WO1991010676A1

WO1991010676A1 - Peptides utilizable for immunomodulation

Info

Publication number: WO1991010676A1
Application number: PCT/US1991/000413
Authority: WO
Inventors: Paul F. Fehlner; Te Piao King; Loucia Kochoumian
Original assignee: The Rockefeller University
Priority date: 1990-01-19
Filing date: 1991-01-19
Publication date: 1991-07-25
Also published as: AU7316191A

Abstract

Amphiphilic carrier molecules having 21 to 26 amino acid residues, in which at least two of the first four to six or the last four to six amino acid residues are basic, are good immunogens as they contain a T cell epitope and a B cell epitope. Also hapten groups can be attached to such amphiphilic carrier molecules for production of hapten-specific antibodies which are useful as diagnostic agents or vaccines.

Description

PEPTIDES UTILIZABLE FOR IMMUNOMODULATION

Related Applications

This application is a continuation in part application of copending application serial number 07/467,386 filed January 19, 1990.

Background Of The Invention

Vaccines often comprise an antigen on a natural adjuvant such as a protein, a carbohydrate, a. lipid or a liposome. Such vaccines are useful and have been employed for many years. There are however a number of art recognized problems with them. Several of these problems are related to the adjuvant. Since they are isolated from natural sources, they are often not of uniform quality. Additionally, despite expensive and arduous purification efforts, it is difficult, and often impossible, to provide products completely free of natural contaminants. Such contaminants may themselves be antigenic. These antigens often cause the undesirable side reactions associated with the use of vaccines, particularly fevers and tissue swelling. Additionally, the concentration of antigen may vary from one batch to another because the amounts of antigen which react with the adjuvant or are absorbed on its surface are not uniform. Melittin is a peptide isolated from bee venom. It is a cationic amphiphilic peptide in which amino acid residues 1 to 20 are predominantly hydrophobic and residues 21 to 26 are hydrophilic and basic. The structure of mellitin may be represented :

GIGAVLKVLTTGYPALISWIKRKRQQ-C0NH

In this specification the standard one letter abbreviations for amino acid residues will sometimes be employed. For convenience, the abbreviations are shown below:

A Alanine M Methionine

B Aspartic Acid/Asparagine N Asparagine

C Half cystine P Proline

D Aspartic Acid Q Glutamine

E Glutamic Acid R Arginine

F Phenylalanine S Serine

G Glycine T Threonine

H Histidine V Valine

I Isoleucine W Tryptophan

K Lysine Y Tyrosine

L Leucine Z Glutamic Acid/Glutamine

At other times the standard three letter abbreviations will be used since it is sometimes easier to visualize a chemical structure when they are utilized.

A number of naturally occurring peptides of similar structure are known which can be employed in this invention, although melittin and its derivative as described below are presently preferred. For convenience, the invention will be principally described as applied to melittin and its derivatives.

Recently it has been recognized that peptide fragments, some of relatively low molecular weight, when attached to a suitable carrier will provide protection against a number of serious mammalion infections. Some of the antigenic peptides which are currently available either commercially or by known synthetic or isolation techniques are listed in Table 1. The table lists the peptides which are segments of proteins associated with the disease or pathogen identified in the second column. The references identify the publications which describe the peptides and how to obtain them. These and other antigenic peptides containing from about 1 to about 25 amino acid residues can be employed in this invention.

TABLE I

PEPTIDE SEQUENCES SUITABLE FOR DEVELOPMENT

OF VACCINES

Pathogen/ Peptide Disease (protein) Ref

A. H-(ASN-Ala-ASN-Pro) n -OH n>3 Malaria, ccs protein of

Plasmodium falciparum

B. H-(Gly-Asp-Arg-Ala-

Asp-Gly-Gln-Pro-Ala) Malaria, ccs protein n -OH n>2 of Plasmodium vivax

C. Glu-Gln-Asn-Val- Malaria, Pf 155 of Glu-His-Asp-Ala Plasmodium falciparum

D. Asn-Ala-Glu-Asn- Malaria, Merozoite surface Lys-Glu-Glu-Leu- protein of Plasmodium Thr-Ser-Ser-Asp- falciparum 4 Pro-Glu-Gly-Gen- Ile-Met

