COMPOSITION AND METHOD FOR IMMUNOLOGICAL CASTRATION AND SPAYING

Abstract: Composition for the production in animals of antibodies specific for LHRH. The composition comprises two or more different carriers individually coupled to LHRH or analogues of LHRH, in amounts sufficient to elicit an immune response against LHRH. The compositions are useful for suppressing male social and sexual behaviour and oestrous cyclicity and ovulation in domestic animals.

Description: COMPOSITION AND METHOD FOR IMMUNOLOGICAL CASTRATION AND SPAYING The present invention relates to the suppression of social and sexual behaviour of male domestic animals and the suppression of ovulation and oestrous cyclicity of female domestic animals, more specifically to a composition and method for immunological castration and spaying. Normal sexual behaviour in male animals is maintained by androgens, or male sex hormones, these being produced mainly in the testes. One of the principal androgens is testosterone. It is common practice for farmers to eliminate sexual activity and to reduce aggressive behaviour of their stock by surgical castration. However, a number of disadvantages are associated with surgical castration. These include haemorrhage, infection and stress associated weight loss (Schanbacher, 198<Λ. Furthermore, castration reduces both growth rate and the rate of lean tissue deposition (Robertson et al, An alternative to surgical castration is active immunization against luteinizing hormone releasing hormone (hereinafter referred to as LHRH) or analogues thereof. LHRH is/released from the hypothalamus into the pituitary portal vessels. It has been suggested that LHRH binds to the gonadotrophs of the anterior pituitary to stimulate secretion and release of luteinizing hormone (LH) . LH acts on the Leydig cells in the testis to increase the production of testosterone. Active immunization against LHRH disrupts the communication between the hypothalamus and pituitary. It is thought that the site at which antibodies to LHRH might inhibit reproductive function is in the hypophyseal portal blood vessels (Schanbacher, 1984). Active immunization against LHRH leads to marked changes in the function and morphology of the anterior pituitary; this results in a reduction in the weight, gonadotrophin content and LHRH binding of the pituitary in several species including the rat (Kock, 1977; Fraser et al., 1982), rabbit CAri ura et al.; 1974) and monkey ((Chappel et al., 1980). In addition, there is a reduction in the secretion of testosterone and involution of the testes (ratt Fraser et al., 1982 * J marmoset monkey: Hodges and Hearn, 1977 j ram: Schanbacher et al., 1982; Jeffcoate et al., 1982; bullr Robertson and others,- 1979, 1981, 1982, 1984). The rate of live weight gain in bulls actively immunized against LHRH is significantly greater than steers (Robertson et al., 1982). Immuno-castrates also convert food more efficiently, have a higher dressing out percentage, leaner carcasses and are shorter in height at the withers and thinner around the forgirth than steers (Robertson et al., 1982) . Bull calves that respond well to active immunization against LHRH show reduced secretion of gonadotrophins (Jeffcoate et al., 1982) and testosterone and a reduction in testicular size (Robertson and others, 1979, 1981, 1982, 1984). From subjective assessments of behaviour it appears that immuno-castrates resemble steers being more docile and easier to handle than bulls, (Robertson and others, 1979, 1981, 1984). When immunized bulls were permitted to serve an oestrous cow or an artificial * vagina three out of the five animals that had high antibody titres did not attempt to mount while the other two attempted to mount and one of these achieved an ejaculation (Robertson et al., 1982). In an on-going study, Robertson et al. (1984) compared non-immunized bulls to animals that had responded well to immunization against LHRH and found that the immuno-castrates were less active than the bulls with regard to riding, heading and licking and moving. Active immunization against LHRH has been reported to suppress ovulation and oestrous in female sheep (Clarke, Fraser and McNeilly, 1979) and cause • gonadal atrophy in rabbits (Arimura et al, 1973), hamsters (de la Cruz et al., 1976) and rats (Fraser and Baker, 1978) . In the studies of Clarke et al, (1978) in sheep, Fraser et al. (1974) in rats and McCormack et al. (1977) in " rhesus monkeys, anti-LHRH treatment resulted in a marked reduction in plasma LH concentrations. Thus it would appear that anti-LHRH treatment has a similar effect on plasma LH levels and gonad function in females as in males. The study of Clarke et al. (1978) is particularly relevant as the ewes showing evidence of raised LHRH antibodies did become anovulatory and did not show oestrous cyclicity for two consecutive seasons. Although active immunization against LHRH has been successfully achieved in bull calves and other animals, there has been considerable variation between individual animals in their response to immunization. In most studies, only about half the bull calves immunized against LHRH responded well to treatment having