MXPA00011947A - Methods for suppressing reproductive behavior in animals - Google Patents

Abstract

Methods for achieving suppression of reproductive behavior and/or fertility in a vertebrate subject are disclosed. The methods us e compositions which include GnRH immunogens, GnRH ananlogs such as GnRH agonists and antagonists, and GnRH antibodies. The methods are useful for the prolonged suppression of testicular function in males and ovarian function in females.

Description

METHODS TO DELETE ANIMAL REPRODUCTIVE BEHAVIOR TECHNICAL FIELD The present invention relates generally to compositions and methods for hormonal modulation. More particularly, the invention is directed to the active and passive immunization of GnRH to achieve prolonged suppression of reproductive behavior and / or fertility. The invention also relates to the use of GnRH agonists and antagonists to reduce the circulating levels of GnRH.

BACKGROUND OF THE INVENTION In the adult animal, the gonadotropin releasing hormone (GnRH) is produced in the hypothalamus (Schally et al., Science (1973) 179: 341-350) and causes the release of gonadotropin hormones, the hormone leuteinizante (LH) and the hormone of stimulation of the follicle (FSH) from the pituitary gland. These, in turn, control the gonadal production of sex steroids; testosterone in males and estrogen and progesterone in females. Therefore, in adults, the hypothalamic-pituitary-gonadal axis (H-P-G) is responsible for sexual function.

The beginning of puberty seems to be activated by the hypothalamus, however, the detailed mechanisms of this process are not well understood. In the pre-pubertal animal, the H-P-G axis is functional and seems to play a key role in the onset of puberty. The first clinical signs of puberty are preceded by pulsatile, increased secretion of GnRH, followed by increased sensitivity of the pituitary to GnRH (Apter, D., Ann, NY Acad. Sci. (1997) 816: 9-21). The secretion of gonadotropin from the pituitary gland, particularly the secretion of LH, progressively increases during the peripubertal period, eventually resulting in gonadal stimulation, the secretion of sex hormones and the progressive appearance of sexual activity and physical maturation, which are recognized as the events collectively referred to as puberty. Treatment with GnRH analogues before puberty has been shown to delay the onset of puberty in some species. However, the mechanism by which this occurs has not been well established. In humans, long-term administration of GnRH agonists has been shown to be effective in the treatment of precocious puberty by inhibiting the progress of puberty (Neely et al., J. Ped. (1992) 121: 634-640 ). In pre-pubertal rams, continuous administration of a GnRH agonist using an implant or minipump for 16 weeks immediately before puberty inhibited the development of sexual behavior, reduced plasmatic concentrations of testosterone, delayed testicular and epididymal development, and inhibited growth rates for at least 2 months after the normal age at which puberty is expected (Tilbrook et al., Hormones and Behavior (1993) 27: 5-28). In female dogs, chronic infusions of GnRH agonists between four months of age and pubertal pro-estrus at 8-12 months of age, suppressed serum concentrations of FSH and LH (Concannon et al., J. Reproduc. Fertile. Supp. (1993) 47: 3-27). However, none of these studies showed a permanent effect on reproductive function once treatment was discontinued. Active or passive immunization against GnRH has also been shown to delay, but not permanently stop, the onset of puberty in several species. In steers, for example, multiple immunizations with a conjugate vaccine of GnRH analogues (human serum albumin-Cys-Gly-GnRH) induced and maintained sufficient anti-GnRH titres to delay puberty for 175 days (Prendiville et al., J. Animal Sci. (1995) 73: 3030-3037). However, the continuous suppression of reproductive function was not demonstrated against the decline in antibody titers. Additionally, this