E. Met-Gln-Trp-Asr- Hepatitis, pre S(l) Ser-Thr-Ala-Phe- His-Gln-Thr-Leu- Gln-Asp-Pro-Arg- Val-Arg-Gly-Leu- Tyr-Leu-Tyr-Leu- Pro-Ala-Gly-Gly

F. Asp-Pro-Arg-Val- Hepatitis, pre S(2) Arg-Gly-Leu-Tyr- Phe-Pro-Ala-Gly- Gly-Ser-Ser-Ser- Gly-Thr-Val G. Cys-Thr-Lys-Pro- Hepatitis Surface antigen 7,8 Thr-Asp-Gly-Asn- Cys-Thr-Cys

H. Asn-Phe-Ser-Thr- Streptococcus pyogenes, Ala-Asp-Ser-Ala- M-protein 9

Lys-Ile-Lys-Thr-

Leu-Glu-Ala-Glu-

Lys-Ala-Asp-Leu-

Gly-Lys-Ala-Leu- Gly-Ala

I. Tyr-Ser-Thr-Leu- Poliovirus, replicase

Tyr-Arg-Trp-Leu- protein 10

Asp-Asp-Ser-Phe

J. Asn-Ala-Pro-Ser- Poliovirus, replicase Lys-Thr-Lys-Leu- protein 10

Glu-Pro-Ser-Ala- Phe

K. Lys-Lys-Pro-Asn- Poliovirus, VPg 11

Val-Pro-Thr-Ile- Arg-Thr-Ala-Lys-

Val-Gen Gly-Ser-Gly-Val- Foot and Mouth Disease 12 Arg-Gly-Asp-Ser- VPI Gly-Ser-Leu-Ala- Leu-Arg-Val-Ala- Arg-Gln-Leu-Pro

M. Arg-His-Lys-Gln- Foot and Mouth Disease Lys-Ile-Val-Ala- VPI 13 Pro-Val-Lys-Gln- Thr-Leu

N. Gly-Leu-Phe-Gly- Influenza, HA2 Ala-Ile-Ala-Gly- Hemagglutinin protein 14 Phe-Ile-Glu

O. Arg-Ser-Lys-Ala- Influenza, Hal Phe-Ser-Asn-Cys- Hemagglutinin protein Tyr-Pro-Tye-Asp- Val-Pro-Asp-Tyr- Ala-Ser

P. Arg-Ile-Leu-Ala- HIVI, envelope glyco- Val-Glu-Arg-Tyr- protein of human 15 Leu-Lys-Asp-Gln- T-lymphotropic virus type Gln-Leu-Leu-Gly- III Ile-Trp-Gly-Cys- Ser REFERENCES

1. Zavala, et al Science (1985) 228: 1436

2. McCutchan, et al Science (1985) 230: 1381

3. Udomsangpetch, et al Science (1986) 213: 57

4. Ravetch, et al Science (1984) 227: 1593

5. Neurath, et al Science (1984) 224: 392

6. Itoh, et al Proc. Natl. Acad. Sci. USA (1986) 83_.:9174

7. Prince, et al Proc. Natl. Acad. Sci. USA (1982) 79.:579

8. Bhatnager, et al Proc. Natl. Acad. Sci. USA (1982) 79:4400

9. Beachey, et al J. Biol. Chem (1983) 258: 13250

10. Baron, et al Journal of Virology (1982) 43.: 969

11. Baron, et al Cell (1982) 2£: 395

12. Bittle, et al Naure (London) (1983) 298_.: 30

13. Atassi, et al Proc. Natl. Acad. Sci. USA (1982) (): 840

14. Muller, et al Proc. Natl. Acad. Sci. USA (1982) 2£. 569

15. Wang, et al Proc. Natl. Acad. Sci. USA (1986) 83: 6159

In the science of immunology, the term "carrier" refers to the portion of an immunogenic molecule which contains a region (epitope) recognized by the T lymphocytes, i.e. T cells of a mammal, including humans. A carrier molecule having a T cell epitope, in cooperation with a major histocompatibility complex molecule by pathways as presently understood by those skilled in the art will bind to the antigen receptor of a T cell to elicit an immunogenic response which can result in the production of protective antibodies by the B lymphocytes or B cells.

