AU602187B2 - Anti-reproductive hormone - Google Patents
Anti-reproductive hormone Download PDFInfo
- Publication number
- AU602187B2 AU602187B2 AU11017/88A AU1101788A AU602187B2 AU 602187 B2 AU602187 B2 AU 602187B2 AU 11017/88 A AU11017/88 A AU 11017/88A AU 1101788 A AU1101788 A AU 1101788A AU 602187 B2 AU602187 B2 AU 602187B2
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- Prior art keywords
- lhrh
- gly
- peptide
- tyr
- conjugate
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- 230000003442 weekly effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/23—Luteinising hormone-releasing hormone [LHRH]; Related peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0006—Contraceptive vaccins; Vaccines against sex hormones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Description
la~ia~p aD nssaa PCr AU-AI_1-101 /88 r~ O WORLD [NTELLECTuAwP 0 NIZA1O Inten 8 7 a. INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 88/ 05308 A61K 39/385, C07K 7/06 Al (43) International Publication Date: 28 July 1988 (28.07.88) (21) International Application Number: PCT/AU87/00448 (22) International Filing Date: 30 December 1987 (30.12.87) (31) Priority Application Number: PH 9868 (32) Priority Date: (33) Priority Country: 14 January 1987 (14.01.87) (71) Applicant (for all designated States except US); COM- MONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION [AU/AU]; Limestone Avenue, Campbell, ACT 2601 (AU).
(72) Inventors; and Inventors/Applicants (for US only) RIGBY, Robin, Deshon, Gerald [AU/AU]; 43 Singles Ridge Road, Winmalee, NSW 2777 HUYNH, Van, Long [AU/AU]; 22 Becky Avenue, North Rocks, NSW 2151 HOSKINSON, Ronald, Milton [AU/AU]: 28 Carcoola Crescent, Normanhurst, NSW 2076 (AU).
MATTNER, Phillip, Edward [AU/AU]; 1 Rose Street, Epping, NSW 2121 (AU).
(74) Agent: F.B, RICE CO.; 28A Montague Street, Balmain, NSW 2041 (AU).
(81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (European patent), FR (European patent), GB (European patent), IT (European patent), JP, LU (European patent), NL (European patent), SE (European patent),
US.
Published With international search report.
aL5R 15 SEP 1988
AUSTRALIAN
10 AUG 1988 PATENT OFFICE i Ck4-J<- J A* I 't ai' i iiC ~d i otn j (54) Title: ANTI REPRODUCTIVE HORMONE (57) Abstract Immunoneutering of mammalian animals is achieved by vaccinating the animals with a composition comprising an immunogenic protein such as bovine serum albumin, conjugated with a peptide selected from the group comprising any continuous 5, 6 or 7 amino-acid fragment of the decapeptide pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 (being lutenising hormone releasing hormone) and a suitable immunoadjuvant such as diethylaminoethyl dextran, emulsions of diethylaminoethyl dextran in a mineral oil, The peptide fragments containing the pGlu from the N terminal t.r the Gly-
NH
2 from the C terminal of the decapeptide are preferred for use in such immunogenic compositions.
ii I WO 88/05308 PCTA87/00-40a 13 WO 88/05308 PCT/AU87/00448 ANTI REPRODUCTIVE HORMONE TECHNICAL FIELD This invention deals with immunological methods that may be used to block or suppress sexual activity in animals and with vaccines that induce specific kinds of hormone autoimmunity to achieve the aims of the invention.
BACKGROUND ART A large body of knowledge exists concerning the endocrine factors that act together to regulate the sexual activity of both male and female mammals. At one level of regulation, the decapeptide comprising mammalian luteinizing hormone releasing hormone (hereafter designated LHRH(1-10)) is secreted from the hypothalamus of the brain and acts at the pituitary gland to cause the pituitary gland in *urn to secrete the well known gonadotrophic hormones, luteinizing hormone (LH) and/or follicle stimulating hormone (FSH). The gonadotrophins then exert important biological actions at the level of the gonads to cause these glands to secrete oestrogenic hormone in the female particularly, and androgenic hormones in the male particularly.
Finally, the oestrogenic and the androgenic hormones participate in feedback mechanisms on the brain tu regulate their own secretion so that only those concentrations of these hormones that are allowed by homeostasis occur in the circulation of animals. Both oestrogens and androgens are known to act biologically at the level of the central nervous system to regulate sexual behaviour in both male and female animals. They may also act at peripheral sites to affect such important processes as growth rate and nutrient partitioning between muscle and adipose tissue. The important relevant consideration in this complex series of biological processes is that the secretion of gonadal oestrogens or androgens is ultimately under the control of LHRH (1-10).
i SUSTITlUTE
SHEET
WO 8/Pi308 PCT/AU87/00448 2 It is well known that when animals are made immune to LHRH the sequence of endocrine events that causes the gonads to secrete either oestrogen or androgen is blocked and, in consequence, sexual activity is blocked and sex-hormone-dependent physiological process are either blocked or suppressed.
This occurs because LHRH (1-.10)-specific antibody provoked in the immune response binds to endogenous LHRH (1-10) and prevents or inhibits the decapeptide from binding to its receptors and expressing its biological activity. The term "immunocastrate" has been coined to describe the physiological state of LHRH-immune animals because of the similarity with surgically castrate animals residing in the inability of the former to express sexual behaviour. A better term might be "immunoneutered" to take account of the fact that LHRH (1-10) specific antibody will block sexual activity in both male and female mammals.
Various attempts have been made to develop commercial anti-LHRH (1-10) vaccines with the objective of achieving an immunological method of controlling sexual activity in animals.
In the prior art, such anti LHRH (1-10) vaccines have been prepared by chemically conjugating LHRH (1-10) to an immunogenic protein and formulating the immunogenic conjugate so formed together with an immunoadjuant.
Nevertheless for numerous perceived applications of such a vaccine the cost of synthesising the LHRH (1-10.) decapeptide has been a seriously limiting factor partly explaining the slow pace of technological development in this area. This invention is concerned, with discoveries that substantially simplify the chemical nature of the immunogenic conjugate necessary to stimulate an anti LHRH (1-10) antibody response and that enhance the scope for 3, anti LHRH (1-10) vaccines to be applied to the 3 immunoneutering of animals.
DISCLOSURE OF THE INVENTION The present invention consists in a composition for use in the immunoneutering of mammalian animals comprising a peptide conjugated with an immunogenic protein, the composition being characterised in that the peptide includes at its free end a sequence selected from the group comprising any contiguous 5, 6 or 7 amino acid fragment of the decapeptide pGlu-His-Trp-Ser-Tyr-Gly-Leu- Arg-Pro-Gly-NH 2 As hereinafter defined, the word contiguous refers to any specific 5, 6 or 7 amino acid fragment wherein the amino acids occur in the same order of sequence as they occur in the decapeptide identified in the preceding paragraph.
15 In another aspect the present invention consists in a vaccine for immunoneutering mammalian animals comprising a composition according to this invention together with an immunoadjuvant.
In a still further aspect the present invention consists in a method for the immunoneutering of male or female mammalian animals comprising administering to the .animal an effective amount of vaccine according to the present invention.
For the purpose of this invention LHRH (1-10) is defined to be the decapeptide represented by the amino acid sequence: 9 pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 1 2 3 4 5 6 7 8 9 Wherein the symbols, according to convention, represent YI thai amino acids as follows: pGlu (pyroglutamic acid), His (histidine), Trp (tryptophan), Ser (serine), Tyr (tyrosine), Gly (glycine), Leu (leucine), Arg (arginine), Pro (proline). Likewise, according to convention, pGlu is located at the N terminus of the decapeptide and Gly-HN 2 locilted at the C terminus, it being noted that at the N -3a terminus the cyclic form of the pyroglutamic acid means that there is no free amino function at that terminus and at the C terminus, amidation of the glycinie moiety means that therj is no free carboxyl function at that terminus.
*oaf 4, Ag. lf _ICC _I WO 88/05308 PCT/AU87/00448 4- Except for glycine which is not optically active, all amino acids are of the L configuration. The numbering of the amino acids in the docapeptide as shown above is as commonly used in the art,, that is, pGlu, at th4 N terminus is designated the first aimino acid residue rith subsequent numbering proceeding sequentially toward the C terminus.