high, antibody titres. This was reported for bull calves immunized at 12 weeks of age (Robertson and other, 1979, 1981), 20 weeks of age (Robertson et al., 1984) and 28 weeks of age (Robertson et al,, 1982). Jeffcoate et al., (1982) actively immunized one month old bull calves against LHRH and reported that antibody titres rose after the animals had received two injections of immunogen but began to fall after a third injection. This necessitated a fourth injection to restore the antibody titres. The phenomena of a lack of immune response to an antigen stimulus is well documented. For example in a text-book on Veterinary Immunology by Herbert (1976)) it is stated on pp. 199-200 that "In general, antibody titres found in members of a vaccinated population will be distributed on a statistical normal curve, a few animals having high titres and another small group failing to repond at all." In another section it is stated that "If a large enough number are tested (with antigen) some amongst those vaccinated will be found to be unprotected. Errors of technique may account for a few of these but others may be quite incapable of achieving a state of protection immunity however many times the antigen is administered, or whatever other steps (i.e. the use of adjuvants) are taken to stimulate an immunity in them". LHRH is a small peptide of 9 ami o acids. In the field of immunology, it is well established that small immunogens elicit a poor immune response when injected into a host. Consequently, in each of the above studies, LHRH has been chemically conjugated to one particular carrier protein, for example, Human Serum Albumin. The present applicants have surprisingly found that a synergistic interaction occurs when animals are immunised with a combination of two or more different carriers coupled to LHRH or analogues of LHRH. Combinations of LHRH or analogues of LHRH conjugated with carriers elicit an effective immune reponse -. (measured in terms of antibody titre) in a greater proportion of animals than if LHRH or analogues of LHRH conjugated to a single carrier are administered. According to one aspect of the present invention there is provided a composition for the production in animals of antibodies specific for LHRH or analogues of LHRH, said composition comprising two or more different carriers individually coupled to LHRH or analogues of LHRH. Analogues of LHRH are peptides where additions, deletions, modifications or alterations have been made to the natural sequence of LHRH. Alterations to the natural sequence include the addition of one or more amino acids to the amino or carboxy terminus of LHRH. Domestic animal species falling within the scope of the invention include, cattle, sheep, goats, cats, guinea pigs, pigs, dogs, reindeer, horses and primates including women. Carriers of any type which are useful immunogens may be utilised. Preferably the carriers are proteins and more preferably the proteins are soluble in aqueous solution although precipitated protein may be used. Examples, of preferred proteins are Diptheria Toxoid (DT), Human Serum Albumin (HSA), staphyloccus protein A (P.A.), Keyhole Limpet Haemocyannin (KLH), and Tetanus Toxoid, (TTJ . ., Generally, any combination of two or more di ferent carriers may be used. Preferably the carriers are proteins such as, HSA and P.A. or KLH and TT, each of these proteins being conjugated to LHRH. However, especially preferred are two or more carriers where each carrier coupled to LHRH and separately administered to an animal elicits a generally good titre response. Especially preferred are compositions where one of the carriers is Diptheria toxoid (DT) . Other carriers that may be used in the present invention include lipopolysaccharides, polysaccharides, glycopeptides, muramyl peptide analogues, peptidoclycans [from bacteria cell walls], liposomes, lecithin like materials, and bacterial proteins for example purified protein derivatives from tuberculin. Conventional techniques may be used in conjugating LHRH and analogues of LHRH to various carriers. For example, heterobifunctional agents such as SPDP (Pharmacia, Piscataway New Jersey) , carbodiimide, glutaraldehyde or biotin/avidin systems are useful. Conditions are generally selected so that LHRH or analogues of LHRH are maintained in its biological active conformation when coupled to the carrier. Preferably the composition contains an adjuvant. Examples of adjuvants which may be used are aluminium hydroxide, Freunds incomplete adjuvant, Freunds complete adjuvant, DEAE dextran, levamisole, PCG and polyA, polyC or polyU. Especially preferred as adjuvants are materials that do not cause local inflammation. One example of such a material is a mineral oil composition that includes bacterial cell wall material such as peptidoglycans, or a synthetic derivative of such a cell wall material. Such a synthetic material is known as muramyl dipeptide. The composition may be optionally buffered to physiological pH using buffers such as TRIS-HC1 , Hepes, pipes or other appropriate buffers. According to a further aspect of the present invention there is provided a method for the suppression of male