The term "hapten" refers to the region (epitope) recognized by the B cell antigen receptor, i.e. antibody. T and B cell epitopes are operationally defined, and it is possible that the same epitope may be recognized by T and B lymphocytes.

T cell epitopes of a peptide or protein can be delineated as follows. T cells can be harvested from blood, lymph nodes or spleen samples of immunized mammals. Fragments of the peptide or protein are considered as T cell epitopes if such T cells proliferate during in vitro incubation in the presence of the fragments.

B cell epitopes of a peptide or protein may be delineated by testing the specificity of antibodies from immunized mammals. Fragments which bind such antibodies are B cell epitopes as antibodies are the antigen receptors of B cells.

Using these procedures, it has been determined that a T cell epitope is found on residues 7 to 19 and a B cell epitope on residue 20-26 of melittin.

As will be described more fully hereinafter, a principal utility for the carriers of this invention is for attachment to haptens for use as vaccines. However, there are other important utilities for the carrier/hapten products of this inventions. These utilities arise because of the ability of the selected carriers of the invention to combine with small molecules which normally do not elicit an antibody response thereby to produce antibodies to the small molecules.

Thus, the selected carriers of the inventions can be covalently bound to products such as vitamins, antibiotics, antidiabetics, steroids, cardiovascular or other physiologically active agents. The resulting product can be utilized to generate antibodies and these antibodies employed as test reagents for the presence of the vitamin, antibiotic, steroid or similar agent in body tissue.

Such products of the invention are particularly useful for testing for metabilites of abuse drugs or of pesticides. The metabolites are normally small molecules which by themselves do not generate antibodies. However, when combined with a carrier of the invention the carrier/hapten product can be used to generate antibodies to the hapten in a rabbit, goat or other animal. These antibodies can then be employed to test for the presence in body tissue the metabolite which was employed as the hapten in the production of the product of the invention.

The products of this invention will be employed in standard immunological testing procedures including radioimmunoassay, precipitation, complement fixation, agglutination and enzyme linked immunoassay. In such tests the hapten may be labelled with a detectable label or caused to react with a labelled substrate to produce a detectable reaction. Useful labels include fluorescent labels such as fluorescein, rhodamine or auramine. Radioisotopes such as 14C, 131I, 125I and 35s may be employed. Enzyme labels which may be utilized include, for example, horse radish peroxidase, β --D - glucosidase, 3- D - galactosidase, urease, glucose oxidase and acid phosphotase. Methods for labelling are well known and need not be described.

The invention also provides a fully guanidinated derivative of melittin in which the epsilon-amino groups of the lysine residues at the 7, 21 and 23-positions are converted to guanidino groups and the ε - amino group at the carboxy end of the molecule is blocked, e.g. by an acyl group. This compound can be prepared by a modification of the proceudre of Habermann and Kowallek, Z. Physiol Chem. 351:884 (1970).

To effect complete guanidation and produce the novel fully guanidated melittin, 2mm α , N-aσetyl- melittin was treated with 0.5M 0-methyl isourea sulfate in 2M urea in water at pH 10.3 and 4°C for 4 days to produce the desired product which was isolated by chromatography on a reverse phase column with a 2- propanol gradient in 0.1% trifluoracetic acid. In contrast to the prior procedure which converted only about 83% of the available amino groups, this procedure permitted complete conversion of the amino groups to guanidino groups because the presence of the urea caused denaturation of the native melittin conformaiton to make all of the amino groups available for reaction.

The product was characterized by amino acid analysis and mass spectrosσopy. Upon trypsin treatment no cleavage was observed at any of the three modified lysine residues.

Any peptide of this invention with unreactive positively charged basic residues and a free carboxyl group can be converted to an active ester, e.g. p- nitrophenyl or N-hydroxysuccinimide ester. Such active ester compounds are especially useful because of their stability, thus permitting storage until needed. They permit facile reaction with a hapten, which has an amino group, in accordance with procedures well known to the skilled artisan.

Those skilled in the art will recognize that the conversion of the ε -amino group of the lysyl residue, in fact produces compounds of the invention containing homoarginyl residues. This illustrates the important fact that the basic residue or residues in the compounds of the invention may be lysyl, homoarginyl or any other basic residue including arginyl, ornithyl, histidyl and mixtures thereof. These may be substituted at the free amino group with any of a number of selected groups such as lower alkyl, particularly methyl or ethyl so long as such substitution does not destroy the basic character.