Throughout the description of this invention the symbol, LHRH where x and y are numbers chosen from 1 to 10, is used to represent the parent LHRH decapeptide or a designated LHRH fragment peptide. The values of x and y define the first and last amino acids in the peptide sequence. Thus, for example the peptide LHRH is composed of the amino acids numbered 1 to 7 in the above formula.
The peptide fragments that can be used to form the novel peptide: protein conjugates of the invention and that can thus become haptens in such conjugates are comprised of any five or any six or any seven amino acids linked as they occur contiguously in endogenous mammalian LHRH (1-10) with or without an additional amino acid or sequence of amino acids acting to link the peptide to the immunogenic protein.
Two kinds of knowledge in the prior art beat: on the present invention. On one hand it was known from in.vitro measurements that antibodies raised against small chemically synthesised peptides are capable of binding to large protein molecules where such peptide sequences occur at conformationally mobile sites. On the other hand, in the particular case of LHRH itself a relatively small hormonal peptide rather than a protein, there are numerous examples of penta, hexa or heptapeptide fragment sequences of that molecule that in vitro will not bind or will only poorly bind to antibodies specifically raised against LHRH see for example the following: Arimura et al, Acta Endocrinologica, 1975, I8, 1 WO 88/05308 PCT/AU87/00448 222-231; Nett et al, Journal of Clinical Endocrinology and Metabolism, 1973, Vol. 36, No. 5, p.880-885; Koch et al, Biochemical and Biophysical Research Communications, 1973, Vol. 55, No. 3, p. 616-622; Jeffcoate et al, Immunochemistry 1974, 11, 75-77; Pique et al, Immunochemistry 1978, 15, 55-60.
This fact suggests that such peptides do not frequently adopt the conformation of LHRH (1-10) that provoked the LHRH (1-10) specific antibodies. While contradictory in vitro studies of this kind in the prior art pose interesting questions about the nature of peptide and protein folding, they throw little light on the biological consequence that might ensure in vivp when the LHRI (1-10) hormone molecule is confronted with the choice of binding either to its natural substrate, the LHRH receptor protein, or to an invoked antibody molecule having a specificity that is not primarily directed toward LHRH (1-10) but to a fragment thereof.
In these circumstances where LHRH (1-10) is subject to competitive binding between two possible substrates and where the relative binding affinities to the two substrates may differ in an unknown manner, the new art provides that its particular antibodies cross react with LHRH (1-10) in such a manner that effective concentrations of the hormone are denied to its endogenous receptor and LHRH (1-10) mediated biological p'ocesses are blocked.
While in the prior art (USP 4608251) it was known that a particular nonapeptide and a particular decapeptide, each bring derived from the single octapeptide LHRH (3-10) could be used to form immunogens that would induce mammals to produce antibodies that react with LHRH the surprising discovery of the present invention is the diversity of much smaller peptides that can be used directly as haptens to achieve the same WO 88/05308 PCT/AU87/00448 6 effect. The recognition of this diversity greatly increases opportunities for the design and manufacture of anti LHRH vaccines and simultaneously provides substantial improvements in the cost: benefit ratio of such vaccines.
The diversity extends to peptides that are primarily representative of the N-terminus or of the C terminus or of the mid molecule structure of the parent LHRH (1-10) molecule. Individual anti LHRH vaccines might be formed by using any of the nominated penta, hexa or heptapeptides but generally preferred peptides are those that retain the pyroglutamic acid at the N-terminus or that retain the glycine amide at the C-terminus. A good example of a preferred peptide is the heptapeptide comprising LHRH This substance combines the properties of a relatively cost-effective synthesis, ease of conjugation to immunogenic proteins and high biological potency when used as hapten.
The novel immunogens of the invention are formed by chemical conjugation of the designated peptides to any of the immunogenic proteins known in the art, -xemplified by but not limited to serum albumins, thyroglobulin, ovalbumin, gelatin, haemocyanin, serum globulin and the like. The chemical processes used to form the immunogenic conjugates may be any of those known in the art to be capable of inducing a covalent bond between the peptide and protein including the use of water-soluble carbodiimide reagents, solvent-soluble carbodiimide Sreagents particularly in combination with N-hydroxysuccinimide or N-hydroxybenztriazole, and the use of glutaraldehyde or alkyl and aryl diisocyanates. Those skilled in the art of peptide to protein conjugation chemistry will recognise that conjugation to proteins of various of the peptides designated in this invention will be facilitated if they be chemically modified at either end with reagents such as the amino acid cysteine to
II
i -r i WO 88/05308 PCT/AU87/00448 -7 introduce thio! functionality or the amino acid lysine to introduce amino functionality. Immunogens formed by the device of firstly adding an extra chemically reactive amino acid, or a group of amino acids terminating in such a reactive amino acid, to either N or the C terminus of the designated peptides and then using conjugation procedures to link such peptides to carrier proteins fall within the scope of the invention. Likewise, designated peptides having a free amino terminus may be linked to thiol-containing proteins by first reacting them with reagents capable of introducing an activated carbon to carbon double bond into the peptide. Such reagents are exemplified by N-succinimidyl-3-maleimido benzoate and immunogenic peptide:protein conjugates formed by this device fall within the scope of the invention. The essential feature of the immunogens claimed herein is that they are proteins bearing the designated peptides as chemically-linked haptens and formed by any of the procedures known in the art. The vaccines of this new art are comprised of any of its novel immunogens used in combination with an immunoadjuvant including typically but not limited to polycationic and polyanionic polyelectrolytes, alhydrogel, mineral oil emulsions or combinations thereof particularly emulsions of a polyelectrolyte and a mineral oil. The vaccination methods of the invention are any of those known in the art to confer anti-hapten immunity on vaccinated animals.
The amino acid sequence of LHRH (1-10) is a highly conserved peptide sequence between classes of mammals and, accordingly, the vaccines of the invention are capable of inducing immunoneutering of male and female animals among a wide class including those that may be farmed for fibre, meat, skin or milk, or among those animals that are widely regarded as companion animals. Whilst, in the first instance dependent upon the formation of anti LHRH (1-10)- WO 88/05308 PCT/AU87/00448 8 binding i tibodies, the biological consequences of immunoneutering livestock are many and diverse and include the prevention of estrous behaviour, prevention of ovulation, suppression or fertility, involution of adult testes, suppression of male libido, prevention or suppression of testicular development in growing prepubertal males as well as the suppression of gonadal sex stojroid secretion. It is shown herein that the vaccines of this new art, using its novel immunoglens, are capable of achieving all these effects thereby creating an opportunity for a new generation of anti LHRH vaccines with diverse applications. The peptides used in the following examples to illustrate this invention are: LHRH comprising pGlu-His-Trp-Ser-Tyr-OH LHRH comprising pGlu-His-Trp-Ser-Tyr-Gly-OH LHRH comprising pGlu-His-Trp-Ser-Tyr-Gly-Leu-OH LHRH comprising H-His-Trp-Ser-Tyr-Gly-Leu-Arg-OH LHRH comprising H-Trp-Ser-Tyr-Gly-Leu-Arg-OH LHRH (4-10) comprising H-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 LHRH (5-10 comprising H-Tyr-Gly-Leu-Arg-Pro-Gly-HN 2 Symbols used herein to represent the immunogenic conjugates of the invention are illustrated, for example, by LHRH (1-5):HSA or LHRH (3-8):BSA wherein the former is intended to represent a chemical conjugate of LHRH with human serum albumin and the latter to represent a chemical conjugate of LHRH with bovine serum albumin. Those skilled in the art will recognise that such symbols fail to represent the fact that depending on Sthe carrier protein may be possible to conjugate as little as 1 and as many as about 50 molecules of LHRH-fragment peptide to that carrier protein, the exact number being an expression of the epitope density when the epitope is the LHRH fragment peptide. This invention places no limits on the epitope density to be achieved in the immunogenic conjugates of its methods and processes, -7 I WO 88/05308 PCT/AU87/00448 4 -9although it is already well known in the art that the epitope density can influence the immunological properties of the conjugate.
To achieve the biological effects of immunoneuting livestock it is necessary to cause them to produce antibodies that bind to LHRH (1-10) and thereby block its biological activity. A common procedure for demonstrating the presence of such antibodies in the sera of immune animals is to perform an in vitro measurement of the anti LHRH (1-10) antibody titre. Such measurements can be used to confirm the presence or absence of antibodies that bind with LHRH (1-10).
