NZ215999A - Vaccine for inducing antibodies to lhrh - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £15999 # '<J Priority Complete r>or;cif..:oTt?n Fii C«: Mmst/p.?-:, C?7t<l/2.0 1 •1. f.; 3 0 HAY 1988 7 308'''.''.''.

NO DRAWINGS 215999 Patents Form No 5 Number PATENTS ACT 1953 Date COMPLETE SPECIFICATION LHRH ANALOGUES USEFUL IN STIMULATING ANTI-LHRH ANTIBODIES AND VACCINES CONTAINING SUCH ANALOGUES jt/WE PITMAN-MOORE, INC., P 0 Box 344, Bear Tavern Road, Washington Crossing, New Jersey 08560, United States of America,a corporation organised under the laws of the State of New Jersey, United States of America sj do hereby declare the invention for which 1/vve pray that a Patent may be granted to m«/us, and the method by which it is to be performed, to be particularly described in and by the following statement. 1 (followed by page la) 21 599 ia- Background of the Invention Luteinizing Hormone Releasing Hormone ("LHRH") is secreted by the hypothalamus and carried to the pituitary gland where it stimulates secretion of follicle stimulating hormone and luteinizing hormone which, in turn stimulate 10 ovarian follicle development, the conversion of ovarian follicular to corpus luteum, tubule development in the testicles and production of progesterone and testosterone. Thus, release of LHRH causes ovulation and formation of corpus luteum in females and spermatogenesis in males.

LHRH is a decapeptide of the following structure: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-nly-NHj wherein, according to convention, the amino group of each amino acid appears to the left and the carboxvl to the right with the hydroxyl of the carboxyl of the terminal Gly being replaced by an NH2 group. The conventional abbreviations for the amino acids are: Glu (glutamic 25 acid), pGlu (pyroglutarnic acid), His (histidine), Trp (tryptophane), Ser (serine), Tvr (tyrosine), Gly (glycine), Leu (leucine), Arg (arginine), Pro (proline), Lys (lysine) and Cys (cysteine). Except for glycine which has no optical center, all amino acids are of the L-30 configuration unless otherwise indicated. LHRH may be produced as described in U.S. Patents 4,159,980 and 4 ,213 ,895.

Analogues of LHRH have been prepared which act as agonists 35 or antagonists of LHRH, i.e., which tend to diminish or accentuate the action of LHRH in the body. Such analogues ^ 1 U KJ IJ , 2 > -i \ IS 20 25 are described in U.S. Patents 3,880,825; 3,941,763; 4,034,082; 4,072,668; 4,075,192; 4,143,133; 4,143,135; 4,211,769; 4,234,571; and 4,263,282. These analogues nay be administered to the animal or patient in amounts such as 2 to 200 micrograms per kilogram of body weight to yield an immediate effect on the reproductive cycle as described in U.S. Patent 4,010,261. A second type of treatment is the administration to the patient or animal of an LHRH analogue as an antigen, i.e., inmunogen, whereby the analogue acts as a vaccine and the host mammal generates antibodies to the analogue which also act against the body's own LHRH. Thus, the analogue's eEfect will persist after the analogue itself has been metabolized or excreted. This second treatment is described for various LHRH analogues or LHRH itself by A. Arimura et al. in Endocrinology 93:1092-1103 (1973); by H.M.

Fraser et al. in the Journal of Endocrinology 63:399406 (1974); by S.L. Jeffcoate et al. in Immunochemistry Vol. 11, p. 75-77 (1974); by I.J. Clarke et al. in the Journal of Endocrinology 78:39-47 (1978); by L. Pique et al. in Immunochemistry Vol. 15 pages 55-60 (1978); and in U. S. Patent 3,963,691.

An object of the present invention is a vaccine containing an immunogen which prevents the function of LHRH when administered to a male or female mammal. At present there are 43 million dogs and 31 million cats in the U.S. and their numbers increase daily. -Stray dogs and cats along with wild animals such as skunks and raccoons are known to be major sources of rabies transmission to domestic animals and humans. Surgical removal of reproductive organs, e.g., spaying and castration, is presently a \ C' •r.