THE DRAWING

The figure shows the structures for certain compounds studied in connection with this invention.

THE INVENTION

It has now been discovered that immunogenic, amphiphilic peptides containing from about 21 to about 26 amino acid residues, at least two of the first four to six residues or the last four to six residues being basic amino acid residues, and at least one T cell epitope are useful carriers for haptens which are B cell epitopes. The carrier must contain at least one T cell epitope so that it can elicit an immunogenic response. It may additionally contain a B cell epitope. However, the B cell epitope on the product of the invention will normally be a peptide or other selected hapten such as those listed above. This product is useful as described above and is particularly useful because it will elicit the production of antibodies for use as reagents for immunoassays or for protection against specific diseases. These latter products may be employed as vaccines to prevent infections of mammals including humans.

In addition to the requirement that the peptide has a T cell epitope, it has been observed that it must also be amphiphilic, i.e. have a hydrophilic and a hydrophobic segment and surface. There may be, but need not be a hinge region between the two segments. There is, for example, in the middle of the melittin molecule a Gly-Leu-Pro region which may act as a hinge.

The amphiphilicity of a carrier may be readily determined by any of a number of known tests. For instance, high salt concentration will cause a tryptophan fluorescence blue shift if tryplophan is present and a higher apparent molecular weight in gel permeation chromatography in amphiphilic molecules due to self association. Amphiphilic molecules also associate with lipid bilayers as shown by hemolysis, formation of ligands or voltage-gated channels. These and other biophysical means may be used to detect the interaction of amphiphilic peptides with lipid bilayers using procedures known to those skilled in this art. For gel permeation chromatography studies, peptide solutions (0.4 mM in 0.5 ml of buffer) were applied to a

50 cm x 0.9 cm column of G-50 Sephadex (Pharmacia) and eluted with 0.15 M NaCl or 1.0 M NaCl in 0.05 M NH 4.Ac buffer, pH 4.75. Eluted peptides were detected by ultraviolet absorption at 230 n . The column was calibrated for molecular weight at each salt concentration with bovine plasma albumin (MW 65,000) horse cytochrome-C (MW 13,400), insulin B-chain (MW 3,400) and synthetic octamer peptide (MW 980). Self- association was also determined by tryptophan fluorescence shift in 0.05 M NH.Ac (pH 4.75) solution with increasing NaCl concentration on a Perkin-Elmer 650-40 spectroflourometer. Tryptophan undergoes a blue shift in its fluorescence maximum as a result of localization of the side chain in a less polar environment.

Hemolysis assay was performed by incubating serially diluted peptide with about 4 x 10 urine red blood cells/ml for 30 min. at 37°C. The cells were spun down and 20 ul aliquots of supernatant were diluted in 1 ml of 0.05 M Tris HC1, pH 8.0. A 100% hemolysis standard was prepared by diluting 20 ul of cell suspension in 1 ml of 0.05 M Tris-HCl. Baseline hemolysis was corrected and peptide concentration resulting in 50% hemolysis (HD_5Q) was determined graphically. Immunization, antibody response assays and specificities were carried out as follows:

Immunization

Groups of four BALB/c mice or DBA/2 (female, 6-12 weeks old, Jackson Labs or Charles River) were immunized i.p. with 2 n ol of peptide absorbed to 1 g alum in 0.2 ml of 0.05 M phosphate buffer (pH 6.0). Mice were immunized on weeks 0, 2, 4, 6, and bled by retro-orbital plexus puncture on weeks 0, l,

3, 5, 7 and 9 and sera from each group were pooled. Mice were kept at the Rockefeller University Laboratory Animal Research Center and handled by humane procedures.