study did not evaluate whether the steers were able to become pregnant and reproduce. Pre-pubertal immunization with an ovalbumin-GnRH analog conjugate vaccine impaired the function of the testes and affected the development of social and sexual behavior of young bulls up to 17 months of age (Jago et al., J. Animal Sci. (1997) 75: 2609-2619). After 17 months of age, testicular function and reproductive behavior returned to normal and immunized bulls could not be differentiated from untreated, normal bulls of the same age. A study in sheep showed significant delay in testicular growth and function during 115 weeks of age when the rams were actively immunized against an ovalbumin-GnRH analogue conjugate vaccine immediately after birth (pre-pubertal) or around puberty ( peripubertal) (Brown et al., J. Reproduc.Fértil. (1994) 101: 15-21). However, 83% of the peripubertally immunized rams had normal reproductive function and hormones for 115 weeks. In the rat, pre-pubertal passive immunization against GnRH resulted in a decrease in the size of the testes, cellularity of the seminiferous tubes and reduced fertility at 100 days of age. However, there was no difference in coupling behavior between the immunized and control groups at 100 days of age (Bercu et al., Endo (1977) 101: 1871-1879). In sheep, pre-pubertal vaccination with an ovalbumin-GnRH analog conjugate resulted in hypogonadotropic hypogonadism in 3 of 4 animals at 4 years of age. However, only anatomical and hormonal changes were reported. The parameters of reproductive performance in this study were not assessed (Brown et al., J. Reproduc.Fértil. (1995) 103: 131-135). In contrast, immunization of young berracos pre-pubertally at approximately 21 and 50 days of age has been shown to coaduce the development of detectable GnRH antibody titers with no effect on serum testosterone levels as the animals reached puberty and sexual maturity (Manns and Robbins, Proceedings of the EAAP Working Group (1997), EEAP Publication No. 92: 137-140). In this way, the long-term effects of pre-pubertal GnRH immunization on sexual function and sexual behavior have not been consistent or predictable. Only one study done in male rats showed reduced fertility. No study has shown reduced fertility in females of any species. Additionally, no study has reduced fertility in cats, dogs, horses or deer using GnRH immunization in the pre-pubertal period as a long-term sterilization method. Additionally, none of the reported studies previously demonstrated biologically effective GnRH antibody titers or a reduction in sexual function and sexual behavior after an individual immunization. The ability to achieve permanent sterilization by pre-pubertal or peripubertal immunization of GnRH would be very useful. The pre-pubertal surgical neutralization of animals, in particular puppies and kittens less than 9 weeks old, has been extensively reviewed in the United States of America (Goeree, G., Can.Vet. J. (April, 1998) 39: 242-243). Studies have compared dogs and cats neutralized pre-pubertal against the peri-pubertal form and found no differences in the subsequent growth, bone density or personality between the two groups. The pre-pubertal neutralized male cats, which may predispose some data to urinary calculi finally in life, did not show a difference in the urethral diameters compared to those of the neutralized cats from 6 to 12 months of age (Goeree G. , Can. Vet. J. (1998) 39: 242-243). No difference was evident in the morbidity or mortality associated with the surgical or anesthetic procedure, between the two groups. However, surgery is expensive, requires special equipment and includes the risk of anesthetic death or post-surgical complications including bleeding, wound formation and infection. Long-term sterilization achieved by blocking the action of GnRH in a pre-pubertal or peripubertal fashion, including active or passive immunization against GnRH, would be an effective and humane alternative to surgical castration.