Ptt-46 21599 commonly used method for preventing reproduction in mammals. However, surgery is relatively costly, tine consuming and impractical when used with wild or stray animals. A vaccine which immunizes the animal against its 5 own LHRH would prevent conception for extended periods and would he a cost-effective method of pooulation control. A further object is a vaccine form which can be used in dart-guns or drug-containing bullets for the immunization of wild mammals. An object of the invention is a vaccine 10 for population control of a large population of animals such as deer, wild horses and burros and animals kept in zoos.

A further object of the invention is a vaccine containing 15 an immunogen for the treatment of male mammals for the undesired effects of LHRH in such animals. For example, cryptorchidism is a condition where one or both testicles of a male mammal have not descended from the abdomen making castration a difficult surqical procedure. A 20 vaccine which prevents LHRH from transmitting signals to produce male hormones would be, in effect, a "immunological castration" for male mammals and could be used to render cryptorchid stallions docile.

Summary of the Invention The invention comprises an immunogenic vaccine which contains the nonapeptide Lys-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-MH2 or the decapeotide Cys-Lys-Trp-Ser-Tyr-Hly-Leu-30 Arg-Pro-r,ly-NH 2 • When administered to a mammal, the vaccine induces in vivo production of antibodies to the host's LHRH whereby the natural secretion of LHRH is neutralized. The vaccine can be used to immunize the mammal against conception or any other conditions which 35 are directly or indirectly influenced by secretion of PM-46 „ rT^*^*7V^^»aT»J*TF^|nnrt.nM-.t|W^j __ 21599 LHRH. por example, the vaccine can he used in the treatment of prostate cancer in nen.

Detailed Description of the Invention The vaccine of the invention contains as the immunological agent, a conjugate between a protein and a peptide selected from: i) Lys-Trp-Ser-Tyr-Clly-Leu-Arq-Pro-Gly-NH 2» or ii) Cys-Lys-Trp-Ser-Tyr-niy-Leu-Arg-Pro-Gly-NH2 or mixtures of i) and ii). The vaccine can be adminis-15 tered as the conjugate per se in solid form as a lyo-philized solid or may be micro- or macroencapsulated. Preferably, the vaccine is used as a liquid emulsion, most particularly as a water-in-oil emulsion with the conjugate in the aqueous phase. The emulsion may be described as an 20 adjuvant, the protein as a carrier protein and the peptide as an LHRH analogue.

Peptides i) and ii) are written above using conventional abbreviations where the amino group of each amino acid 35 appears to the left and the carboxyl group to the right. The last R amino acids of both compounds are the same and in the same order as the last 8 amino acids of LHRH. The individual amino acids making up peptides i) and ii) above and iii) described below are preferably each of the L-30 configuration in view of their lower cost compared to the D-configuration. However, the invention comprises peptides wherein each or any of the amino acids are of the D-configuration. Peptides i) and ii) may be obtained from Peninsula Laboratories, Inc. of fill Taylor Uay, Belmont, 35 CA 94002 or other commercial custom peptide synthesizers, * in is .n ^ ( i * - i- ui* j: including Bachem Inc. of 3132 Kashiwa Street, Torrance, CA 90505 and Vega niochemicals of Tucson, Arizona 35734. Further, they may be prepared by conventional methods known in the art such as by solid phase synthesis using benzhydrylamine resin, protected amino acids, a coupling reagent such as <1 icyclohexylcarbodiinide (DCC), removal of protecting groups with liquid hydrofluoric acid and purification by counter current distribution, Cie column high pressure liquid chromatography and gel chromatography. Such techniques are described in the text by John Stewart and Janice Young entitled "Solid Phase Peptide Synthesis", W.H. Freeman S Co., San Francisco, 1969 and in 'J.S. Patent 3,941,763. Benzhydrylamine resin may be prepared as described by P. Rivaille et al. in Helvetica Chimica Acta, Vol. 54 pages 2772-2775 (1971). Peptide synthesis may also be accomplished by solid phase synthesis and segment condensation synthesis.