social and sexual behaviour of domestic animals comprising the administration of a composition to a domestic animal said composition comprising two or more different carriers individually conjugated to LHRH or analogues of LHRH. In an alternative aspect there is a method of suppressing the ovulation and oestrous cyclicity of female domestic animals by the administration of the above described compositions. Administration may be parenterally, for example, subcutaneous, and/or intramuscular or intravenous injection, orally or by absorption through the skin or by a mini-pump either implanted in the animal or attached to the outside of the animal. In the case of bovine, bull calves are preferably immunised with the composition at between 8 to 40 weeks of age. In the case of sheep, ram lambs are preferably immunised at 8 to 24 weeks. Booster injections are preferably given eight weeks apart and preferably 1 to 3 booster injections are given. In the previously defined method of the present invention, within the scope of the method is a treatment where the administration of the substance (e.g. LHRH) coupled to individual carriers is separate but the administration takes place over a relatively short space of time. A period of minutes or hours would usually work be satisfactory, although it would be obviously inconvenient compared to a single administration of the substance coupled to at least two carriers. The present invention has particular utility in the immunological castration and spaying of domestic pets or companion animals, particularly cats and dogs. Conventional surgical treatments performed by veterinarians may be expensive, and often traumatic for the animal involved and its owner. An increase in the proportion of animals responding; may also occur where other substances that elicit an immune response, other than LHRH or analogues of LHRH, are conjugated to at least two different carriers and combinations of these conjugates are subsequently administered to animals. Examples of such substances which elicit an immune response include peptides and proteins such as adrenocorticotrophic hormone, substance P, human chorionic gonadotrophin, somatostatin, epidermal and insulin—like growth factors; steroids including androstenedione (fecundin), testosterone and other substances such as melatonin and the prostaglandins. In the descriptions the following terms have meanings set forth below: LHRH: Luteinizing hormone releasing hormone is a specific substance involved in stimulating luteinizing hormone and follicle stimulating hormone from the adenohypophysis. Carrier: Any substance, especially protein which, when coupled to small substances, can elicit a 'immune response to animals. Antibody titre: The dilution of antisera which binds 50% of 0.15 nCi 131 I-labelled LHRH for 16 h at 4°C. Precipitation of the gamma globulins was achieved using polyethylene glycol at a final concentration of 14.5%. DT: Diphtheria toxoid. HSA: Human serum albumin. KLH; Keyhole limpet haemocyanin. TT: Tetanus toxoid. CP: Corynebacterium parvum. P.A. : - Protein A from Staphylococcus aureus. Carb: l-ethyl-3 (3-dimethyl aminopropyl) carbodiimide. Glut: Gluteraldehyde. The present invention will now be described by way of example only with reference to the following experimental procedures, examples and tables. Figures 1, 2 show Antibody Titre (Log Scale) plotted against various treatment groups for ram lambs and bull calves respectively. Figures 3 and 4 show mean testicular volume of ram lambs given Corynebacterium Parvum (CP), Keyhole Limpet Haemocyanim (KLH), Diphtheria Toxoid (DT) , Testanus Toxoid (TT) and a combination of these carriers coupled to LHRH; CP, KLH, DT in Figure 4 and KLH, TT and DT in Figure 3. A. EXPERIMENTAL PROCEDURES 1. Preparation of LHRH conjugates (a) Tetanus toxoid - LHRH LHRH (240 g) and tetanus toxoid (35.5 ml, 248 mg) was dissolved with 20 ml distilled water and the pH of the mixture adjusted to 6.0 with 0.1 N NaOH. A freshly prepared solution of 0.4 mg 1-ethyl—3 (3-dimethyl amino propyl) carbodimide hydrochloride (ECDI) in 2 ml distilled water has added dropwise and the reaction allowed to proceed at room temperature (22 C) overnight. The solution was dialysed against 3 changes of saline (5 litres). To estimate the number of moles of LHRH bound to tetanus toxoid 3nCi of T31I labelled LHRH was added into the reaction mixture. Using this procedure it is possible to determine the apparent incorporation of labelled LHRH on to the carrier protein by counting the quantity of 131I - LHRH in the solution before and after extensive dialysis. In this example 39 moles of LHRH were coupled to each mole of tetanus toxoid. (b) Preparation of LHRH - tetanus toxoid immunogen using the gluteraldehyde coupling method. - An amount of 10 mg LHRH and 1200 μl (13.6 mg) tetanus toxoid was dissolved in 34 ml water and 0.1 M NaOH added to give a final pH 8.0. Gluteraldehyde (4 ml of 25% v/v) was then added dropwise and the reaction allowed to proceed for 18 h at 25 β C. The solution was dialysed as described in (a) . Calculation of 131I - LHRH activity before and after dialysis gave a value of 38 moles LHRH per mole