It would be expected from the prior art that the novel vaccines of the invention would be cable of provoking anti peptide antibodies when the peptide is any of the peptide fragments designated herein. That property alone would not be expected to achieve the biological effects of immunoneutering because none of the designated peptide fragments are known to possess the biological activity of LHRH It is a feature of the invention that the antibody provoked by its vaccines produce, in addition to anti-peptide fragment antibodies, anti LHRH (1-10) antibody titres as well that are frequently of the same order of magnitude as those which can be achieved when LHRH (1-10) itself is used as hapten. Because of this fact the biological effects caused by the novel vaccines can be understood, In general there will be four major influences that determine the outcome of an immunoneutering vaccination with the vaccines of the invention. These will be: the sex of the target animal; (ii) the species of the target animal; (iii) the particular peptide fragment used to formulate the vaccine; and (iv) the magnitude of the anti LHRH (1-10) antibody titre provoked by the vaccine.
The invention recognizes that, to most efficiently achieve WO 88/05308 PCT/AU87/00448 the biological effects of i.munoneutering of a given sex, either within or between species, it may be necessary to optimize both the nature of the peptide fragment used in vaccine preparation and the level of anti LHRH (1-10) antibody titre it provokes, both these last two variables being subject to experimentation.
Examples 1-10 illustrate procedures for the formation of the immunogenic conjugates of the new art using a well known water soluble carbodimide reagent to effect chemical condensation of designated peptides with carrier proteins in an entirely aqueous reaction procedure.
Examples 11-13 illustrate procedures for the formation of the immunogenic conjugates of the new art using a solvent-soluble carbodiimide reagent to e~ffct peptide:protein conjugation in a mixed solvent system.
Example 14 describes the method used for the measurement cf anti LHRH (1-10) antibody titre in the plasma of vaccinated animals.
Example 15 illustrates two kinds of new art vaccines that are effective in inducing anti LHRH (1-10) antibodies in vaccinated animals.
In Examples 16-19 and Table 1 it is shown that the new art of the invention is effective in inducing anti LHRH (1-10) antibodies and in blocking the oestrous cycles of immuno neutered Merino ewes.
Examples 20 and 21 together with Tables demonstrate that the new art in comparison with prior art is effective with adult Merino rams including the induction of anti LHRH (1-10) antibodies, reducing the serving capacity, causing involution of the testes and diminishing the plasma testosterone concentration of immunoneutered rams.
Examples 22-25 together with Tables 6-9 demonrtrate that the new art is effective with mice in provoking anti LHRH (1-1C) antibodies, in blocking both male and female WO 88/05308 PCT/AU87/00448 11 fertility and in suppressing plasma testosterone concentration in vaccinated males..
Examples 26-30 and Table 10 demonstrate that the new art is effective with growing immunoneutered crossbred ram lambs in provoking anti LHRH (1-10) antibodies and in suppressing prepubertal testicular development.
Examples 31-33 and Table 11 demonstrate that the new art is effective with immunoneutered, adult, seasonally anoestrous ewes in provoking anti LHRH (1-10) antibodies and in suppressing both the oestrous and ovulatory response that accompanies the sudden introduction or rams into a flock of seasonally anoestrous ewes.
SExample 34 and Table 12 demonstrate that with immunoneutered female cattle, the new art is effective in inducing anti LHRH (1-10) antibodies, in suppressing oestrous behaviour and, as can be inferred from the suppressed concentrations of plasma progesterone, it is Seffective in suppressing ovulatory capability. These i examples illustrate the scope of the invention that extends to all mammals whose reproductive activity is regulated by LHRH (1-10).
Throughout the following examples given to illustrate the invention the values of measured anti LHRH (1-10) antibody titre are presented merely to co Bt such antibodies are in fact formed in response v in- ination.
Where mean values for experimental variablea presented in the Tables in the form a t b, (a and b being numbers) these values represent treatment group mean values plus o minus the standard error of the mean.
EXAMPLL.1 Preparation of an LHRH (1-5 Human Serum Albumin Conjugate with a Water Soluble Carbodiimide co donsina Reagent LHRH (100mg) was dissolved in deionized water ml) and added to a stirred solution 6f human serum albumin (HSA, CQO dissQlved in detanied water
W-
h I WO 88/05308 PCT/AU,87/00448 12 ml). To the combined solutions at ambient temperature was added ethyldimethylaminopropyl carbodiimide hydrochloride (1,0 g) which immediately dissolved. The solutior was immediately adjusted to pH 6.5 and maintained at pH 6.5 by the metered addition of sodium hydroxide solution (4 Mol 1 1 for 5 h. The product was dialysed against 2 litres of deionized water (3 changes per day fox 4 days). Finally, lyophilization yielded the immunogenic
I
LHRH (1-5):HSA conjugate.
EXAMPLE 2 An example according to Example was used instead of LHRH EXAMPLE 3 An example according to Example was used instead of LHRH EXAMPLE 4 An example according to Example was used instead of LHRH EXAMPLE An example according to Example was used instead of LHRH EXAMPLE 6 An example according to Example was used instead of LHRH EXAMPBL 7 An example according to Example 1 wherein LHRH (1-6) 1 wherein LHRH (1-7) 1 wherein LHRH (2-8) 1 wherein LHRH (4-10) 1 wherein LHRH (5-10) 5 wherein bovine serum albumin (BSA) was used as carrier protein instead of human serum albumin.
EXAMLB.
An example accirding to Example 3 wherein bovine serum albumin was used instead of human serum albumin.
EXAMPLE 9 An example according to Example 4 wherein bovine serum albumin was used instead of human serum albumin.
ii I WO 88/05308 PCT/AU87/00448 13 An example according to Example 1 wherein LHRH (3-8) was used instead of LHRH and bovine serum albumin was used insted of human serum albumin.
EXAMPLE 11 To LHRH (100 mg) was added with stirring a solution of N-hydroxy succinimide (50 mg) in dry dimethylformamide (1.1 ml). To the resulting solution was added a solution of dicyclohexylcarbodiimide (140 mg) in dry dimethylformamide (0.7 ml) and the mixture allowed to stand at ambient temperature for 3 hours. To the solution was then added with stirring and cooling a solution of human serum albumin (HSA, 100 mg) in phosphate buffer (0.05 Mol 1- 1 pH 7.8, 4 ml). The pH was immediately readjusted to 7.5 with saturated sodium carbonate solution and the mixture kept for 16 hours at ambient temperature.
The reaction mixture was then diluted with water, centrifuged to remove insoluble products and the supernatant liquid extensively dialysed against deio ized water (10 litres, 3 changes per day) for 4 days and then lyophilized to yield the immunogenic LHRH conjugate.
EXAMPLE 12 An example according to Example 11 wherein LHRH (1-6) was used instead of LHRH free acid.
EXAI1PLE.13 An example according to Example 11 wherein LHRH (1-7) was used instead of free acid.
EXAMPLE 14 Measurement of Anti LHRH (1-10) Antibody Titre by Radioimmunoassay Anti LHRH (1-10) antibody titre was determined in an assay similar to that reported by G.E. Abraham in Acta Endocrinologica, Volume 75, Supplement 183, Pages 7-42 (1974) for steroid specific antibodies. Plasma was I i WO 88/05308 PCT/AU87/00448 14 serially diluted with sodium phosphate buffer (0.05 Mol 1 pH 7.4) containing gelatin sodium chloride and sodium azide To 0.1 ml of diluted plasma was added radiolabelled I1' 2 -LHRH (1-10) (3.7 TBq/mole, 10000 dpm) in 0.9 ml phosphate buffer. The mixture was kept at 4 0 C for 16 h after which was added a suspension of dextran-cCated charcoal (0.2 ml consisting of 2% (w/v) decolourizing charcoal (Ajax Chemicals Pty. Ltd.) suspended in phosphate buffer containing 0.1% (w/v) dextran T-70 (Pharmacia Pty. Ltd). The solution was allowed to equilibrate for 1 h with the added charcoal to allow adsorption of the free radioligand and was then centrifuged (1000 g, 15 min) to pellet the charcoal. The supernatant solution was removed by suction and the radioactivity on the charcoal pellet measured by gamma counting. The antibody titre is defined as the dilution of antiserum which binds 50% of the I125-LHRH (1-10) available and is expressed as the reciprocal. The between assay coefficient of variation was 16%.