The carrier protein used in the invention is preferably one with a molecular weight of at least about 40,000 dalton and more preferably at least about 60,000 dalton. In a particular aspect of the invention, the protein may be of human origin such as to heighten the immune response when the vaccine is administered to an animal while rendering the vaccine less danqerous to a human if it is accidently given to a human. Peptide i) contains alpha- and epsilon-NH2 groups for conjugation to the carrier protein. Except at very low pH, peptide ii) dimerizes quickly through the SH groups of Cys to the peDtide of the following formula iii): V 21 599 iii) SCH2CH( MH2 )CO-Lys-Trp-Ser-Tyr-Gly-Leu-Arq-Pro-Gly-MH 2 SCH2CH(NH2)CO-Lys-Trp-Ser-Tyr-Gly-Leu-Arq-Pro-Gly-NH2 Formation of the dimer makes conjuqation through the SH —^ qroup uncertain. However, the d isul f ide-bonded dimer iii) may conjuqate to the carrier protein through either -'JH 2 group to provide the advantage of 2 LHRH analogue determi-10 nants through one bond between an -NH2 qroup of the analoque and one -COOH group of the carrier protein. Carrier proteins which may be used in the invention include albumin, such as from bovine, baboon, doq, chicken eqq, turkey eqq, qoat, quinea piq, hamster, human, mouse, 15 piqeon, porcine, rabbit, rat, sheep or other sources, immunoqlobulin from such sources or hemocyanin such as from Keyhole Limpets such materials being available from Siqma Chemical of St. Louis, Mo. Keyhole Limpet Hemocyanin (KLH), e.g., as obtained from Cal Biochem of La 20 Jolla, CA, is preferred in view of its high immunogeni-city.

Conjuqation between the peptide and the carrier protein may be carried out as described by J. H. Kennedy et al. in,. 25 Clinica Chimica Acta, Vol. 70, pages 1-31 (1976) with conjugating agents such as glutaraldehyde or a water soluble carbodiimide, e.g., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (ECDI).

% The conjugate may be administered per se as a vaccine or preferably micro- or macroencapsulated. Alternatively, the conjuqate is provided with an adjuvant for administration to the mammal. This is preferably a water-in-oil emulsion, it beinq found that an oil-in-water 35 emulsion qives markedly inferior results. Thus, an aqueous solution of the pentide-protein conjugate of the PM-46 ---f—ir'TtnT'drr' 'i 'frmwwfr » ,1,, 2 1 59 ~> > j | -7- | invention is formulated into a stable water-in-oil emul- ! sion usinq an oil phase consisting of an oil such as f j mineral oil and a non-ionic emulsifier. Suitable mineral oils include those having a viscosity of about B to 5 20 centistokes at 40°C, e.g., Drakeol 6 obtained from the Penreco Division of the Pennzoil Co. Nonionic emulsifier include Montanide obtained from Seppic of 70 Champs Flvees, Paris, France. The non-ionic emulsifier may be used with the oil in a weight ration of about 1:6 to 1:17, 10 e.g., about 1:9. For a water-in-oil emulsion, the aqueous phase is slowly added to the oil phase in a honogenizer after which the mixture is emulsified to yield an emulsion having a viscosity of about 200 to ^00 centistokes at 40°C. Viscosity measurements may be taken on a Cannon-15 Ubbelohde Viscometer, available from Cannon Instruments Co. of State College, PA as described in U.S. Patent 2,305 ,57-0 . The thus-produced vaccine emulsion is stable for at least 1 month at 37°C.

The emulsion vaccine may be administered parenterally to a mammal. Vaccine in the liquid form, e.g., in a water-in-oil emulsion, may be injected by syringe. In the solid form, e.g., lyophilized conjugate, may be used in a ballistic implant or dart gun arrangement as described in 25 U.S. Patents 3,948,263 and 3,9R2,536 which are incorporated by reference. Such devices are available from BallisiVet Inc. of iThite Rear Lake, Minnesota. The amount of conjugate to be administered to the mammal to achieve production of anti-LHRH antibodies essentially equivalent 30 to be host's production of LHp.H will depend on the deqree ^ of conjugation between peptide and protein and the size and species of the host. In general, about 0.2 to 1.0 mg of conjugate per kilogram of body weight is administered and is given twice at a 3 to 6 week interval. Annual 35 booster administrations of the same dose is recommended P*1 -46 215999 i recommended for a continued effect. Since the antibody i titer will decrease gradually, the effect of the invention vaccine will diminish and is thus reversible, which is an advantage of the invention over prior surgical methods.