DESCRIPTION OF THE INVENTION Accordingly, the present invention is directed to methods for reducing GnRH levels in a pre-pubertal vertebrate, the methods result in prolonged reduction in reproductive performance and / or fertility, for example, a prolonged suppression of Testicular function and / or development in males, or ovarian function and / or development in females. The invention is particularly suitable for use with domestic animals such as cats, dogs and horses and wildlife such as deer, and provides a viable and desirable alternative to commonly used surgical forms of sterilization. Long-term immunosterilization can be achieved by active or passive immunization against GnRH during the pre-pubertal or peripubertal period. Without being desired to be bound by a particular theory, it seems to be a critical period in early life during which normal and sustained gonadal development requires gonadotropic stimulation, particularly by LH of the pituitary gland. Inhibition of the HPG axis by active or passive immunization against GnRH or by treatment with agonists or antagonists of the GnRH gene (collectively referred to herein as GnRH therapy) in a pre-pubertal or peripubertal fashion), results in a reduction in the concentrations in GnRH circulation which in turn can cause the decelerated regulation of GnRH receptors of the pituitary and a reduction in gonadotropins during this critical time period. This can lead to long-term deterioration of gonadal development and function due to the permanent inability of the pituitary gland to respond to GnRH. Accordingly, in one embodiment, the present invention is directed to a method for prolonged suppression of reproductive performance and / or fertility. The method comprises administering to the vertebrate subject, in a pre-pubertal manner, a composition comprising an effective amount of a GnRH immunogen, GnRH analog, or antibodies that cross-react with the endogenous GnRH of the vertebrate subject. The composition optionally includes an immunological adjuvant, preferably comprising an oil and dimethyldioctadecylammonium bromide. In certain embodiments, the method results in a long-term, prolonged suppression of gonadal development and / or gonadal function, such as a suppression of testicular development and / or male functions or long-term, prolonged suppression of development and / or function. ovarian in females. The method may further comprise administering to the vertebrate subject a second composition comprising an effective amount of a GnRH immunogen. The GnRH immunogen may be the same or different in the compositions. In another embodiment, the invention is directed to a method for prolonged suppression of reproductive-behavior and / or fertility in a feline, canine, equine or cervino subject. The method comprises administering to the subject, in a pre-pubertal manner, a vaccine composition comprising an effective amount of a GnRH multimer comprising the general formula (GnRH-X-GnRH), wherein: GnRH is a GnRH immunogen; X is one or more molecules selected from the group consisting of a peptide bond, an amino acid spacer group, a carrier molecule and [GnRH] n, where n is an integer greater than or equal to 1; and y is an integer greater than or equal to 1. In yet another embodiment, the invention is directed to a method of prolonged suppression of reproductive behavior and / or fertility in a feline subject. The method comprises: (a) administering to the subject, pre-pubertally, at about 3 to about 15 weeks of age, a first vaccine composition comprising an immunological adjuvant and an effective amount of a GnRH multimer comprising the Formula General (GnRH-X-GnRH) and, wherein: GnRH is a GnRH immunogen; X is one or more molecules selected from the group consisting of a peptide bond, an amino acid spacer group, a leukotoxin polypeptide and [GnRH] n, where n is an integer greater than or equal to i; and y is an integer greater than or equal to l; and (b) administering to the subject about 2 to about 10 weeks after administration of the first vaccine composition, a second vaccine composition comprising an immunological adjuvant of an effective amount of a GnRH multimer comprising the General Formula ( GnRH-X-GnRH) and where :. GnRH is a GnRH immunogen; X is one or more molecules selected from the group consisting of a peptide bond, an amino acid spacer group, a leukotoxin polypeptide and [GnRH] n, where n is an integer greater than or equal to i; and y is an integer greater than or equal to 1. In certain embodiments, the first and second vaccine compositions comprise the same GnRH multimer and the GnRH multimer comprises the amino acid sequence depicted in Figure 6A-6F (SEQ ID.