EXAMPLE Typ~e 1 Vaccines SThe type 1 vaccines used in the examples given herein were formed by dissolving 1 mg of the immunogenic LHRH fragment:HSA conjugate in physiological saline (1 ml) and I 25 emulsifying with Freund's complete adjuvant (1 ml).
Type 2 vaccines The type 2 vaccines used in the examples given herein were formed by dissolving 1 mg of the immunogenic LHRH fragment:HSA conjugate in 0,6 ml of 20% DEAE-dextran (diethylaminoethyldextran) and emulsifying with 1.4 ml of a solution of the oil-soluble surface active agent Arlacel (1 part v/v) Jn a mineral oil (5 parts v/v).
In both Type 1 and Type 2 vaccines the LHRH fragments can be comprised of any of those designated LHRH fragment peptides that can be used to make the immunogenic
I«
WO 88/05308 PCT/AU87/00448 conjugates of the invention.
EXAMPLE 1I Effec' on Anti LHRH (1-10) Antibody Titre and on the Oestrous Cycles of Adult Merino Ewes Following Vaccination aainst LHRH (1-1Q) HSA The following is an example of prior art, presented to allow a comparison with the new art of the invention.
A group of nine adult Merino ewes were vaccinated against LHRH (1-10) using a LHRH (1-10):HSA conjugate in a Type 1 vaccine (Example 15). Vaccination was comprised of a primary treatment given as a 1 ml injection, intramuscularly to each hind leg. A booster vaccination of the same type and by the same route was given 14 weeks subsequently. The flock was maintained at pasture together with a group of 18 randomly selected unvaccinated control ewes, One week post booster vaccination a blood sample was taken by jugular venepuncture for the measurement of plasma anti LHRH (1-10) antibody titre. At this rime three vasectomised rams fitted with coloured marking crayons were introduced to the flock. Once each week the ewes were examined, as is commonly done in the art, for the occurrence of oestrous "marks" left by the ram that are indicative of the occurrence of oestrous behaviour of the ewe. The colour of the marking crayons was changed weekly and the posterior portion of the ewes regularly clipped to allow unambiguous detection of marks. Antibody responses were measured in the vaccinated e es and the occurrence of oestrous cycles measured during a 100 day period during the breeding season when unvaccinated control ewes were expressing normal behavioural oestrous.
The immunoneutering effects of this vaccination are recorded in Table 1.
WO 88/05308 PCT/AU87/00448 -16- EXAMPLE 17 Effect on Anti LHRH (1-10) Antibody Titre and on the Oestrous Cycles of Adult Merino Ewes Following Vaccination Against LHRH An example according to Example 16 wherein LHRH was used as immunogen instead of LHRH (1-10):HSA such that 9 ewes were given an Example 1 type conjugate and 9 others were given and Example 11 type conjugate.
The immunoneutering effects of this vaccination recorded in Table 1 confirm that the new art can block oestrous behaviour.
EXAMPLE 18 Effect on Anti LHRH (1-10) Antibody Titre and on the Oetrous Cycles of Adult MerinQo EwesFollowing Vaccination Against LHRH (1-6)HSA An example according to Example 16 wherein LHRH (1-6):HSA was used as immunogen instead of LHRH (1-10):HSA such that 9 ewes were given an Example 2 type conjugate and 9 others were given an Example 12 type conjugate. The immunoneutering effects of this vaccination recorded in Table 1 confirm that the new art can block oestrous behaviour.
EXAMPLE 19 Effect on Anhi LHRH (1-10) Antibody Titre and on the Oestrous Cyces of Adult Merino Ewes Following Vaccination Against LHRH (1-7):HEA An example according to Example 16 wherein LHRH (1-7):HSA was used as immunogen instead of LHRH (1-10):HSA such that 9 ewes were given Example 3 type conjugate and 9 others were given an Example, 13 type conjugate. The immunoneutering effects of this vaccination recorded in Table 1 confirm that the new art can block oestrous behaviour.
4 WO 88/05308 P CT/A U87/00448 17 Antibody response and thae incidence Qf oestrous cycles in control. LHIIH-inimune and LHRH fragment-immune ewes urg h reigsao Treatment Immunogen Mean anti Mean number group type LHRH (1-10) of oestrus antibody titre cycles observed Control -4.8 0.3 14I*RH (1-10) immune, Example 1* 1,000,000 0 LHR.H immune Example 1 57,200 0 IJHRH immune Example 11 40,200 0 LHRH immune Example 2 205,000 0 LHRH immune Example 12 18,400 0 LHRH immune Example 3 135,000 0 LHRII immune Example 13 67,900 0
I
This immunogen was made by the methoa of Example 1 except that LHRH (1-10) free acid form was used in~stead of LIIRH EXAMPfLE Effect on Anti LHRH (1-10) Antibody -TitLre. Plasma 2TZstsLrone Concentration. Testis Siz nd Servng Capacity of Adult Merino Rams Following VAccjnation gainst LHRHJ (1-10):,.HSA This is an exaiple of the prior art given to enable a comparison with the new art of the invention.
Four adult Merino rams were vaccinated against I.HRH (1-10) using an LHRH (1-10):HSA conjugate in a Type I.
vaccine (Example 15). Vaccination was comprised of a primary treatment given as a 1 ml intramuscular injection to each hind leg. A booster vaccination of the same type and by the same route was given approximately 14 weeks subsequently. The ramns were maintained at pasture i II WO 88/05308 PCT/AU87/00448 '4 18 together with four randomly selected unvaccinated control rams. One week post booster vaccination a blood sample was taken by jugular venepuncture for the measurement of plasma LHRH (1-10)-specific antibody titre in the boost response. Subsequently, at various intervals, measurements were made of anti LHRH (1-10) antibody titre, testosterone concentration, serving capacity and testis size of all rams in the study. The effect of the prior art immunoneutering vaccination on these variables is shown in Tables 2 to EXAMPLE 21 Effect on Anti LHRH (1-10) Antibody Titre, Plasma Testosterone Concentration, Testis Size and Serving Capacity of Adult Merino Rams Following Vaccination Against LHRH (1-7):HSA An example according to Example 20 wherein LHRH (1-7):HSA (Example 3) was used instead of LHRH (1-10):HSA. The effects of this vaccination are given in Tables 2 to 5 and they confirm the immunoneutering 20 capability of the new art.
Table 2 Anti LHRH (1-10) antibody titres in vaccinated adult Merino rams Anti LHRH (1-10) antibody titre in indicated treatment group Day of LHRH (1-10) immune LHRH immune experiment 30 289 98 542 44 111 4310 2136 12437 3213 214 730 139 1222 216 340 226 158 834 444
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i WO 88/05308 PCT/AU87/00448 19 Serving capacity* of adult LHRH (1-10) immune.
LHRH immune and control rams Day Mean serving capacity of rams in indicated treatment group LHRH (1-10) LHRH Control immune immune 0# 7 1.1 6.8 0,8 8.0 1.6 11t 7.5 1.8 4.8 0,8 8.5 12y 4.3 0.9 5.3 1.0 6.5 157 4.0 0.8 3.3 0.8 8.3 178 2.8 0.9 a 2.3 0.5 a 7.0 214 2.3 0.5 2.5 .0a 6.3 1.1 309 0.5 0 3 b 1.5 1 .0h 5.8 0.3 Mean number of services given to oestrous ewes.
Calculated by taking the mean of the combined number of services given during opportunities on each of 2 consecutive days. Measurements were made by the procedure of P.E. Mattner and A.W.H. Braden reported in the Australian Journal of Experiment Agriculture and Animal Husbandry, Volume 15, Page 330 (1975).
Primary vaccination given on day 0; booster vaccination given on day 101.
a,b Values in the same row with different superscripts differ; a, P(0.05; b, P<0.01.
WO 88/95308 PCT/A U87/00448 20 Testis size* of LHRH (1-10) immune, LHRH immune and control rams Day Mean testis size of rams in indicated treatment group LHRH (1-10) LHRH Control immune immune 157# 50 15 a 38 5 98 3 178 3 5 7b 2 4 2b 100 214 43 8 b 3 1 2b 100 Testis size is expressed as a percentage of the pre vaccination volume and estimated by the comparative palpation procedure of C.M. Oldham, N.R. Adams, P.B.