The vaccine of the invention, as described above, may be used to treat any condition in nan or other mammals which / is brought on or aggravated by LHRH. The vaccine is t^us an effective contraceptive agent in males and females, an > 10 agent to treat sexual hyperactivity in males and females, ;<t e.g., for the treatment of cryptorchidism in male mammals 3 such as horses, and the treatment of cancers and other ) conditions which are stimulated by sexual hormones. For J example, cancer of the prostate gland is believed to be i advances by male hormones and removal of male gonads or injection of antagonistic female hormones is often used for treatment. The anti-LHRH vaccine of the invention may be used to treat prostate cancer by preventing LHRH from signaling the secretion of male hormones.

Example 1 Preparation of Peptide ii) Synthesis of peptide ii) was carried out by the solid phase method useing para-metvylbenzhydrylamine resin with the projected amino acids, in order of coupling: Boc-Oly, Hoc-Pro, Doc-Arg(Tos), Boc-Leu, Boc-C.ly, Boc-Tyr(BrZ), Boc-Ser(OBzl), Boc-Trp, Boc-Lys(CIZ) and Boc-Cys(MHz 11 .

The coupling reagent was DCC (dicvclohexylcarbodiimiie), Boc (butyloxycarbony1) removal by trifluoroacetic acid and neutralization by triethylamine. After the synthesis on a Beckman Model 990 Synthesizer, the peptide was removed from the resin and all protecting groups by liquid HF.

Purification was by counter current distribution and gel chromatography.

PM-46 2159 >: In more detail, the cycle of steps used for the addition of each amino acid is as follows, where washes are for one * • minute each, unless otherwise stated. 1. Methylene chloride, three tines 2. 40* trifluoroacetic acid in methylene chloride, once for two minutes ' 10 3. 40% trifluoroacetic acid in methylene chloride, once for 25 minutes 4. Methylene chloride, once 15 5. Ethanol, once for two minutes 6. Methylene chloride, twice 7. 10% triethylamine in methylene chloride, once for two 20 minutes R. 10% triethylamine in methylene chloride, once for 10 minutes 9. Methylene chloride, three times After this series of washes, the appropriate amino acid derivative and dicyclohexylcarhodiimide are added at three-fold excess, and coupling to the qrowing peptide 30 proceeds for the next two hours. The first amino acid residue is incoroorated by coupling Boc-glycine to the benzhvdrylamine styrene resin. Serine is added as the benzyl ester and cysteine as the S-methoxybenzyl derivative. Tyrosine is added as Roc-Tyr(Br-2), arginine as 35 Boc-Ar(os) and lysine as Boc-Lys(Cl-Z). After the peptide has been synthesized, it is given a final deprotection ">*« -AC / 215999 ; with trifluroacetic acid and is washed with methanol and I dried. The peptide is removed from the dry resin with | anhydrous hydroqen fluoride, using anisole to minimize side reactions. This reaction requires about 45 minutes 5 at 0 degrees C. The resin is then dried under vacuum. The peptide-polvstyrene mixture is washed with ether and the peptide is extracted with 10% acetic acid. The £ peptide solution is lyophilized to qive the crude peptide.

Three grams of crude peptide were applied in two lots to a 10 countercurrent distribution apparatus, using the solvent system described below for peptide i). Each run gave Zj 0.9 grams of partially purified peptide. The 1.8 grams of \] peptide was applied to a large P-2 column in 10% acetic j acid. The peptide was easily soluble in 10% acetic acid and the eluate was collected at 4 drops per second and 15 ml/tube. Tubes 27-35 contained 1.005 grams of pure peptide.

PreDaration of PeDtide i) Synthesis of peptide i) was carried out as peptide ii) above with protected amino acids, in order of coupling, Boc-Oly, Boc-Pro, Boc-Arq(Tos), Hoc-leu, Boc-Gly, Boc-Tyr(Br-7.), Boc-Ser(OBzl), Boc-Trp and Boc-Lys(Z). 25 Purification was by countercurrent distribution and C.0 column purification.

In more detail, peptide i) was made by coupling the first amino acid, glycine, as Boc-glycine in BHA resin. From 30 this point the synthesis of the crude peptide was as above, as was the HF cleavage. Three grams of the crude peptide were applied to a counter-current distribution apparatus using the solvent system 4:1:5 of butanol:acetic acid:water. Fractions 8R to 109 contained 0.9 grams of a 35 partially purified peptide. These fractions were pooled and lyophilized. The martially purified peptide was DM— .1 C. 2 15 9 9 9 -ii- applied to a C-19 column where the initial solvent >/as 0.1* acetic acid and the final solvent was 30* aceto-nitrile containing 0.1% acetic acid. A linear qradient was used with 700 ml of each solvent. Peptide fractions 5 that appeared oure by TLC were pooled and submitted to quality control.