NO:), or an amino acid sequence with at least about 75% sequence identity to it. In another embodiment, the invention is directed to a method for prolonged suppression of reproductive performance and / or fertility in a feline subject. The method comprises: (a) administering to the subject, pre-pubertally in about 5 to about 12 weeks of age, a first vaccine composition comprising an immunological adjuvant comprising a light mineral oil and dimethyldioctadecylammonium bromide and an amount effective of a GnRH multimer comprising the amino acid sequence depicted in Figures 6A-6F (SEQ ID NO:), or an amino acid sequence with at least about 75% sequence identity thereto; Y (b) administering to the subject about 2 to about 10 weeks after administration of the first vaccine composition, a second vaccine composition comprising an immunological adjuvant comprising a light mineral oil and dimethyldioctadecylammonium bromide and an effective amount of a GnRH multimer comprising the amino acid sequence depicted in Figures 6A-6F (SEQ ID NO:), or an amino acid sequence with at least about 75% sequence identity thereof. In a further embodiment, the invention is directed to a method for suppressing reproductive performance and / or fertility in a feline, canine, equine or cervino subject for at least 10 months. The method comprises administering to the subject, a first composition comprising an effective amount of a GnRH immunogen. In other embodiments, a second vaccine composition comprising an effective amount of a GnRH immunogen is administered. The GnRH immunogen in the first and second compositions can be the same or different. In certain embodiments, both the first and second compositions are administered pre-pubertal or post-pubertal, with the first composition being administered pre-pubertal and the second composition administered post-pubertal. In other embodiments, the method comprises originally administering additional compositions comprising an effective amount of a GnRH immunogen to at least about 6 to about 12 months subsequent to the previous administration. In particular embodiments, the additional compositions are administered at approximately annual intervals. These and other embodiments of the present invention will readily be presented to those skilled in the art in view of the description herein.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 depicts the relationship between the GnRH antibody titers (mean ± SEM) and serum testosterone in cats immunized with GnRH vaccines. Figure 2 shows GnRH antibody titers (mean ± SEM) in kittens immunized at 8, 12 and 16 weeks of age using 100 μg of a GnRH fusion protein described in the examples. The GnRH antibodies were measured as% binding in a 1: 5000 dilution of serum. The arrows represent immunization times. Figure 3 shows the effect of treatment with leuprolide acetates in serum LH concentrations in 10-month-old kittens immunized at 8, 12 and 16 weeks of age with a GnRH vaccine. Figure 4 shows GnRH antibody titers (mean ± SEM) in kittens immunized at 6 and 10 weeks of age with 10 μg of a GnRH fusion protein described in the examples. The GnRH antibodies were measured as the% binding at 1: 5000 dilution of serum. The arrows represent immunization times. Figures 5A and 5B show the nucleotide sequence and amino acid sequences of the GnRH constructs used in the chimeric GnRH leukotoxin-gene polypeptide functions herein. Figure 5A depicts an individual copy of a decapeptide of GnRH. Figure 5B depicts a molecule with four copies of a GnRH decapeptide when n = 1 and eight copies of GnRH with n = 2, etc. Figures 6A through 6F show the nucleotide sequence and predicted amino acid sequence of the chimeric LKT-GnRH protein from pCB130. Figure 7 depicts serum testosterone concentrations in kittens immunized at 8, 12 and 16 weeks of age by a GnRH fusion protein described in the examples. The arrows represent the immunization time. Figure 8 depicts serum concentrations of estradiol in kittens immunized at 8, 12 and 16 weeks of age with a GnRH fusion protein described in the examples. The arrows represent immunization time.

DETAILED DESCRIPTION The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology and immunology, which are within the skill of the art. These techniques are fully explained in the literature. See, for example, Sambroo, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual; DNA Cloning, Vols I and II (D.N. Glover ed.); Oligonucleotide Synthesis (M.J. Gait ed.); Nucleic Acid Hybridization (B. D. Hames &S.J. Higgins eds.); B. Perbal, A Practical guide to Molecular Cloning; the series, Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D.M. Weir and C.C. Blackwell eds., Black ell Scientific Publications). Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or process parameters since they can of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting. 1. Definitions In describing the present invention, the following terms will be employed, and it is proposed that they be defined as indicated below. The term "gonadotropin releasing hormone" or "GnRH" refers to a decapeptide secreted by the hypothalamus that controls the release of both leuteinizing hormone (LH) and follicle stimulating hormone (FSH) in vertebrates (Fink, G., British Medical Bulletin (1979) 35: 155-160). The amino acid sequence of GnRH is highly conserved among vertebrates and especially in mammals. In this regard, GnRH derived from most animals including human, bovine, porcine and ovine GnRH (formerly designated LHRH) has the amino acid sequence pyroGlyu-His-Trp-Ser-Tyr-Gly-Le-Arg-Pro -Gly-NH2 (SEQ ID NO: 1) (Murad et al., Hormones and Hormone Antagonists, in The Parmacological Basis of Therapeutics, Sixth Edition (1980) and Seeburg et al., Nature (1984) 311: 666-668) . As used herein, a "GnRH polypeptide" includes a molecule derived from a native GnRH sequence, as well as recombinantly produced or chemically synthesized GnRH polypeptides having the amino acid sequence that are substantially homologous. to native GnRH and that remain immunogenic, as described below, although they have the ability to act as GnRH agonists or antagonists that bind and eventually decelerate to GnRH receptors, or otherwise block the action of GnRH. GnRH such as competing for GnRH receptors. In this manner, the term encompasses GnRH derivatives and analogs including any addition, substitution and / or single or multiple deletion of amino acids that occurs internally or at the amino or carboxy terminus of the peptide. Accordingly, under the invention, a "GnRH polypeptide" includes molecules having the native sequence as well as GnRH analogues. Representative GnRH analogs include an analog with an N-terminal Gln or Glu residue instead of a residue of pyroGlu, an analog having Asp at position 2 of amino acids instead of His (see Figure 5B); a GnRH analog with an N-terminal addition such as Cys-Gly-GnRH (see for example, Prendiville et al., J. Animal Sci. (1995) 73: 3030-3037); a carboxyl-containing GnRH analog (see for example, Jago et al., J. Animal Sci. (1997) 75: 2609-2619; Brown et al., J. Reproduc. Fertile. (1994) 101: 15-21. ); the GnRH analogue (D-Trp6-Pro9-. ethylamide) GnRH (see for example, Tilbrook et al., Hormones and Behavior (1993) 27: 5-28) or (D-Trp6) GnRH (see for example, Chaffaux et al., Recueil de Medicine Veterinaire (1985) 161: 133-145); GnRH analogs with the first, sixth and / or tenth amino acids that occur naturally replaced by Cys and / or where the N-terminus is acetylated and / or the Cys is amidated (see, for example, State Patents) United Nos. 4,608,251 and 4,975,420); the GnRH analog pyroGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Gly-Y-Z (SEQ ID NO:) wherein X is Gly or a D-amino acid, and Y is one or more amino acid residues which may be the same or different, preferably 1-3 Gly residues, and Z is Cys or Tyr (see, publication of U.S. Patent No. GB 2196969); GnRH analogue described in U.S. Patent No. 5,688,506, including the GnRH analogue Cys-Pro-Pro-Pro-Pro-Ser-Ser-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg- ProGly (SEQ ID NO:), pyroGlu- His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Ser-Ser-Pro-Pro-Pro-Pro-Cys (SEQ ID NO:), pyroGlu- His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyArg-Pro-Pro-Pro-Pro-Cys (SEQ ID DO NOT: ); the GnRH analog known as Deslorelin, commercially available from Apeptech (Australia), and Ovuplant ™; and molecules with other additions, substitutions and / or deletions of amino acids that retain the ability either to produce the formation of antibodies that cross-react with the naturally occurring GnRH, or molecules that act as agonists or antagonists of GnRH . Representative GnRH agonists include the Lupron Depot ™ compounds, both available from TAP Pharmaceuticals, Inc. (Deerfield, IL), with the chemical formula 6-Oxo-L-propyl-L-histidyl-L-triptophyl-L-acetate. seryl-L-tyrosyl-D-leucyl-L-leucyl-L-arginyl-N-ethyl-L-prolinamide, Zoladex ™, a goselin acetate implant, available from Zeneca Pharmaceuticals (Wilmington, DE). Representative antagonists include analogs modified at the N-terminus, such as those described in U.S. Patent No. 5,413,990; the analogs described in U.S. Patent No. 4,740,500 including an analogue with the formula Ac-ß-D-NAL-R2-D-3-PAL-Ser-Arg-R6-Leu-Arg-Pro-RIO, in where R2 is Cl-D-Phe, FD-Phe, N02-D-Ph_e, Br-D-Phe, 3,4C12-D-Phe or Ca Me-Cl-D-Phe; R6 is D-3PAL, D-Trp, For-D-Trp, N02-D-Trp, (imBzl) D-His, D-Tyr or (3-D-NAL, and RIO is Gly-NH2, NHCH2CH3, NHNHCONH2 or D-Ala-NH2, 0-D-NAL is the D-isomer of alanine which is substituted by naphthyl in the β-carbon atom and D-3PAL is D-alanine which is substituted by pyridyl in the β-atom of carbon with the bond that is at position 3 in the pyridine ring, NML is a substitution of methyl in the alpha-amino