Gheradi, D.R. Lindsay and J.B. Mackintosh reported in the Australian Journal cf Agriculture Research, Volume 29, Pages 173-9 (1978).
Day 157 is 56 days post booster vaccination a,b Values in rows with different superscripts differ; a, P< 0.05; b, P<0.01.
Table Mean testosterone concentration (ng/ml) in the plasma of LHRH (1-10) immune. LHRH immune and control rams Day Mean testosterone* concentrations (ng/ml) in indicated treatment grotup LHRH (1-10) LHRH Control immune immune 111 0.2 0.07 a 0.08 0.088 10.8 1.8 214 0.2 0.24 0.12 0.07 a 4.3 1.3 Day 111 is 10 days post booster vaccination.
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WO 88/05308 PCT/AU87/00448 21 Values in the same row with different superscripts differ; (P<0.01).
EXAMPLE 22 Effect on Anti LHRH (1-10) Antibody Titre and the Fertility of Male Ouackenbush Mice Following Vaccination Against LHRH (1-10):HSA This is an example of the prior art given to enable a comparison with the new art of the invention.
Eight male Quackenbush mice having proven fertility were vaccinated against LHRH (1-10):HSA using Type 1 vaccine (0.2 ml; Example 15) intraperitoneally. A booster vaccination of the same type and by the same route was administered 4 weeks subsequently. Two weeks post boost the mice were bled retro occularly to obtain an 0.1 ml plasma sample for the measurement of anti LHRH, (1-10) I antibody titre. At this time each vaccinated male mouse was boxed together for 6 weeks with a single female of the same strain that had proven fertility. The incidence of pregnancy among the females during this period was observed. Immediately following the 6 week breeding opportunity all females that had failed to conceive were isolated for 7 days and then given a second opportunity (7 days only) to breed with a different non vaccinated control male that had proven fertility. The incidence of pregnancy among the females was again observed. In this manner it was established that the failure of any female to breed with an LHRH (1-10) immune male was directly attributable to male infertility induced by the effects of the vaccination. Thus the statistical comparison that is possible in this study is the incidence of fertility (or infertility) in the group of LHRH (1-10)-immune male mice compared with that of unvaccinated controls when given the opportunity to breed with a single group of proven fertile females.
WO 88/05308 PCT/AU87/00448 -22- Throughout the study, water and food were available adLlib~itum. The anti fertility effects of this vaccination are shown in Table 6.
For the purpose of Examples 22 to 25 the concept of "proven fertility", that, is, the ability to breed, means that each fertile female was known to have previously delivered at least three litters and each fertile male was known to have previously sired at least three litters.
Fertility in Tables 6-9 means the percentage of animals in a treatment group capable of breeding.
Anti LHgH (1-1n) antibody titre and fertility of LHRH (1-10) immi1ne and control Quackenbuash male mice Treatment Group Antibody titre Fertility() ~LHRH (1-10) Immu~ne 64450 21240 O Control 0 100 a Significantly different to control (P<0.001).
EXMLE2 Effect on Anti--LHRH (1-10) Antibody Titre, Plasma Testosterone Concentration and the Fertility of Male Otackenbush Micle Followding, Vaccinati ai~ns- LHRHi (1-7)*:ESA seven male Quackenbush mice of previously proven tartility were vaccinated against LH-RH (l-7)**fSA using Type I vaccine (0.2 ml, Example 15) intraperitoneally, A booster vaccina~tion of the same type and given by the same route was administered 4 weeks subsequently, Two weeks post boost the mioe were bled retro occularly to obtain a 0.3. ml sample of Oldama for the measurement of specific af~ti LI-R1I (1-10) antibody titre and of plasma testosterone Ooncentration, At this timne each male. LHRHi-imnune mouse a 9 I i WO 88/05308 PCT/AU87/00448 23 was boxed together with a single female of the same strain and having previously proven fertility, for a period of 6 weeks. The incidence of pregnancy among the females during this perj,od was observed. Immediately following the 6 week breeding opportunity all females that had failed to conceive were isolated for 7 days and then given a secondary opportunity (7 days only) to breed with a different nonvaccinated control male of previously proven fertility, In this manner it was established that the failure to breed with an LHRH (1-10) immune male was directly attributable to male infertility. Throughout the study water and food were available ad libitum. The anti fertility effects of this vaccination are shown in Table 7.
Table 7 Anti LHRH (i-10) antibody titre, plasma testosterone concentration and fertility in LHRH immune and control Ouackenbush male mice Treatment group Antibody titre Plasma testosterone Inn f/mi\ Fertility LHRH immune 3190 1130 2.3 0.78 a Control 0 14.2 A 3.5 100 a Significantly different to controls, P< 0.02 b Significantly different to control, P <0.001.
EXAMPLE 24 Effect on Anti LHRH (1-10) Antibody Titre and the Fertility of Female Quackenbush Mice Following Vaccination Against LHRH (1-7):HSA Seven female QuackenbuSh mice of previously proven fertility were vaccinated against LHRH (1-7).HSA using a LL. iY WO 88/05308 PCT/AU87/00448 24 Type 1 vaccine (0.2 ml, Example 15) intraperitoneally. A booster vaccination of the same type and by the same route was given 4 weeks subsequently. Two weeks post boost the mice were bled retro occularly to obtain a 0.1 ml sample of plasma for the measurement of anti LHRH (1-10) antibody titre. At this time each female LHRH-immune mouse was boxed for 6 weeks together with a single male of the snme strain that had previously proven fertility. The incidence of pregnancy among the females in this period was observed and any pregnancy used to confirm the fertility of that female. Three months following the j first booster vaccination a second booster vaccination was given to each female and the opportunity to breed with a fertile male repeated as previously. Again any pregnancy among the females was used to confirm the fertility of that female. Throughout the study water and food were available ad libitum. The anti fertility effects of this vaccination are shown in Table 8, Table 8 Antibody titre and fertility in female Quackenbush mice vaccinated against LHRH (1-7):1HA Time during treatment Antibody titre Fertility Pre vaccination 100 Post first boost vaccination 5126 1783 28.$Q Post second boost vaccination 10175 j 2271 Q0 a Significantly different to prevaccination fertility (P<0.01)
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WO 88/05308 PCT/A U87/00448 EXAMPLE Effect on Anti LH il).,Antibody Titre, Plasma Testosterone Concfefltri ,'nd the .Fertility of Male Ouackenbush !aly lowing Vaccination An example according to E'ample 23 except that mice were vaccinated against XLHIk and 16 weeks following the first booster vaccination the vaccinated males were given a second booster vaccination and then given an opportunity to breed with fertile females. The occurrence of pregnancy in any female was taken as proof of the fertility of her male mate. The anti fertility effects of this vaccination are shown in Table 9.
Table 9 Anti LHRH (1-10) antibody tit-e. plasma testosterone concentration and fertility of LHRH immune and control Ouackenbush male mice Variable Treatment group LHRH immune Control 14 days post first boost vaccination Anti LHRH antibody titre 4531 a 1310 Nil Testosterone concentration (ng/ml) 1.7 0.86 a 16.9 4,1 14 days post second boost vaccination Anti LHRH antibody titre 3353 923 Nil Testosterone concentration (ng/ml) 1.5 1.1 a 15.6 3,6 Male fertility 25 a 100 a Significantly different to controls P<.0.01 I C C-a WO 88/05308 PCT/AU87/00448 26 EXAMPLE 26 Effect on Anti LHRH (1-10) Antiboy Production, and Testicular Development in Growing Crossbred Ram Lambs vaccinated Against LHRH (4-10):BSA Fifteen crossbred ram lambs, the progeny of Poll Dorset siral and Border Leicester cross Merino dams, were vaccinated against LHRH (4-10) using a LHRH (4-10):BSA conjugate (Example 7) in a Type 2 vaccine formulation (Example 15). A primary vaccination (2 ml) was given subcutaneously in the neck region when the lambs had a mean age of about 3 weeks. Six weeks subsequently a blood sample was taken by jugular venepuncture for the measurement of anti LHRH antibodies. At th.s time a measurement of testicular volume was made by the palpation procedure. Additionally, a booster vaccination of the same kind as the primary was given in the same volume antd i by the same route. Seven days following the boost a further blood sample was taken for measurement of the antibody response. Twenty one dayei following the boost, testicular volume was again measured and the rams were weaned from their mothers. Throughout this study ewe mothers and ram lambs were kept at pasture. Control lambs used to enable statistical comparisons with the foregoing treatment comprised a comparable group of untreated sexually entire ram lambs (N Table 10 reports the effect of the vaccination against LHRH (4-10) on anti LHRH antibody production and on the retardation of testicular development in these growing crossbreed lambs.