tetanus toxoid. (c) Preparation of LHRH - Protein A immunogen using the carbodiimide coupling method. An amount of LHRH (10 mg in 10 ml water) was added to 500 μl Protein A (50 mg) and 10 ml distilled water. The pH was adjusted to 6.5 with 0.01 M NaOH or HC1 and 200 mg ECDI added in 10 ml distilled water. The reaction was incubated at 25°C for 18 h as described in part (a) . Incorporation of I - LHRH to Protein A was estimated to yield a LHRH/protein A ratio of 1:36 after dialysis against three changes of physiological saline. (d) Preparation of LHRH - Protein A immunogen using the gluteraldehyde coupling method. An amount of 10 mg LHRH and 500 μl (50 mg) protein A was dissolved in 34 ml distilled water and the pH adjusted to 8 using 0.01 M NaOH. To the mixture was added dropwise 4 ml of a 25% gluteraldehyde solution and the solution incubated at 25°C for 18 h. Following dialysis against three changes of saline the amount of incorporation of LHRH with protein A was calculated using the iodinated LHRH method as described previously in part A. Results showed that the apparent LHRH/protein A mole ratio was 1:34. (e) Preparation of LHRH - Keyhole limpet haemocyanin (KLH) using carbodiimide as the coupling reagent. To 10 mg LHRH and 20 mg KLH in 24 ml distilled Was added 0.01 M HC1 to give a final pH 6.5. ECDI (10 ml of a 20 mg/ml solution) was then added to the mixture and incubated at 25°C for 18 h. The solution was dialysed against three changes of saline as described in part (a) . Addition of I - LHRH to the above solution indicated a LHRH/KLH mole ratio of 1:750 following dialysis. (f) Preparation of LHRH - KLH using the gluteraldehyde as the coupling agent. An amount of 5 mg LHRH and 10 mg KLH was dissolved in 12 ml distilled water, and the pH adjusted to 8.Θ with 0.01 M NaOH. So this mixture 2 ml gluteraldehyde (25% w/v) was added dropwise and the solution incubated at 25°C for 18 h. The mixture was then dialysed against 3 changes of physiological saline as described in part (a) . 131 Addition of -LHRH to the solution indicated * that the apparent mole ratio of LHRH/KLH was 849. (g) Diphtheria toxoid - LHRH LHRH (200 mg) and diphtheria toxoid (137 ml, 219 mg) was dissolved in 10 ml distilled water and the pH of the solution adjusted to 6.0 with 0.1N NaOH. A freshly prepared solution of 0.6g ECDI in 2 ml distilled water was added dropwise and the reaction allowed to proceed at room temperature (22 C) overnight. The solution was dialysed against 3 changes of saline (5 litres). (h) Corynebacterium parvum - LHRH LHRH (100 mg) and corynebacterium parvum (60 mg) were dissolved in 5 ml distilled water and the pH adjusted to 6.0 using 0.1 N NaOH. A freshly prepared solution of 0.2 g ECDI in 2 ml distilled water was added dropwise and the mixture incubated at room temperature overnight. -The solution was dialysed against 3 changes of saline (5 litres). (i) Keyhole limpet haemocyanin - LHRH LHRH (120 mg) and keyhole limpet haemocyanin (80 mg) were dissolved in 10 ml distilled water and the pH adjusted to 6.0 using 0.1N NaOH. A freshly prepared solution of 04g ECDT in 4 ml distilled water was added dropwise and the mixture incubated at room temperature overnight. The solution was dialysed against 3 "changes of saline (5 litres). The effectiveness of coupling, that is the moles of LHRH bound to carrier proteins 0.15 nCi 12S I-LHRH was added to add of the above mixtures. After each reaction the percent bound 125 I-LHRH was calculated based on the counts obtained before and after dialysis. Generally the effectiveness of coupling was good. B. RESULTS Experiment 1. A flock of 42 Merino ram lambs were used in the study. The rams were selected by liveweight into 7 groups of 6 rams. (However during the course of the experiment some rams were lost; deaths were attributed to pneumonia) . Treatments are shown below: Group 1. Entire rams injected with the carrier proteins KLH, protein A and tetanus toxoid. 2. Rams surgically castrated at 10 weeks of age. 3. Rams immunized against a LHRH - tetanus toxoid conjugate. 4. Rams immunized against a LHRH-Protein A conjugate. 5. Rams immunized against a LHRH-KLH conjugate. 6. Rams immunized against a LHRH-HSA conjugate. 7. Rams immunized against LHRH-TT, LHRH - Protein A, and LHRH-KLH conjugates. < U The dose, composition and time of injection of conjugates is shown in Table 1. Each individual immunogen solution was emulsified with equal volumes of Freund's complete adjuvan β The 5 ram lambs were then given 2 ml of the mixture in 4 separate subcutaneous sites in the back leg adjacent to the groin. The combined immunogens were injected with 8 ml of the emulsion. Booster injections were made using identical quantities of freund 8 s incomplete i adjuvant. Blood -samples were collected by venepuncture at two weeks following the second booster injection. The antdLbody titres for this experiment-, are illustrated in Table 3. 15 Experiment 2 A herd of 35 bull calves of predominently Friesian breed were used for the experiment. The calves were raised on skim milk and weaned onto grass 20 at 10 weeks of age. The bulls were divided into 5 groups of 7 animals using liveweight as the basis of selection (during the course of the experiment three bulls were lost due to pneumonia) . Treatments are shown below. 