group of Leu, the GnRH analog known as Abaralix (Garnick et al., ENDO '98, p. 108: OR43-I) and the analog PPI-149 (Praecis, Cambridge, MA) Thus, the term "GnRH polypeptide" includes a GnRH molecule that differs from the reference sequence by having one or more substitutions. deletions and / or amino acid adhesions and having at least about 50% amino acid identity to the reference molecule, more preferably about 75-85% sequence identity and more preferably about 90-95 % identity om ás, to the relevant portion of the native polypeptide sequence in question. The amino acid sequence will have no more than about 1-5 amino acid substitutions, or no more than about 1-3 amino acid substitutions. Particularly preferred substitutions in general will be of a conservative nature, i.e., those substitutions that take place within a family of amino acids. In this regard, amino acids are generally divided into four families: (1) acid. -spartate and glutamate; (2) basic.- lysine, arginine, histidine; (3) non-polar.- alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar; glycine, asparagine, glutamine, cystine, serine, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine are sometimes classified as aromatic amino acids. For example, one can reasonably predict an isolated replacement of glycine with isoleucine or alanine, or vice versa, a threonine with a serine or vice versa; or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a major effect on activity. The proteins that have substantially the same amino acid sequence as the reference molecule, but possessing minor amino acid substitutions that retain the desired activity, therefore fall within the definition of a GaRH polypeptide. A "GnRH polypeptide" also includes peptide fragments of the reference GnRH molecule, so long as the molecule retains the desired activity. GnRH epitopes are also captured by definition. In particular, GnRH multimers including repetition sequences of GnRH polypeptides such as multimers including 2, 4, 8, 16, 32 copies, etc., of one or more GnRH polypeptides are contemplated herein. optionally including spacer sequences, such as those described in International Publication Nos. WO 98/06848 and WO 96/24675 and shown in Figure 5B herein. These multimers are described more fully below. For purposes of the present invention, a GnRH polypeptide can be derived from any of the several known GnRH sequences, described above, including without limitation, GnRH polypeptides derived from human, bovine, porcine, ovine, canine subjects , felines, cervinos, rodents such as hamsters, guinea pigs, gerbils, marmots, gophers, lagomorphs, rabbits, ferrets, squirrels, reptiles and birds. A "GnRH peptide" is a GnRH polypeptide, as described herein, that includes less than the full length of the reference GnRH molecule in question and that includes at least one epitope as defined below. In this manner, a vaccine composition comprising a GnRH peptide will include a portion of the full-length molecule but not the complete GnRH molecule in question. Particular GnRH peptides for use herein include, for example, GnRH peptides with 5, 6 or 7 amino acids, particularly those peptides that include the amino terminus or the carboxy terminus, such as the GnRH peptides include amino acids 1 -5, 1-6, 1-7, 2-8, 3-8, 3-10, 4-10 and 5-10 of the native sequence (see, for example, International Publication No. WO 88/05308. "GnRH multimer" means a molecule having more than one copy of a selected GnRH polypeptide, GnRH immunogen, GnRH epitope peptide, or multiple tandem repeats of a selected GnRH polypeptide, GnRH immunogen, peptide or GnRH epitope The GnRH multimer may correspond to the molecule with repeat units of the General Formula (GnRH-Xg) and where GnRH is a GnRH polypeptide, X is one or more molecules selected from the group consisting of a bond peptide, an amino acid separating group, a molecule Carrier cell and [GnRH] n, where n is an integer greater than or equal to one, and is an integer equal to or greater than 1, and additionally wherein "GnRH" can comprise any GnRH polypeptide. And therefore it can define 1-40 or more repeating units, preferably, a junction 30 repeating units and more preferably, 1-20 repeating units. Additionally, the selected GnRH sequences may all be the same, or they may correspond to different GnRH analogs, variants or epitopes, preventing them from retaining the ability to produce an immune response. Additionally, if the GnRH units are linked chemically or recombinantly to a carrier, the GnRH molecules can be linked to either the 5 'end, the 3' end or they can flank the carrier in question. Additionally, the GnRH multimer can be located at sites internal to the bearer. The GnRH multimers are further discussed in detail below. The term "GDRH immunogen" refers to the GnRH polypeptides, as described above, that produce an immune response without an associated