I I 0 00 00 00
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AntibodvJJIRH 1-10) LibDdy -response and testicular volumes in vaccinated- crossbred lambs Variable Day of Treatment group Experiment LHRH (4-10) LHRH LHRH(2-8) LHRH LHRH (1-10) Untreated control Anti LHRI (1-10) antibody titre 42 105 36 2020+ 560 230C± 750 2160 830 1190± 220 0 Mean Testicular volume (cubic centimetres) 42 22 3 a 22 3+ 5a 29+ 6 27± 4 48 4 Anti LHRH (1-10 antibody titre 49 2724 580 7200+ 1100 5568± 1530 3800 800 3255 410 0 Mean Testicular volume (cubic centimetres) 63 16 ±+0.7a 20 5 i' 61 ±78 55 ga 38 12a 125 17 a Significantly different to controls (P 0.05) J-3 Q at 0 tr 0 ia.
C) C o t-4 ::t Lii
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r1 WO088/05308 PCT/A U87/00448 -28- Effect on Anti LHRH (1-10) Antibody Production and iji Testicular Develop~ment in Growing Crossbred Lambs Vaccinated Against LHRH (1-7)*BSA An example according to Example 26 wherein LHRH 4(1-7):BSA (Example 8) was used as immunogen instead of LHRH (4-lO):BSA. Table 10 reports the anti gonadal effect of this vaccination.
EXAPLE28 Effect on Anti LHRH (1-10) Antibody Production and Testicula Development in Growing Crossbred Lambs Vaccinated Aainst T.LN 2jfH.
0 An example according to Example 26 wherein LHRH (2-8):BSA (Example 9) was used as immunogen instead of LHRHT (4-10):BSA. Table 10 reports the anti gonadal effects of this vaccination.
EXAMLE 29 Effect on Anti LITRH (1-10) Antibody Production and Te-sticular Development in Growing Crossbred Lambs Vaccinated Against LHRH (3-8):BSA An example according to Example 26 wherein LHRH :BSA (Example 10" was used as imnunogen instead of LURH Table 10 reports the anti gonadal effects of this vaccination.
Efect opAnti LURE (1-10) Antibody Resnons _juld Testicular Development in Growing Crossbred Lambs Vaccinated Against LHJRH (1-10':HSA An example according to Example 26 wherein LHRH (1-10):HSA was usedi a immunogen instead of LHRH This is an example of Itht prior art and it is included to allo1' a comparison with the new art. The anti gonodal effects of, this vaccination are recorded in Table~ r WO 88/05308 PCT/AU87/00443 -29- EXAMPLE 31 Effect of Vaccination Against LHRH (5-10):HSA on Anti LHRH Antibody Production and on the Ram-induced Ovulation and Oestrous Responses in Seasonally Anoestrus Merino Ewes Twenty five adult Merino ewes were isolated from rams by a distance of several kilometres and were vaccinated against LHRH (5-10) using a LHRH (5-10):HSA conjugate (Example 6) in a Type 1 vaccine (Example 15). A primary vaccination (2 ml) was given subcutaneously in the neck region. A booster vaccination of the same type and by the same route was given 7 weeks subsequently. At this time, which was late in the anoestrous season of the ewes, and immediately prior to the boost, a blood sample was taken by jugular venepuncture to allow for the measurement of the primary anti LHRH (1-10) antibody titre. One week following the boost a second blood sample was taken for measurement of the secondary antibody response. At this time 4 vasectomized rams harnessed with marking crayons were introduced to the flock to induce an ovulation response and oestrous behaviour. Eighteen days post-boost the incidence of fresh ovulations in the ewes following introduction of the rams, was determined at laparoscopy.
Thirty five days post-boost the flock was examined for "ram marks" indicative of oestrous behaviour in the ewes. Two groups of control ewes used to enable stati4stical comparisons with the foregoing treatment comprised a comparable group (N 25) of Merino ewes vaccinated against LHRH (1-10):HSA with a Type 1 vaccine (ExtL.ple according to the same protocol and (ii) a group (N of untreated ewes. Throughout the study all ewes were maintained together at pasture and were kept isolated from rams until these were introduced to the flock as scheduled.
The effects of these vaccinations on the suppression of ovulation and of oestrou behaviour are shown in Table 11.
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Table 11. Antibody response, incidence of ramn-induced oestrous behaviour and ovulation response in control and immunized seasonally anoestrus Merino ewes following introduction of ramns Variable Day of Treatment group Expe LHRH (5-10) LHRH (4-10) LI{!i LHRH (1-10) Untreated immune immune immune immune controls Anti LURH (1-10) antibody titre 49 5000 2200 15900 14200 Nil Anti LHRH (1-10) antibody titre 56 53400 42800 43230 57800 Nil Z Ewes ovulating 67 Nil 3 Nil 3 29a Nil 3 83 Oestrous behaviour confirmed 84 Nila Nil 3 24a 9a 88 days post booster vaccination Day 1 is regarded as the day of primary vaccination Significantly different to controls (P 0.001) :J WNO 88/05308 PCT/AU87/00448 31 EXAMPLE 32 Effect of Vaccination Against LHRH (2-8):HSA on Anti LHRH (1-10) Antibody Production and on the Ram-Induced Oestrus and Ovulatory Response in Seasonally Anoestrus Merino Ewes An example according to Example 31 wherein the LHRH (2-8):HSA (Example 4) was used as immunogen instead of LHRH (5-10):HSA. The oestrus-suppressing and ovulation-suppressing effect of this vaccination is reported in Table 11.
EXAMPLE 33 Effect of Vaccination Against LHRH (4-10):HSA on Anti LHRH (1-10) Antibody Production and on the Ram-Induced Oestrus and Ovulatory Response in Seasonally Anoestrus Merino Ewes An example according to Example 31 wherein the LHRH (4-10):HSA (Example 5) was used as immunogen instead of LHRH (5-10):HSA. The oestrus-suppressing and ovulation-suppressing effect of this vaccination is shown is Table 11.
EXAMPLE 34 Effect of Vaccination Against LHRH (1-7):HSA on the Anti LHRH (1-10) Antibody Response, Oestrous Behaviour and Progesterone Concentrations in Crossbred Heifers Nine crossbred heifers were vaccinated against LHRH using an immunogenic conjugate of LHRH (1-7):HSA (Example 3) in a Type 2 vaccine formulation (Example A primary vaccination of 5 ml was administered subcutaneously at one site either side of the neck. Seven weeks subsequently a blood sample was taken by jugular venepuncture for the measurement of the primary antibody response. At this time a booster vaccination of the same type and volume as the primary was administered by the same route as the primary. In addition in intramuscular
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I ii 1; n WO 88/053081 PCT/A U87/00448 -32 injection of a synthetic prostaglandin was given to each heifer to synchronize oestrous cycles of the herd (Estrumate, 1200 Twelve days post boost a further blood sample was taken for measurement of the booster antibody response. Additionally a second administration of the synthetic prostaglandin (1200 intramuscular) was given and KaMarr heat mount detection pads attached to the back of each heifer in the manner than is well known in the art. Nineteen days post boost the incidence of oestrus among the heifers was recorded. Throughout this study a group of 8 control heifers were managed identically except that they remained unvaccinated. All cattle were kept at pasture. The anti-reproductive effects of this vaccination shown in Table 12 are seen to be the significant reduction in oestrous behaviour that is also accompanied by a significant reduction in mean plasma progesterone concentratiopn in the immune heifers compared to controls.
I1 WO 88/05308 P CT/A U87/00448 33 Anti T11Rhi antibody. response. oestrou"Qbhaviour and progesterone concentration -in heifers variable Day of Treatment group experiment LHREI Untreated immune controZ.