25 Group Treatment 1. Entire bull calves were immunized against the carrier proteins, tetanus toxoid, keyhole limpet haemocyanin and protein A 30 (n=6) . TABLE 1 Composition, dose of immunogen and booster schedule for LHRH immunization in ram lambs Immunogen Primary First Second Third Booster Booster Booster Time after birth 10 weeks 18 weeks 26 weeks 34 weeks Tetanus toxoid - LHRH (Carb*) 400μg 400μg 400μg 400μg Protein A - LHRH (Carb) 900μg 900μg 900μg 900μg KLH - LHRH (Carb) 900μg 900μg 900μg 900μg HSA-LHRH (Carb) 500μg 500μg 500μg 500μg Tetanus toxoid-LHRH + Protein A-LHRH + KLH-LHRH: Tetanus toxoid - LHRH (Carb) - 400μg ' 400μg 400μg 400μg Protein A - LHRH (Carb) 900μg 900μg 900μg 900μg KLH - LHRH (Carb) 900μg 900μg 900μg 900μg ._ TABLE 1 (continued) Composition, dose of immunogen and booster schedule for LHRH immunization in ram lambs Immunogen Primary First Second Third Booster Booster Booster Time after birth 10 weeks 18 weeks 26 weeks 34 weeks Tetanus toxoid + KLH + Protein A: Tetanus toxoid 400μg 400μg 400μg 400μg KLH 400μg 400μg 400μg 400μg Protein A 900μg 900μg 900μg 900μg *Carbodiimide used to couple LHRH to carrier protein. 2. Bull calves surgically castrated at 14 weeks of age (n=6) 3. Bull calves immunized against a LHRH-HSA conjugate (n=6) . 4. Bull calves immunized against a LHRH-tetanus toxoid conjugate (n=6) . 5. Bull calves immunized against LHRH-coupled to tetanus toxoid, keyhole limpet haemocyanin and Protein A (n=6) . The composition, dose and immunization schedule for the experiment is outlined in Table 2. For the immunization procedure the immunogen solution was emulsified in an equal volume of Freund's complete adjuvant. For all individual treatment groups a total of 2 ml of the emulsion solution was injected into 4-5 sites. For the combined immunogen group the bull calves were injected into 4-5 sites with 8 ml of the emulsion solution. Injections were given subcutaneously near the front shoulder and the brisket of the calves. For booster injections the immunogens were emulsified using Freund's incomplete adjuvant. Blood samples were collected into heparinized containers at two weeks after the second booster injection. The antibody titres for this experiment are given in Table 4. A test, was developed to study the social and sexual behaviour of the bulls. This involved exposing each bull calf individually to a non-oestrous female for 15 minutes and observing and recording the social TABLE 2 Composition, dose of immunogens and booster schedule for LHRH immunization in bull calves Immunogen Primary First Second Third Booster Booster Booster Time after birth (14 weeks) (22 weeks) (30 weeks) (38 weeks) - Tetanus toxoid -LHRH (Carb)* 360μg 200μg 200μg SOOμg -LHRH (Glut)** 360μg 200μq 200μg 500μg KLH - LHRH -(Carb) 360μg 200μg 200μg SOOμg -(Glut) 360μg 200μg 200μg SOOμg Protein A -LHRH (Carb) 900μg 650μg; . 650μg 1500μg -LHRH (Glut) 900μg 650μg 650μg 1500μg HSA - LHRH -(Carb) 350μg 200μg 200μg 500μg -(Glut) 350μg 200μg 200μg SOOμg Tetanus toxoid-LHRH + KLH-LHRH + Protein A-LHRH: Tetanus toxoid -LHRH (Carb) 360μg 200μg 200μg . SOOμg -LHRH (Glut) 360μg 200μg 200μg SOOμg TABLE 2 (continued) Composition, dose of immunogens and booster schedule for LHRH immunization in bull calves Immunogen Primary First Second Third Booster Booster Booster Time after birth (14 weeks) (22 weeks) (30 weeks) (38 weeks) KLH - LHRH -(Carb) 360μg 200μg 200μg 500μg -(Glut) 360μg 200μg 200μg 500μg Protein A -LHRH (Carb) 900μg 650μg 650μg 1500μg -LHRH (Glut) 900μg 650μg 650μg 1500μg Tetanus toxoid + KLH + Protein A: Tetanus toxoid 360μg 200μg 200μg SOOμg -KLH 360μg 200μg 200μg SOOμg -Protein A 360μg 200μg 200μg 500μg * Carbodiimide used for coupling LHRH to carrier protein. ** Gluteraldehyde used for coupling LHRH to carrier protein. - " TABLE 3 Antibody titres in ram lambs immunized against various LHRH conjugates Carrier Keyhole Tetanus Human Protein A Combined Proteins' limpet toxoid serum » KLH + TT KLH, TT a d and protein A Protein A haemocyanin albumin unconjugatec 1 (KLH) (TT) (HSA) <1:50 <1:50 <1:50 1:2000 1:1400 10 <1;50 <1:50 <1:50 1:1700 <1:50 1:600 <1:50 <1:50 1:1900 1:400 1:200 1:20000 <1 50 1:200 1;6000 <1:50 1:100 1:2500 <1:50 1;100 1:3000 1:2000 <1:50 1:6000 15 Mean 1 • » * • titre <1J50 <1:150 1:2200 1:1050 1:130 1:5146 Range NA <1:50 <1:50- <1;50- <1?50- 1:600- 20 -1:200 1;6000 1:2000 1:200 1:20000 NO . 2/5 3/5 3/4 2/3 6/6 animε.ls responding TABLE 4 Antibody titres in individual bull calves immunized against various LHRH conjugates. Carrier ' Tetanus Human Serum Combined <1:10 1:500 1:170 1:150 <1:10 <1:10 1:180 1:400 10 <1:10 1:25 <1:10 1:30 r <1:10 1:65 <1:10 1:50 <1:10 <1:10 1:10 1:120 <1:10 <1:10 <1:10 1:280 15 Mean <1:10 1:98 1:58 1*170 Animals 0/6 3/6. 