Example 2 Peptide i)-BSA Conjugate Into 1 ml of 0.35% NaCl solution made with distilled water was dissolved 20 mg of bovine serum albumin (TSA). 10 mg of peptide i) was dissolved in 1 ml of distilled water and 15 slowly added to the BSA solution with constant mixinq. 100 mg of l-(3-dimethvlaninopropyl)-3-ethylcarbodiiniie hydrochloride (ECDI) was dissolved in 0.5 ml of distilled water and slowlv added to the HSA solution with constant mixinq. The mixture is then incubated for 6 hours at room 20 temperature with constant stirring in a shaker. The mixture was then dialyzed against 2 to 3 liters of phosphate buffer saline (PDS) for about 16 to 24 hours at 5°C. The dialyzing process was repeated twice. The dialyzed conjugate was then filter sterilized and assayed 25 for the degree of conjugation.

Example 3 Peptide i)-HSA Conjuqate The procedure of Example 2 was repeated substituting human serum albumin (HSA) for DSA to obtain a peptide i)-HSA conjugate.

PM-46 215999 Examnle 4 \ Peptide i)-KLH Conjugate An aqueous solution of about 1.5 to 2% Key Hole Limpet (KLH) was dialyzed against a 0.85% MaCl saline solution for about 36 to 48 hours with two changes of saline f- solution. The KLH solution protein concentration was then determined by the niuret Reaction Method using a HSA 1 10 standard. The Biuret method is described by A. G. Gornall *?j et al. in the Journal of biological Chemistry, Vol. 177, page 751 (1949). As determined by the Biuret method, 20 ng of KLH was then conjugated to 10 mq of peptide i) as I ; described in Example 2 to yield a oeptide i)-KLH i conjugate.

Example 5 Peotide ii)-HSA Conjuqate A conjugate or peptide ii) and HSA was prepared by the method described in Example 3 substituting peptide ii) for peptide i).

Example 6 ♦ Peptide ii)-KLH Conjugate % The procedure of Example 4 was repeated substituting 30 peptide ii) for peptide i) to obtain a conjugate between peptide ii) and KLH.

PM-4 6 2159 Example 7 Peptide ii)-HgG Conjuqate Twenty rng of hnian gamma globulin (HgG) was dissolved in a mixture of 2 ml of PBS and about 0.25 ml of dimethylformamide into which had been dissolved 5 mg of n-maleimidobenzoyl-N-hydroxysuccinimide (MBS). The thus-produced mixture was allowed to react at roon temperatjre for .75 minutes. The activated HgG was then separated from unreacted materials bv adding PHS and subjecting the mixture to gel filtration on SEPHAOFX* G 25 gel available from Sigma Chemical Co. of St. Louis, MO. To the activated HqO in PHS was 'added in mg of peptide ii) which was then incubated for 2 hours at room temperature. The mixture was dialyzed as in Example 2, filter sterilized and assayed for the degree of conguation.

EXAMPLE 8 Determination of Degree of Conjugation The degree of conjugation of peptide i) or ii) to the carrier protein in Examples 2-7 was estimated by the niuret method. In this procedure, the total weight of protein in the unconjugated protein is determined along with the protein weight in the conjugate, after dialyzinq the conjugate to remove any unconjugated peptide i) or ii). The difference in protein weight is the conjugated peptide and from this, the degree of conjugation of peptide to carrier protein can be determined.

TIsinq the biuret technique, it was found that the deqree of conjugation of the conjugates of Examples 2-7 was about 10 to 40 peptides per 100,000 dalton of molecular weight of the carrier orotein. 2159 Exanples 9-14 i Adjuvant Vaccines The conjugates produced in Examples 2-7, respectively* were individually diluted with phosphate buffer saline ^ ^ (PBS) to make 5 to 15 tnq/ml solutions. Thionersal (1 part - per 10,000 by volume) was added as a preservative and the material was sterilized either by filter sterilization, 10 e.g., through a 0.2 urn disc, or by qanma irradiation. 1 This constitutes the aqueous phase of the water-in-oil adjuvant vaccine. The oil phase was made by mixing 1 Dart sj of the nonionic emulsifer Montanide with 9 parts of _j Drakeol 6 light white mineral oil followed by filter 1 15 sterilization. The sterile oil phase is placed in the ! proper size emulsifying or homogenizing flask equipped ! with side tubelettes. A Virtis homoqenizer Model 23 or 45 l ! made by Virtis of Gardiner, N.Y. 12525 is started at low speed and an equal amount of the aqueous phase was added I j 20 slowly. After the entire aqueous phase was added, the mixture was emulsified at a higher speed until an effective water in-oil emulsion was made.

