide conjugated to a non-immunogenic amphipathic peptide. 9. The immunogenic composition according to claim 4, wherein the mixed peptide comprises a non-immunogenic neutral peptide conjugated to a non-immunogenic amphipathic peptide. 10. The immunogenic composition of claim 5, wherein the one or more additional peptide-lipid covalent agents comprise a non-immunogenic hydrophilic peptide cross-linked to a lipid. 11. The immunogenic composition according to claim 5, wherein the one or more additional peptide-lipid covalent agents comprise a non-immunogenic neutral peptide cross-linked to a lipid. 12. The immunogenic composition according to claim 1, wherein the lipid in the peptide-lipid covalent substance is phosphatyldiethanolamine. 13. The immunogenic composition according to claim 2, wherein the lipid in the peptide-lipid covalent substance is phosphatyldiethanolamine. 14. The immunogenic composition according to claim 3, wherein the lipid in the peptide-lipid covalent substance is phosphatyldiethanolamine. 15. The immunogenic composition according to claim 4, wherein the lipid in the peptide-lipid covalent substance is phosphatyldiethanolamine. 16. The immunogenic composition according to claim 5, wherein the lipid in the peptide-lipid covalent substance is phosphatyldiethanolamine. 17. The immunogenic composition according to claim 2, wherein the sterol is cholesterol. 18. The immunogenic composition according to claim 4, wherein the sterol is cholesterol. 19. The immunogenic composition according to claim 5, wherein the sterol is cholesterol. 20. An immunogenic composition comprising a mixture of two or more of the immunogenic compositions according to any one of claims 1-5. 21. (A) (i) a covalently bound peptide-lipid covalent substance comprising a non-immunogenic amphipathic peptide conjugated to a lipid; (ii) one or more lipids and one or A peptide-lipid covalent substance comprising a non-immunogenic amphipathic peptide conjugated to a lipid associated with a mixture of a plurality of sterols, and (iii) a hybrid peptide conjugated to a lipid A peptide-lipid covalent substance comprising: (a) a non-immunogenic hydrophilic peptide, (b) a non-immunogenic neutral peptide, (c) a non-immunogenic amphipathic peptide, And (d) the peptides (a), (b) and (c)
From a mixed peptide conjugated to said lipid consisting of a non-immunogenic amphipathic peptide conjugated to a peptide selected from the group consisting of hybrid peptides consisting of any two or more combinations of A peptide-lipid covalent bond consisting of: (iv) a hybrid peptide conjugated to a lipid associated with a mixture of one or more lipids and one or more sterols. The substance, wherein the hybrid peptide is (a) a non-immunogenic hydrophilic peptide, (b) a non-immunogenic neutral peptide, (c) a non-immunogenic amphipathic peptide, and (d) the peptide ( a),
The above-mentioned one or more consisting of a non-immunogenic amphipathic peptide conjugated to one peptide selected from the group consisting of a hybrid peptide consisting of a combination of any two or more of (b) and (c) A peptide-lipid covalent binding substance comprising a mixed peptide conjugated to a lipid associated with a mixture of a plurality of lipids and one or more sterols; and (v) a first peptide-lipid covalent binding substance. One or more additional peptide-lipid covalently bound substances associated with a mixture of one or more lipids and one or more sterols, wherein the first peptide is (1) conjugated to a lipid A non-immunogenic peptide that is chemically bound, and (2) a hybrid peptide that is conjugated to a lipid, the hybrid peptide being (a) a non-immunogenic hydrophilic peptide, (b) a non-immunogenic medium Sex peptide (C) non-immunogenic amphipathic peptide, and (d) said peptide (2) (a), (2) (b) and (2)
From the group consisting of hybrid peptides conjugated to said lipids consisting of amphipathic peptides conjugated to peptides selected from the group consisting of hybrid peptides consisting of any two or more combinations of (c) The one or more additional peptide-lipid covalently bound substances consisting of a member selected from the group, which is associated with the mixture of said one or more lipids and one or more sterols is (1) conjugated to the lipid Non-immunogenic hydrophilic peptide bound to, (2) non-immunogenic neutral peptide conjugated to lipid, (3) non-immunogenic amphipathic peptide conjugated to lipid, and ( 4) A hybrid peptide conjugated to a lipid, wherein the hybrid peptide is (a) a non-immunogenic hydrophilic peptide, (b) a non-immunogenic neutral peptide, (c) a non-immunogenic amphipathic agent. Sex peptide,
And (d) conjugated to a peptide selected from the group consisting of mixed peptides consisting of any two or more combinations of the peptides (4) (a), (4) (b) and (4) (c). The first peptide-lipid share consisting of one or more members selected from the group consisting of hybrid peptides conjugated to the lipid consisting of non-immunogenic amphipathic, hydrophilic or neutral peptides bound to One or more selected from the group consisting of a binding substance and one or more additional peptide-lipid covalent binding substances associated with a mixture of said one or more lipids and one or more sterols; A pharmaceutical composition comprising a mixture of an immunogenic composition and (B) a pharmaceutically acceptable carrier, diluent or excipient, which selectively induces or enhances antibody production. object. 22. The pharmaceutical composition according to claim 21, wherein the lipid in the peptide-lipid covalent bond substance is phosphatidylethanolamine. 23. The sterol in the immunogenic composition (ii), (iv) and (v) is cholesterol.
The described pharmaceutical composition.