2/6 6/6 responding per total animals and sexual behaviour of the animals. The results from this test are presented in Table 5. TABLE 5 Effect of immunization Against LHRH on Social and Sexual Behaviour of Bulls After the Second Booster Injection - TREATMENT Percentage of time in test engaging in: Entire Castrate Comb. TT HSA Social-sexual inter¬ actions of a non- aggressive-nature 1.01 0.31 0.61 0.68 1.41 Sexual interactions 0.61 0.09 0.39 0.48 0.90 Social and sexual investigation 0.72 0.31 0.35 0.46 0.70 Aggressive interaction 1.43 3.83 3.33 2.80 3.50 The antibody titres shown in Tables 3 and 4 for ram lambs and bull calves immunized respectively with various LHRH — conjugates and combinations thereof respectively, are depicted graphically in Figures 1 and 2. In Figure 1, as expected, none of the ram lambs immunized with the carrier protein alone showed any immune response in the assay system. Lambs immunized with a single LHRH conjugate gave variable results in both antibody titre and the number of animals which responded.. With KLH, only 40% of animals responded. The mean titre of this response is 1:130. A similar result was obtained with protein A, where although 66% of animals tested gave an immune response (2 of the animals in the experiment died from pneumonia) , the mean titre of this response was 1:130. The best results using a single conjugate were obtained for tetanus toxoid, (TT) where 60% of animals showed an immune response with a mean titre of 1:2200. The results obtained on immunization with a mixture of LHRH-TT, LHRH - Protein A and LHRH - KLH conjugates illustrate the efficacy of the present invention. All of the lambs tested gave a substantial immune response with a mean titre of 1:5146, with the antibody titre of individuals varying from 1:600 to 1:20,000. These results are clearly superior and are statistically significantly better (p<0.05 on X 2 test of proportion of animals responding) to those obtained using a single LHRH - conjugate. Similar results, including the same statistical significance to the above are show in.Figure 2 where all of the bull calves immunized with a mixture of LHRH coupled to tetanus toxoid, keyhole limpet haemocyanin and Protein A, showed a good immune response with a mean titre of 1:170, this being in •a* contrast with the results obtained with LHRH coupled to a single carrier protein. The behavioural data presented in Table 5 show that control animals spent more time involved in social and sexual behaviour and in social and sexual investigation than animals in the castrate, combination (LHRH individually coupled to KLH, TT and PA) , and tetanus toxoid (TT coupled to LHRH) treatments. As expected, the castrated males spent the least time involved in social and sexual behaviour. The time spent involved in social and sexual behaviour by bulls in the combination treatment was more than that spent by the castrates but was less than that spent by the controls. This suggests that active immunization against LHRH may influence the social and sexual behaviour of the bulls. Although the central animals spent less time involved in aggressive interactions than either the castrates or the combination animals it is encouraging to note that this attribute was similar for the castrates and for the animals in the combination group (it is possible that submissive test animals may be associated with aggressive interactions) . Again, this suggests that the immunization may affect aggression. It is clear that there was a greater effect on the behaviour of the animais in the combination treatment than on the animals injected with tetanus toxoid-LHRH and HSA-LHRH conjugates. Experiment 3 A flock of 80 Merino X Corredale rams at 5 months of age were used in this experiment. The rams (16 weeks of age) were allocated to 8 groups of 10 rams. One group were surgically castrated at 16 weeks and another group were left as entire animals. The remaining 60 rams were treated with a number of LHRH antigens. Various LHRH - carrier protein conjugates were prepared as described previously. The antigens used were LHRH coupled separately to diphtheria toxoid, tetanus toxoid, keyhole limpet haemocyanin and corynebacterium parvum. Two other groups were injected with two different combinations of the individual LHRH complexes. Treatments are shown in Table 6, together with the total amounts of conjugates used. Rams were injected subcutaneously in the neck with these single LHRH carrier proteins using Freund's complete adjuvant. Eight weeks following the booster injection pen tests were carried out on all animals to determine w h et h er they were sexually active. This involved presenting each male with 4 oestrous females in an arena (5m X 5m) for 15 minutes and recording the bouts of anogenital investigation, bouts of courtship, number of mounts and number of ejaculations performed by the males. Six males were observed concurrently and all the animals were observed on the same day. The animals were k ept in the testing arenas for 12 hours before the pen tests to allow them to become familiar with the testing situation. An animal was considered to be sexually active i f he mounted and/or ejaculated during test. TABLE 6 TREATMENT AND DOSE RATES Group No. of Treatment Dose Ewes. I 1 0 Control 2 1 0 Castrate 3 1 0 LHRH- (TT) Img 4 1 0 LHRH- (DT) Img 5 10 LHRH- (CP ) 1mg 6 10 LHRH-( LH ) 1mg (method 1 ) 7 10 LHRH-DTT + 8 10 LHBH-DT + ; 3mg TABLE 7 LIVE EIGHT CHANGES IN CONTROL CASTRATE AND TREATED