Anti LHRI antibody titre Anti LHRH antibody titre Oestrus heifers confirmed confirmed by KaMarr detection Mean plasma progesterone concentration (ng/ml) Mean plasma progesterone concentrat ion tna/mi Nil 301 146 1 3a Nil Nil 0.1 +0.06 2.9 +0.8 ~iJ 57 0.2 +0.1 67 0.6 0 4 b a b Significantly different Significantly different to untreated control 0.01) to untreated control (P 0.
Claims (11)
1. An immunogenic peptide-protein conjugate for use in the immunoneutering of mammalian animals comprising a peptide conjugated with an immunogenic protein, the coMP0 idbeing characterized in that the peptide is a sequence selected from the group comprising any contiguous 6 or 7 amino acid fragment of the decapeptide pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2
2. A conjugate as claimed in claim 1 in which the peptide includes the pyroglutamic acid from the N-terminus or the glycine amide from the C-terminus of the decapeptide.
3. A conjugate as claimed in claim 1 in which the peptide includes at its end proximal t the immunogenic protein an additional amino acid or sequence of amino acids such that the resultant peptide is not homologous with the decapeptide.
4. A conjugate as claimed in claim 3 in which the additional amino acid is a chemically reactive amino acid at either the N-terminus or the C-terminus of the defined f.e peptide sequence.
5. A conjugate as claimed in any one of claim 1 to 4 in which the peptide is selected from the group comprising pGlu-His-Trp-Ser-Tyr-OH pGlu-His Trp-Ser-Tyr-Gly-OH pGlu-His-Trp-Ser-Tyr-Gly-Leu-OH S' H-His-Trp-Ser-Tyr-Gly-Leu-Arg-OH S H-Trp-Ser-Tyr-Gly-Leu-Arg-OH H-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 H-Tyr-Gly-Leu-Arg-Pro-Gly NH 2
6. A conjugate as claimed in any one of claims 1 to 5 in which the immunogenic protein is selected from the group comprising serum albumin, thyroglobulin, ovalbumin, gelatin, haemocyanin and serum globulin.
7. A conjugate as claimed in claim 1 in which the |I I I u I rtyf WO 88/05308 PCT/AU87/00448 35 peptide is selected from the group pGlu-His-Trp-Ser-Tyr- Gly-Leu-OH and H-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 and the protein is selected from the group comprising bovine serum albumin, human serum albumin, ovalbumin and serum globulin.
8. A vaccine for immunoneutering mammalian animals comprising at least one composition as claimed in any one of claims 1 to 7 and an immunoadjuvant.
9. A vaccine as claimed in claim 8 in which the immunoadjuvant is selected from the group comprising a diethylaminoethyl dextran or another polycationic polyelectrolyte, a polyanionic polyelectrolyte, a mineral oil, or an emulsion of a polycationic or polyanionic polyelectrolyte and a mineral oil.
A method for the immunoneutering of male or female mammalian animals comprising administering to the animal an effective amount of a vaccine according to claim 8 or Claim 9.
11. An immunogenic peptide:protein conjugate as herein described with reference to any one of Examples 1 to 13; a vaccine for immunoneutering mammalian animals as herein described with reference to Example 15; or a method for the immunoneutering of mammalian animals comprising Sadministering to the animal a composition according to A claim 1 as herein desribed with reference to Example or a method for the immunoneutering of mammalian animals comprising administering to the animal a composition according to claim 1 as herein described with reference to any one of Examples 16 to 34o "^A INTERNATIONAL SEARCH REPORT gnternsionag Autilcition No PCT/AU 87/00448 I. CLASSIPICATriam sunac SUE i ATT11R1 I t 004'1 0111111?CII4OR I$IMUOII 3201. 11111 ACCeodens to Intofnational Patent Classificaggon (IFCI of 6 both Natina ClassiiCaioln 4"d IPC Int. Cl. 4 A61K 39/385, C07K 7/06 IL, PIRLDS 211APICH1 minimu~m Oocumontstion Seavcnod f Classification System Clsi Uata minoofe IPC A61K 39/385 OoCumefll~tion flaClid othaf thn, Minim~um 06CUM~nO11110 th 98111" Moqat ucM DOCUM41011 ON. I"Clued In IRO 1 144111 AU IPC as above; Australian Classification 87,11:1O0 (SF 114) 111, 00CUMINTS CONS10SNI 10 5 ASLAVANTO CaI@Oaiy Cilatiofl of 04cument. ii -it"l Indication, where oooarall, at th 1 0115 046149nI baa s I Rolasn to C1aim NO. 'I E X AU,A, 76423/87 (THE STATE OF VICTORIA) 14 January (1-9) 1988 (14.01.8 8) See page 5 lines 14-17, and 29-33 and page 7 lines 15-21 X AUA, 34497/84 (AKZO 26 April 1985 (26.04.85) See example 1 X Us A, 4608251 (Abdos S. MIA) 26 August 1986 (1-9,11) (29408,86) A AM,, 80826/75 (503647) (ALL INDIA INSTITUTE OF MEDICAL SCIENCE) 11 November 1976 (11:11,76) A Chemical Abstracts, Volu,:! 98, No.17, issued 1983, (1-9) Aoril 25 (Columbus, Ohio, ,Donna L. Vogel et all 'Sertoli cell maturation is impaired by neonatal passive immunization with antiserum t. LHRH's abstract No. 137896q So.iaIdaltoros f ctedO@4~it~ii5~ 0 *.at o t 1 and n o f~ iv ii oIn*0dal'on bu.t "A Cotumniif flh6Ime cons o #oiI sis o, the ent 10fld1 Is met 4110d to wndarstarid the atiuntolo of ltm0*fy unestif-nq iri tCtidfd4 Ve of oa5n441f foloennd Inventi'on 404umwisn out oulwar'oC an oe Ith ifthe I tiflln dufm.M Of 04rtigulisI tW~yfOV40 1116 41IM4rie Inwe0At MIMIg date ofnoia omlr'idefov! neyti of ganlt as Itanildos I1 datdfment WhIOI ma ti' oU ~ilt sa orlotiii 4AuOfo v411lIvetieAe WnidncI is 4114d ole 00In, ouglid144111A 441e Of an1190Eo *Y1 decumelil at oertcullo tolowenco:st 11 oi~ot 041aton of olnar 4804141 reson is. tg4ftCannal fit tlist invlve i n Ilonfive oil# WAlIA In# "W d 4 y*MjM tfflpf 10 4Atl 11dw441414111 U44, 0011111041 Of deaumoni I$ torma~lee with one a oto s0 t et $Udlm 4oqu4 almsr M4rio f"60111, 114c oft ~liiil boenl oaenws 14 Wean 511C114 P'ddiumo 44iflloaid ofiof to 11h0 .Jnset" f fifin MII ut I I~~M Amme fI.sm aeifml IV,. CIPITIPICATION 0410 of in. ACIUAI Como141ion 41 tIMO I'11fillseiOitl 3404m1 oilo of Melling of ths Intrialional Seeren R11904M March 1988 (30.03,88) c f4 *9 ?g illI4106l Ssetdf'Vl A4001111l 3 of eAulharie ide~f Australian Patent Office ~PLIET FOk PCIISAWO1 rI d gruel) li44i"111O~ 1ne~atoaiAai~~~f N, PCT/AU 87/00448 #IU~trTH& INFORMATION cONTINUID FROM T141 SICONO SIIT 0US51!VATIO14S WHIME CIRATAIN CLAIMS WIIAI FOUP4O UOSAARqCIIABLE This into~atiomal town~ waort Pas mot boon satabtisned in fesooed of @cqM4i claims undet Aflic@ 11(2) lartie tollowilig tseasn 14: claimn numoffs J1Q, b.oaust May relate to &Utilect rmatner not Isaifard to be Wreeoad by this AuthorirY4 nen'elyt Method of treatment of the animal body by therapy. 