Antibody response assay and specificity

Peptide or peptide-lactoside antibody responses were determined by solid phase enzyme immunoassay (ELISA) . Wells of a 96- well assay plate (Falcon Micro Test III) were

•i coated overnight with 30 ul of 5 uM peptide solutions in 0.05 M Tris-HCl buffer (pH 8.0), and for 30 min with 200 ul of 2g/l fish gelatin in 0.5 M NaCl + 0.05 M Tris-HCl, buffer (pH 8.0), with 0.02% Tween-20. Anti- Sera were serially diluted and 20 ul volume were incubated in the antigen coated wells for 60 min followed by three washes. Plates were incubated for 60 min with 2 ug/ml of rabbit

5 anti-mouse IgG, washed; then incubated with 2 ug/ml of sheep-anti-rabbit Ig coupled to horseradish peroxidase. After washing, enzyme activity was determined after 30 min incubation with 50 ul of 0.25 mg/ml

10 aminoantipyrene, 0.005% hydrogen peroxide, and

8 mg/ml phenol in 0.1 M phosphate (pH 7.0).

Antibody specificity was established by inhibition assays. They were performed by mixing antisera, diluted to 1/2 to 1/3 maximal 15 activity as determined by ELISA, with serially diluted inhibitor solution. These solutions were incubated in wells coated with the appropriate peptide antigen as described above, and developed by the same methods.

20 Specific IgE was determined by passive cutaneous anaphylaxis in rats. Antisera were serially diluted in phosphate buffered saline containing 0.1% bovine serum albumin, (PBS- BSA) and 0.1 ml volumes were injected

25 subcutaneosly in rat skin. After 12 to 24 hours, rats were injected intravenously with 300 nmol of antigen in 2 ml of 5 mg/ml Evan's blue dye in the PBS-BPA. Positive reactions were scored where blue dye was visible; PCA titers represent the highest sera dilution which yield a positive spot.

The peptides of this invention, whether carrier or hapten, may be isolated from natural sources or synthesized by standard solid phase procedures using the protection, deprotection and cleavage techniques and reagents appropriate to each specific amino acid or peptide. A combination of manual and automated (e.g. , Applied Biosystem 430A) solid phase techniques can be used to synthesize the novel peptides of this invention. For background on solid phase techniques, reference is made to Andreu, D. Merrifield, R.B., Steiner, H. and

Boman, H.G., (1983) Proc. Natl. Acad. Sci USA 80, 6475- 6479; Andreu, D. , Merrifield, R.B., Steiner, H. and Boman, H.G., (1985) Biochemistry 24, 1683-1688; Fink, J., Boman, A., Boman, H.G. , and Merrifield, R.B., (June 1989) Int. J. Peptide Protein Res. 33, 412-421; Fink,

J., Merrifield, R.B., Boman, A. and Boman, H.G., (1989) J. Biol. Chem. 264, 6260-6267; each of which being hereby incorporated herein by reference.

The figure shows the structure of melittin and certain of the derivatives or analogs of melittin which were studied in connection with the reduction to practice of this invention. The lactopyranoside moiety is a B cell epitope.

Melittin, fragments 1-19 and 1-22 (chymotrypsin and clostripain digest products respectively) and melittin 1-26 1-24, N, α - acetyl-(6-26) and N, α - acetyl-(8-26) (synthesized, each with a C-terminal carboxyl group) and 1-20-G.Q- and 1-20-D- (synthesized, each with a C- terminal carboxamide group) were purified by. cation exchange and reverse phase chromatography. Peptides were conjugated with p-aminophenyl- , D-lactopyranoside (lac) at the C-terminus. All peptides were characterized by HPLC, UV, amino acid analysis and in most cases mass spectrometry.

When these compounds were studied for immugenicity by the procedures described above, the results shown in Table II were observed.

Table II Immunogenicity of Melittin and Its Analogs

ELISA IgG Titer (1/3 Maximal Activity)

l-20-G₄S₂ l-20-D₆

300

630

130 It will be observed that melittin and the 1-26 lac derivative were highly immunogenic and 1-24 lac was moderately immunogenic, whereas the 1-22 Lac derivative only weakly immunogenic and the 1-19 Lac inactive.

Also it will be observed that 1-20-G.Q- was moderately immunogenic but l-20-D_g was poorly immunogenic.

It was also observed that both of the N, a - acetyl (6-26) and -(8-26) were inactive as immunogens. This observation, coupled with the observed weak membrane activity, indicate that in addition to the T and B cell epitopes, peptide length plays a role in the immunogenicity of the molecule.

Table III shows the relative amphiphilicity of the products.