LAMBS Treatment Time of testing; Group 0 14 wks 1 wles 21 wks 27 wks Control 40.7 54.2 48.4 51.0 210.0 Castrate 40.6 49.6 43.1 46.4 - LHRH - TT 39.2 54.2 45.4 47.5 146.1 LHRH - DT 39.3 53.9 45.6 48.7 83.3 LHRH - CP - 39.3 55.8 46.8 49.3 171.0 LHRH - KLH 38.7 55.3 46.3 48.7 153.3 LHRH-TT+DT+KLH 36.3 54.7 46.5 48.6 71.0 LHBH-DT+CP+KLH 38.7 55.5 44.9 48.6 55.0 Liveweights and testicular size (as determined using a series of calibrated beads) was measured every 4 weeks. Antibody titre to LHRH were measured by both radioimmunoassay (RIA) and ELISA techniques. Plasma samples were serially diluted with phosphosaline buffer (o.OSM, pH 7.6). The diluted samples (100 ul) were added to 100 ul bovine gamma globulin (3V 9w/v) and 100 ul of a buffer solution containing 2nCi 125I-LHRH. The tubes were incubated overnight at 4°C. Free and bound LHRH were separated by adding 1 ml 21% polyethylene glycol 4000 and aspirating the supernatant after centrifugation. The tubes were counted for 1 minute in a clinigamma counter. Liveweights of all rams are shown in Tab l e 7. There were no significant differences among groups although the mean liveweight values for the immunise d rams were higher than the castrated males but lowe than the control rams. The mean testicular volumes for eac h treatment are shown in Table 8 and Figures 3 an d 4. There was considerable variation with each individual antigen treatment. The largest decrease for individual carriers was observed in the rams immunised against LHRH coupled to diptheria toxoid. The next best carriers were tetanus toxoid follwed by keyhole limpet haemocyanin and corynebacterium parvum. The two different combinations of 3 separate antigens gave lower testes volumes than each of the individual antigens. A similar trend in results was observed for the RIA results. The median titres were highest in the two groups with the combined antigens, followed by diphtheria toxoid, tetanus toxoid, keyhole limpet haemocyanin, corynebacterium parvum. The individual RIA and ELISA IgC results are shown in Table 9. TABLE 8 TESTICULAR VOLUME IN CONTROL, CASTRATED AND IMMUNISED RAM LAMBS Treatment 1rime of testing Group 0 14 wks 17 wks 21 wks 27 wks Control 160.0 168.0 183.5 187.0 210.0 Castrate - - - - - LHRH - TT 155.0 135.6 108.3 111.1 146.1 LHRH - DT 150.0 101.7 73.3 59.1 83.3 LHRH - CP 144.0 149.5 162.8 170.5 171.0 LHRH - KLH 153.3 133.5 136.1 127.2 153.3 LHRH-TT+DT+KLH 138.9 81.1 62.2 56.0 71.0 LHRH-DT+CP+KLH 141.1 77.5 67.8 48.3 55.0 TABLE 9 ANTIBODY LEVELS IN RAMS, LAMBS AS MEASURED BY ELISA AND RIA ASSAYS TABLE 9 (CONTINUED) 775 - 128 256 1:28,200 737 32 64 64 1:3,780 738 32 8 16 1:1,320 739 64 256 64 1:2,940 741 8 32 0 1:1,020 742 0 8 64 <1:600 743 0 0 16 <1:600 744 0 0 2 <1:600 745 4 0 2 <1:600 746 0 0 4 <1:600 747 0 0 0 <1:600 748 16 16 32 <1:600 749 - 0 0 4 <1:600 750 - 0 4 <1:600 752 2 8 8 <1:600 721 — 256 256 754 64 64 32 1:1,320 755 0 8 - 756 8 16 16 <1:600 757 16 8 8 <1:600 758 4 16 16 <1:600 759 0 8 0 <1:600 760 16 32 16 1:2,160 761 64 128 1024 1:5,400 762 — 64 16 1:900 783 16 64 64 1 :10,800 1910 16 64 128 1:7,200 785 32 32 32 1:2,400 786 - 128 128 1:9,900 787 64 32 128 1:41,900 788 64 32 128 1 :3,660 1864 0 0 0 1:600 790 16 0 4 <1:600 791 - 32 • 8 4 1 :1 ,200 792 32 32 512 1:27,600 793 16 64 64 1:5,600 794 - 512 256 1:3,660 795 4 8 32 1:1,800 796 0 128 128 1:12,600 797 512 256 256 1:22,200 798 8 32 32 1 :9,000 800 32 32 32 1:1,440 772 4096 1024 256 1 :44,000 TABLE 1 0 EFFECT OF ACTIVE IMMUNISATION AGAINST LHRH ON THE PROPORTION OF ANIMALS MOUNTING AND/OR EJACULATING Treatment Proportion of Animals Mounting Group and/or ejaculating Control 10/10 Castrate 0/9 LHRH - TT 2/9 LH H - DT . 1/9 LHRH - CP 5/9 LHRH - KLH 3/10 LHRH - TT + DT + KLH 0/.10 LHRH - DT + CP + KLH 0/8 In general those rams with high RIA titres had elevated titres as measured by the ELISA assay. However discrepencies were found in individual rams, in some instances high RIA titres were found in samples with low IgC titres. These results suggest that the RIA assay is monitoring a higher number of antibody subclasses than the more specific IgG ELISA assay. The proportion of rams in each group that were sexually active at 2 months after the booster injection are shown in Table 10. Ten out of a total of 10 rams in the control group were showed sexual activity. The best single antigen was diphtheria toxoid with 1 of 9 rams sexually active. The order of effectiveness was diphtheria toxoid, tetanus toxoid, keyhole limpet haemocyanin and corynebacterium parvum. The two groups with the combined antigens were not sexually active. For those rams given a combination of TT, DT and KLH coupled to LHRH there were no rams active out of a total of 10 rams. Similar results were obtained using a different combination of carriers, DT CP and KLH where again there were no rams active out of a total of 8 rams (two rams died during the trial). None of the castrated rams were sexually active. These results indicate that a combination of various carrier protein gives a better physiological response than each individual antigen given separately. This effect was observed for testicular size, RIA titres and for the proportion of animals that were sexually active. Overall these results indicate combinations of LHRH antigens result in a better immune response compared with individual antigens. Other aspects of the present invention and modifications and variations thereto, will become apparent to those skilled in the art on reading this specification, and all such other aspects and modifications and variations are considered as included within the scope of the present invention. 