The stability of the individual emulsions was determined 25 by centrifuqing a sample of the emulsion at 10,000 to 12,000 O for 6 minutes at which no more than 5% separation v"' was detected. The viscosity of the preparation was typically between 260 to 300 centistokes per second. The emulsions were found to be stable for more than one month 30 at 37°C. o PM-46 •H 2 1 5999 EX AMPLE 15 Vaccine Efficacy — Rats The vaccines produced in Examples 9-14 from the conjuqate produced in Examples 2-7, respectively, were tested for blockaqe of the effects of LHRH in rats.

Younq rats were inoculated intramuscularly with 1.0 nl, 10 0.5 ml and/or 0.2 ml of the vaccines produced in Examples 9-14 followed hy a booster injection of the sane dose, 3-1 4 weeks after the first injection. i Serum titer for antibodv against LHRH was determined bv 15 the ELISA test as described by A. Voller et al. in the Hulletin of the World Health Organization, Vol. 53, paqes 55-65 (1976) and in the "Manual of Clinical Immunology", Chapter 69, pages 506-512, American Society of Microbiology (1976). Microelisa plates were coated with 20 synthetic LHRH. Antibody titers were determined at 4-6 weeks intervals.

In addition, the effectiveness of the vaccine was determined by observinq the atrophy of the testicles in 25 the case of males and the uterus and ovaries in the females. The results are shown in the following Table I. o PM-46 215999 -16-TAPLE I RESULTS OF ANTIFERTILITY VACCINE Example Coni uqate Dose Antibody Gonadal Titer Range Atrophy* 7) in 9 in 10 10 n n n i?. 12 12 13 13 13 14 )-RSA 1.0 ml 640 - 6400 7/7 ) —MSA 1.0 ml <50 - 6400 3/4 )—HSA 0.5 ml *00 - 6400 3/4 )—HSA 0.*! ml 50 - 6400 0/5 )-KLH 1.0 ml >6400 4/4 )-KLH 0.5 ml 800 - 5400 3/3 ) -KLH 0.2 ml 50 - 6400 /5 ) -HSA 1.0 ml >6400 /5 )-HSA 0.5 nl 200 - >6400 2/5 ) —HSA 0.2 ml 1600 - >6400 3/5 ) - KLH 1.0 ml 400 - >6400 4/4 ) -KLH 0.5 ml 800 - >6400 4/4 ) -KLH 0.2 ml <50 - 6400 4/5 )-HgG 0.5 ml sno - 6400 4/5 considered to be positive if the size has to 75% or less i of the control qroup.

**Number of vaccinated animals with positive atrophy/number vaccinated.

From the above data, the peptide i)-KLH vaccine is considered to consitute the preferred embodiment of the invention.

P*1-4 6 2159 Example 16 Vaccine Efficacy — Cats The vaccine produced in Example 11 was tested for efficacy in cats. Five female and three male cats were each given a 0.5 ml injection of the peptide i)-KLH vaccine with a second 0.5 ml injection 4 weeks after the first. Immediately before the second injection, the anti-LHRH antibody 10 titer range for the cats was found to be 800-1600 and 4 weeks after the second injection, the range was 12,800-- 51,200.

The female cats did not come into heat 18 months after the 15 first injection even after being exposed to a normal male cat.

The male cats all had gonadal atrophy according to the atrophy definition in Example 15 for at least one vear 20 after the first injections. One male cat showed gonadal reversion to a normal size IR months after the first injection.