2.7 Claim furnaces best they (state to cams of ie4 Informational loolicatiort loot do niot comoly with the 01414(i~ed toqitre "'Itts to sucn en aslant trial no nieeninglUt itterrietiOnal searcol tam go qarriad out, soci~liy 3c CI" 11urs,ow,,w, 5 beaus* ""ry a.00 dci 1 claIr amd are no~t itfted im ICGO(d4ico wittt the4 e$cON 4 ONO (rind Ofadn KCT Au* 11,40). vi,, 003SVATIONS WHKRI UNdITY OF INVII4TIOp4 15 LACKIN'G This Internationial Saaitirlg Avthatirty found muitlol., Invoollome in tisu Inlefnelionel siatidttilft as fgltqwst '2As a1l tseijtd additional soatith 1ee0 warts timeliy P414 by the ea0t46414t this Internatioa*l, 0earco faort coves all 60etchabla, glenm 0f the internitionet *O401404 t7" As only $arm* of too foauira4 qddtlOa4 setclt* wofq itmety cold 41 the soolttamt, this Internationalstei fegin toies only those 4laumiato the 40t0naioiatsola to lf Wh$ih loot were*au cl, ac11iflly 41aIuMte No 1equised additional laatn tots were timeoly caid by the 400114411". e~ei'~y fl Inertiorrat soeth 'aeon ta rtotte~odto toe 11 101 111i 11 NOt R'14tidna4 In the 4lAun'$A( t1l 40064c by 4141M AUiduloci 4 rs- At 1 eAli belin gould 04 esareted wtht 4110M, fijetifying 4A Additional l04, the IMlarnaerrl Se(irti Aimtnouit did not ifeute 0MOcemat 41any 4441116A41 Iq@. Aemort am Protee Q The idd,1jlosic t foottt -06C wau dOi'4ai 11 Of 4041t44nhe 4rolasl. t] meraatest saicampenii0 t'e 4,y 1 ofAdditilue tecrth loe. Foeri JOCtlIAITI (euo'0l4Mntntt 46401 1211 lJeA'f~ AMNEX TO fliE INTERNATICNAL SEARCH REPORT CN INTRTIOAL APPLICATIC&4 ID. PCT/AU 87/00448 This Annex Lists the known "All publication lavel patent family napbers relating to thje patent do~cunnts citw-d in thYe above-mentiored international search report. The Australian Patent Office is in no way liable for these particul~ars wlich are nerely given for the purpose of infonntion. Patent Dovument Cited in Search Report AU 34497/84 Patent Family Menbers AU 34498/84 JP 60172932 EP 142192 JIP 60172933 EP 142193 AU 808,26/75 AR 217615 CA 1054937 CH 614626 DE 2518546 DK 2069/75 Fl 751746 GB 1492445 IL 47177 NEL 7505412 NO 751680 NZ 177422 SE 7505411 US 4161519 IN 140168 US 4608251 EP 181236 EMD OF ANNEX 23K(241) /3
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AUPH9868 | 1987-01-14 | ||
| AUPH986887 | 1987-01-14 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU1101788A AU1101788A (en) | 1988-08-10 |
| AU602187B2 true AU602187B2 (en) | 1990-10-04 |
Family
ID=3771985
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU11017/88A Ceased AU602187B2 (en) | 1987-01-14 | 1987-12-30 | Anti-reproductive hormone |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU602187B2 (en) |
| WO (1) | WO1988005308A1 (en) |
| ZA (1) | ZA88149B (en) |
Families Citing this family (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5484592A (en) * | 1989-03-23 | 1996-01-16 | Stitching Centraal Diergeneeskundig Instituut | Peptide, immunogenic composition and vaccine or medicinal preparation: a method of immunising a mammal against LHRH, and a method of improving the meat quality of pigs |
| NL8900726A (en) * | 1989-03-23 | 1990-10-16 | Stichting Centr Diergeneeskund | PEPTIDE, IMMUNOGENIC COMPOSITION AND VACCINE OR MEDICINAL PRODUCT; METHOD FOR IMMUNIZING A MAMMAL AGAINST LHRH, AND METHOD FOR IMPROVING THE MEAT QUALITY OF PIGS |
| US5403586A (en) * | 1989-08-25 | 1995-04-04 | Biotechnology Australia Ptl Ltd. | LHRH-TraTp fusion proteins |
| PL296623A1 (en) * | 1991-03-01 | 1993-09-20 | Rhone Merieux | Method of improving organoleptic properties of male slaughter animal meat |
| FR2685333B1 (en) * | 1991-12-10 | 1995-03-03 | Rhone Merieux | NEW PEPTIDE IN PARTICULAR FOR THE REALIZATION OF ANTI-LHRH IMMUNOGENIC CONJUGATES, VACCINES INCORPORATING THEM AND VACCINATION PACKAGE RELATING THEREOF. |
| ATE191147T1 (en) * | 1991-07-26 | 2000-04-15 | Commw Scient Ind Res Org | SYSTEM FOR PROVIDING A PEPTIDE-BASED VACCINE THAT FORMES ITS OWN ADJUVANT AND ITS PRODUCTION |
| US6761890B1 (en) | 1995-06-07 | 2004-07-13 | Pepscan Systems B.V. | Peptide, immunogenic composition and vaccine or medical preparation, a method to immunize animals against the hormone LHRH, and analogs of the LHRH tandem repeat peptide and their use as vaccine |
| CU22627A1 (en) * | 1996-12-17 | 2000-12-22 | Ct Ingenieria Genetica Biotech | VACCINE PREPARATION FOR THE REVERSIBLE IMMUNE-CASTRATION OF MAMMALS |
| US6635740B1 (en) | 1997-03-27 | 2003-10-21 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Ligand/lytic peptide compositions and methods of use |
| AUPO776897A0 (en) * | 1997-07-09 | 1997-07-31 | Csl Limited | A method of achieving production gains in livestock and agents useful for same |
| JP2002516875A (en) * | 1998-06-04 | 2002-06-11 | メタモーフィックス，インコーポレイティド | How to control animal reproductive behavior |
| AUPP807399A0 (en) * | 1999-01-08 | 1999-02-04 | Csl Limited | Improved immunogenic lhrh composition and methods relating thereto |
| NZ552736A (en) * | 2004-08-04 | 2009-08-28 | Cytos Biotechnology Ag | Carrier conjugates of GNRH-peptides |
| BRPI0515076A (en) * | 2004-09-10 | 2008-07-01 | Queensland Inst Med Res | peptide, composition, lipopeptide, method of generating an anti-lhrh response in an animal and use of a peptide |
| US8524247B2 (en) | 2008-09-17 | 2013-09-03 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention | Rabies virus-based recombinant immunocontraceptive compositions and methods of use |
| CU23739A1 (en) | 2008-09-30 | 2011-12-28 | Ct Ingenieria Genetica Biotech | PHARMACEUTICAL COMPOSITION USING COMBINATIONS OF VARIANTS OF THE GONADOTROPIN LIBERATING HORMONE (GNRH) AS IMMUNOGEN |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU3449784A (en) * | 1983-10-22 | 1985-04-26 | Akzo N.V. | Preparation of immunogens consisting of antigenic determinants bound to glycoside-containing carriers |
| US4608251A (en) * | 1984-11-09 | 1986-08-26 | Pitman-Moore, Inc. | LHRH analogues useful in stimulating anti-LHRH antibodies and vaccines containing such analogues |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU503647B2 (en) * | 1974-10-14 | 1979-09-13 | All India Institute Of Medical Sciences | Antipregnancy vaccine |
| AU7642387A (en) * | 1986-07-03 | 1988-01-29 | State Of Victoria, The | Composition and method for immunological castration and spaying |
-
1987
- 1987-12-30 WO PCT/AU1987/000448 patent/WO1988005308A1/en unknown
- 1987-12-30 AU AU11017/88A patent/AU602187B2/en not_active Ceased
-
1988
- 1988-01-11 ZA ZA880149A patent/ZA88149B/en unknown
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU3449784A (en) * | 1983-10-22 | 1985-04-26 | Akzo N.V. | Preparation of immunogens consisting of antigenic determinants bound to glycoside-containing carriers |
| US4608251A (en) * | 1984-11-09 | 1986-08-26 | Pitman-Moore, Inc. | LHRH analogues useful in stimulating anti-LHRH antibodies and vaccines containing such analogues |
Also Published As
| Publication number | Publication date |
|---|---|
| AU1101788A (en) | 1988-08-10 |
| ZA88149B (en) | 1988-06-28 |
| WO1988005308A1 (en) | 1988-07-28 |
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