Table III Biophysical Activity of Melittin and Its Analogs

Self Association Membrane Activity

[NaCl]_5Q is the concentration of NaCl in 0.05 M NH.Ac, pH 4.75, required to induce 1/2-maximal shift of the tryptophan-19 fluorescence maximum, which occurs as a result of peptide self- association. GPC (MW) is the apparent molecular weight determined from gel permeation chromatography on G50 Sephadex in 0.15 M Nacl, pH 4.75. For reference, melittin MW is 2840. Fluorescence shift and gel permeation chromatography were performed as in (2) .

2HD_5f) is the concentration of peptide required for 50% hemolysis of a suspension of 3.6 x 10 murine red blood cells. Hemolysis performed essentially as in (6) .

Similarly, it was observed that the N, _α ~ acetyl- (6-26) and N, α- aσetyl-(8-26) fragments had very weak membrane activities, being about 50 and about 180 fold, respectively, less than that of melittin.

Melittin shows the greatest propensity to self- associate, since it requires the lowest concentration of NaCl for half-maximal shift of tryptophan fluorescence maximum and it is eluted as an oligomer by gel permeation chromatography. 1-26-lac and 1-24-lac show similar properties to these of melittin. Although 1- 26-lac is eluted as an apparent monomer under the test conditions, it is eluted as a tetramer in 1.0 M NaCl (data not shown) . The other analogs in Table III show no or little propensity for self association.

Peptides with 2-4 basic residues at the C-terminus showed greater hemolytic activities than those with only neutral residues. Peptide 1-20-D.. with acidic residues (aspartic acid) at the C-terminus showed no lytic activity. Additionally, shortening the peptide at the N-terminal end decreases its hemolytic activity.

Overall the order of amphiphilic behavior is mellitin > 1-26-lac > 1-24 lac > l-20-G₄Q > 1-20-D , 1-22-lac > 1-19-lac > N, α-acetyl (6-26) > N, α - acetyl (8-26) which, it will be observed is substantially the same as the order of immunogenicity.

These findings establish that there is a relationship between immunogenicity and amphiphilicity and that the carrier molecules of this invention must have both.

Although this invention has been described with particular reference to melittin and its derivatives, it is not so limited. It will be apparent from the above, that many other carrier molecules may be employed in the invention. Such products are generally known or can be prepared by known means such as those described above. They should be amphiphilic and include a T epitope sequence. Generally they will contain from about 21 to 26 amino acid residues and at least two of the first four or final four residues are basic. Melittin, shown above, is typical of a carrier with the basic amino acids at the carboxy end. The following structure is one example of a compound of the invention with the basic amino acids at the amino end:

QQRKRKIGIGAVLKVLTTGLPALISW

The novel products of the invention are carriers covalently bonded to haptens such as those described above. The novel products of the invention, therefore, are amphiphilic, immunogenic peptides with at least one T cell epitope containing from about 21 to about 26 amino acid of which at least two of the first four to six or the last four to six amino acid residues are basic residues, which 4 basic are covalently bound to a hapten having at least one B epitope region. Typical examples of products of the invention would be a melittin derivatives in which the terminal carboxyl group is covalently bound to any one of the peptides listed in Table I, above.

The carrier can have more than one T epitope or may, as in the case of melittin have a T and a B epitope. It may in fact have several epitopes. The hapten may have more than one B epitope and additionally may contain one or more T epitopes. Vaccines prepared from the products of the invention may contain all of the same B epitope and be univalent. They may also contain two or more B or T epitopes and be multivalent. This description of the available procedures for producing the products of the invention should be adequate to teach those skilled in the art the basic principles of the technology. It will also teach the skilled artisan the salient features of the novel products, one of the most important of which is that haptens are conjugated to a low molecular weight carrier. The resulting molecular products contain a high proportion of hapten a relative to the carrier. This is in contrast to conventional products used as a basis for vaccines. These conventional products often are composed of a small amount of hapten on a large amount of carrier.

Other important features of the carrier of the invention as supports for hapten are that the exact structure is known; there are no contaminants which may themselves be antigenic, produce tissue irritation or other undesirable reactions; and the exact concentration of the antigen is known. The principal advantage of the products of this invention as the basis for vaccines is that unlike previous systems using natural carriers such as keyhole limpet hemocyanin, tetanus toxoid and bovine serum albumin, the carriers of this invention are fully defined chemical entities to which the haptens are bound in known concentrations. Additionally, the hapten comprises a large part of the molecule not a relatively small and undefined proportion of the molecule as in the case of natural carriers.