32 REFERENCES Ariumura et al. (T974) - Blockage of the preovulatory surge of LH and FSH and ovulation by anti-LHRH serum in rats. Endocrinology 95:323-325. Chappel et al. (1980) — Active immunization of male rhesus monkeys against luteinizing hormone releasing hormone. Biology of Reproduction. 22:333-342. Clarke et al. (1978) - Active immunization of ewes against luteinizing hormone releasing hormone, and its effect in ovulation and gonadotrophin, prolactin and ovarian steroid secretion. de al Cruz et al. (1976) - Effect of administration of anti-serum to luteinizing hormone-releasing hormone on gonadal function during the oestrous cycle in hamster. Endocrinology 98:490—497. Fraser et al. (1974) — Effect of active immunization to luteinizing hormone releasing hormone on serum and pituitary gonadotrophϊns, testes and accessory sex organs in the male rat. J. Endocr. 63:399-406. Fraser et al. (1978) — Changes in the ovaries of rats after immunization against luteinizing hormone releasing hormone. J " . Endocrinology 77:85-93. Fraser et al. (1982) - In: Progress Towards a Male Contraceptive, (pp. 41-78 Ed. Jeffcoate, S.K., and Sandier, M. John Wiley & Sons, Bristol). Herbert, W.J. - In: Veterinary Immunology (Blackwell) Scientific, Pub.). Hodges and Hearn (1977) - Effect of immunization against luteinizing hormone release hormone on reproduction of the mamoset monkey. Nature, 265:746-748. Jeffcoate et al. (1982) - Effects of active immunization of ram lambs and bull calves against synthetic luteinizing hormone releasing hormone. Theriogenology, 18:65-77. Kock (1977) - In: Proceedings of the 5th International Congress of Endocrinology (pp. 374-378. Ed. by James, V.H.T. , July 18-24) . McCormack et al. (1977) - The effect of luteinizing hormon releasing hormone (LHRH) antisera administration of gonadotrophin secretion in the rhesus monkey. Endocinology 100:663-667. Nieschlag and Kleg (1974) - Loss of sexual activity in rabbits actively immunized with testosterone. Experimentia 30, 434,435. Robertson et al. (1970) - Affect of castration and dietary protein level on growth and carcass composition in beef cattle. Journal of Agricultural Science (Cambridge 74: 299-310). Robertson et al. (1979) - Immunological castration in male cattle. Vet. Rec. 105:556-557. Robertson et al. (1981) - Further studies on immunological castration in male cattle. Vet. Rec. 111 :529-531. Robertson et al. (1 84) - Iπrmunological castration of young bulls for beef production. In "Manipulation of growth in farm animals" (Eds. Roche, J.F. and D'Callaghan, D. ) pp. 137-145. Schanbacher (1982) — Responses of ram lambs to active immunization against testosterone and luteinizing hormone releasing hormone. J.Physiol. 242E, 201—205. Schanbacher (1984) - Active immunization against LHRH in the male. In 'Immunological Aspects of Reproduction in Mammals", pp. 345—362. Ed. D.B. Crighton. Butterworths.

Claims: CLAIMS 1. A composition for the production in animals of antibodies specific for LHRH, said composition comprising two or more different carriers individually coupled to LHRH or analogues of LHRH, in amounts sufficient to elicit an immune response against LHRH. 2. A composition as claimed in claim 1, wherein at least two of the carriers are carrier proteins. 3. A composition as claimed in claim 2 wherein at least one of the carrier proteins is Diptheria toxoid. 4. A composition as claimed in any one of claims 1—3 wherein the composition further comprises an adjuvant. 5. A composition as claimed in claim 4 wherein the adjuvant selected is, such the composition causes no inflammation. 6. A method for the suppression of male social and sexual be avour of domestic animals, comprising the administration of a composition as claimed in any one of claims 1 to 5. 7. A method for the suppression of ovulation and oestrous cyclicity of female domestic animals comprising the administration of a composition as claimed in any one of claims 1 to 5. 8. A method as claimed in claim 6 or wherein there are two or more administrations of said compositions. 38 9. A method as claimed in any one of claims 6 to 8, where the animals are selected from dogs, cats, cattle, sheep, donkeys, rabbits and deer. 10. A composition for the production in animals of antibodies specific to a substance which when conjugated to a carrier is capable of eliciting an immune response in the animal wherein the composition comprises a mixture of the substance conjugated to at least two different carriers.

Inventors: FAIRCLOUGH ROBERT JOHN, TILBROOK ALAN JOHN, FAIRCLOUGH, ROBERT, JOHN, TILBROOK, ALAN, JOHN
Applicants: VICTORIA STATE, THE STATE OF VICTORIA
Application year: 1987
Main classification: A61K37
IPC codes: A61K38, A61K39, C07K7