ExamDle 17 Vaccine Efficacy — Horse The vaccine produced in Example 10 was tested for efficacy against cryptorchidism in horses. A crvotorchid stallion 30 was found to have a serum testosterone level of 0.7 nanograms per ml and an anti-LHRH antibody titer of 10 as determined by the Elisa test using LHRH as the antigen. The animal was injected with 3 ml of the peptide i)-HSA vaccine produced in Example 10. A second injection of 35 3 ml of the vaccine was nade 4 weeks after the first.

Just prior to the second injection, the animal's serum PM-46 \

Claims (24)

215999 -18- testosterone level was found to be n.OQ nanoqrans per ni with an antibody titer of ?560. Four weeks after the second injection, these figures were found to be 0.IT5 and 20,480, respectively. ] i 3 i ] PM-4 6 -19- What we claim is:

1. An inmunogen vaccine which induces in a mamnal production of antibodies against LHRH, which vaccine comprises a water in oil emulsion, the water phase comprising a 5 conjugate between a carrier protein and a synthetic pep-tide selected from the group consisting of: i) Lys-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, or 10 ii) Cys-Lys-Trp-Ser-Tyr-Cly-Leu-Arg-Pro-Gly-NH2, or mixtures of peptides i) and ii).

2. A vaccine of Claim 1, wherein said peptide is peptide i). 15

3. A vaccine of Claim 1, wherein said peptide is pept ide i i).

4. A vaccine of Claim 1, wherein said peptide is a 20 mixture of peptide i) and peptide ii).

5. A vaccine of Claim 1, wherein the amino acids of said peptides are L-amino acids. 25

6. A vaccine of Claim 1, wherein said carrier protein has a molecular weight of at least 40,000 dalton. %

7. A vaccine of Claim 1, wherein said peptide ii) is 30 present at least in part as a dimer. *• • > H. a vaccine of Claim 1, wherein said peptide is conjugated to said carrier protein in d ratio of 10 to 40 peptide molecules per 100,000 dalton molecular weight 3S of the s.arriet protein.

PM-46 '.*V •• c' v- V E V .y' *\ -f \ / ■ ~ LbUJ; -20-

9. A vaccine o£ Clain 1, wherein the oil phase further comprises a non-ionic emulsifier.

10. A vaccine of Clain 1, wherein said water phase and 5 said oil phase are present in said emulsion in equal parts by volume.

11. A method of inducing in a non-human animal the production of antibodies to LHRH which comprises administering to the 10 mamnal a vaccine of Claim 1.

12. The method of Claim 11, wherein said vaccine is administered parenterallv. 15

13. A method of preventing ovulation in a non-human animal which comprises administering to the mammal a vaccine of Claim 1. PN-46 30 35

14. A method of treating a non-human animal for cryptorchidism which comprises administering to the mammal a vaccine of Claim 1.

15. The method of Claim 14, wherein said animal is a stallion horse.

16. A peptide selected from the group consisting of: i) Lys-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 r ii) Cys-Lys-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-MH,, and ' £ ■ , /V if'* c fc -^. - ' \ - - p ' '< i i ) ■' 10 tsm-4 6 70 25 1 U b' U .' -21- iii) SCH2CH(NH2)CO-Lys-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-MH2 SCH2 CH(NH2)CO-Lys-Trn-Ser-Tyr-Gly-Lea-Arg-Pro-Gly-»H 2.

17. The peptide of Claim 16, wherein said peptide is i).

18. The peptide of Claim 16, wherein said peptide is

19. The peptide of Claim 16, wherein said peptide is i i i).

20- A conjugate between a carrier protein and a peptide 15 selected from the .group consisting of: i) Lys-Trp-Ser-Tyt—Glv-Leu-Arg-Pro-Gly-riH2 , or i i) Cys-Lys-Trp-Ser-T yr-Gly-Leu-Arg-Pro-Gly-NH 2, or mixtures of i) and ii).

21. A conjugate of Claim 20, wherein said carrier protein has a molecular weight of at least 40,000 dalton.

22. A conjugate of Claim 20. wherein said peptide is peptide i).

23. A conjugate of Claim 20, wherein said peptide is 30 peptide ii).

24. A conjuqate of Claim 20, wherein said peptide is,--'3 * " U' nixture of peptide i) and peptide ii). c- ■i 4^Sro 35 25 • A conjuqate of Claim 20, wherein said peptide i,£d O *' is present at least in part as a dimer. WKT-WALKER, McCABB-per: A-i"" ATTORNEYS FOR THE APPLICANT