The products of this invention can be employed to produce vaccines using any of the procedures known to those skilled in the art. The products can, for example, be suspended in inert oil, suitably a vegetable oil such as sesame, peanut or olive oil. Alternatively, they can be suspended in an aqueous isotonic buffer solution at a pH of about 5.6 or 7.4. Typically, such solutions will be made isotonic with sodium chloride and buffered with sodium citrate-citric acid or with phosphate. The solutions may be thickened with a thickening agent such as methyl cellulose.

Vaccines may also be prepared in emulsified form, either water in oil or oil in water. Any of a wide variety of pharmaceutically acceptable emulsifying agents may be employed including, for example acacia powder or an alkaryl polyether alcohol, sulfonate or sulfate such as a Triton.

Stabilizers such as sorbitol or hydrolyzed gelatin may also be added to any of the above described compositions. It is not unusual to incorporate an antibiotic such as neomycin or other antiinfective agent to prevent infection. Because the products of this invention provide a high concentration of antigen in a small molecular volume, in some instances the vaccines of the invention will be employed without adjuvants. However, if an adjuvant is employed it may be selected from any of those normally employed to stimulate the immune systems of mammals. These include, for example, Freund's adjuvant (complete or incomplete) , Adjuvant 65 (containing peanut oil, mannide monooleate and aluminum monostearate) , and mineral gels such as aluminum phosphate or alum. Freund's adjuvant is no longer used in vaccine formulations for humans or for food animals because it contains nonmetabolizable mineral oil and is a potential carcinogen. It can be used in vaccines for non-food animals. Mineral gels are widely used in commercial veterinary vaccines.

The vaccines of the invention may be defined as comprising a pharmaceutically acceptable excipient, of the general nature described above, together with an amount of a hapten of the invention which is sufficient to produce an immunological response. An effective amount may be very small. It will, as is known, vary with the hapten. With the products of this invention, because of the high concentration of hapten in a low molecular volume, it will be lower than with ordinary vaccines employing the same haptens. The quantity which constitutes an effective amount may vary depending on whether the vaccine is intended as a first treatment or as a booster treatment.

It may be convenient to provide the products of this invention as lyophilized or freeze dried powders ready to be reconstituted with a pharmaceutically acceptable carrier just prior to use.

When the carrier of this invention is used to carry a hapten which makes the final product useful as an . immunogen to generate antibodies useful as diagnostic agents, the hapten may be selected from any of a wide variety of products other than the peptides mentioned above. It is only necessary that the hapten contain or be modified to contain a functional group which can form a covalent bond with a free basic group at the amino end of the carrier or the carboxy group at the carboxy end of the carrier. Such haptens would include, for example, those listed hereinabove.

Those skilled in the art will be aware of many other such haptens.

Claims

What Is Claimed Is

1. An immunogenic molecule containing a T cell epitope and comprising an amphiphilic peptide carrier having from about 21 to about 26 amino acid residues in which at least two of the first four to six or the last four to six amino acid residues are basic residues covalently bound to a hapten which is a B cell epitope.

2. An immunogenic molecule of claim 1 wherein the hapten is a peptide covalently bound to the carboxy end of the carrier.

3. An immunogenic molecule of claim 1 in which the hapten is a saccharide covalently bound to the carboxy end of the carrier.

4. A vaccine comprising a pharmaceutically acceptable vaccine excipient and an amount of immunogenic molecule of claim 1 which is effective to produce an antibody response in a mammal.

5. A vaccine of claim 4 wherein the hapten is a peptide.

6. A vaccine of claim 4 wherein the hapten is a saccharide.

7. Guanidinated melittin in which the carboxamide group at the carboxy terminus is replaced with an active ester group.

8. The p-nitrophenyl ester of quanidated melittin.

9. The N-hydroxysuccinimide ester of guanidated melittin.

10. An immunogenic molecule of claim 1 in which the basic residues are selected from the group consisting of lysyl, arginyl, homoarginyl, ornithyl, histidyl and mixtures thereof.