immunological carrier, adjuvant or stimulant, as well as GnRH polypeptides capable of becoming immunogenic, or more immunogenic, by means of association with a carrier molecule, adjuvant or immunostimulant, or by mutation of a native sequence and / or by incorporation into a molecule containing multiple repeat units of at least one epitope of the GnRH molecule. The term can be used to refer to an individual macromolecule or a homogeneous or heterogeneous population of antigenic macromolecules derived from GnRH. In general, a GnRH immunogen will cause the formation of antibodies that cross-react with the endogenous GnRH that occurs naturally of the vertebrate species to which this immunogen is derived. The term "GnRH immunogen" also refers to nucleic acid molecules, such as DNA and RNA molecules that encode GnRH polypeptides that are capable of expression in vivo, when administered using nucleic acid distribution techniques described subsequently additionally. "Homology" refers to the percent identity between two polynucleotides or two portions of polypeptide. The two DNAs, or two polypeptide sequences are "substantially homologous" to each other when the sequence exhibits at least about 75% and 85%, preferably at least about 90%, and more preferably at least about 95% - 98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences that exhibit complete identity to the specified DNA or polypeptide sequence. The percent "identity" between two amino acid or polynucleotide sequences can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of correspondences between the two aligned sequences, dividing the length of the shortest sequence and multiplying the result by 100. Easily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, MO in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl. 3: 353-358, National biomedical Research Foundation, Washington, DC, which adapts the local homology algorithm of Smith and Waterman (1981) Advances in Appl. Math. 2: 482-489 for peptide analysis. Programs to determine nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wl) for example, the BESTFIT, FASTA and GAP programs that also depend on the Smith and Waterman algorithm. These programs are easily used with the default parameters recommended by the manufacturer and described in the sequence analysis package (Wisconsin Sequence Analysis Package) referred to above). For example, the percent identity of a particular nucleotide sequence to a reference sequence can be determined using the Smith and Waterman homology algorithm with a default scoring table and a separation penalty of six nucleotide positions. Alternatively, the identity can be determined by hybridization of the polynucleotides under conditions that form stable duplexes between the homologous regions, followed by digestion with individual strand-specific nuclease (s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a low Southern hybridization experiment, for example, severe conditions, as defined for that particular system. The definition of the appropriate hybridization conditions is within the skill of the technique. See, for example, Sambrook et. al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.

An "epitope" refers to any portion or region of a molecule with the ability or potential to produce and combine with a specific GnRH antibody. For purposes of the present invention, a polypeptide epitope will usually include at least about 3 amino acids, preferably at least about 5 amino acids, of the reference molecule. There is no critical upper limit to the length of the fragment, which may comprise close to the full length of a protein sequence, or even a fusion protein comprising two or more epitopes of a protein in question. Epitopes on polypeptide molecules can be identified using any number of epitope correlation techniques, well known in the art. See for example Mapping Epitope Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes can be determined for example by concurrently synthesizing large numbers of peptides on solid supports, peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still bound to the supports. This technique is known in the art and is described in for example, U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc. Nati Acad. Sci. USA 81: 3998-4002; Geysen et al. (1986) Molec. Immunol. 23: 709-715. Similarly, conformational epitopes can be easily identified in determining the spatial conformation of amino acids such as for example by x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, supra. Computer programs that formulate hydropathy scales from the amino acid sequence of the protein, using the hydrophobic and hydrophilic properties of each of the 20 amino acids, as described for example in: Kyte et al., J. Mol. Biol. (1982) 157: 105-132; and Hopp and Woods, Proc. Nati Acad. Sci. USA (1981) 78: 3824-3828, can also be used to determine antigenic portions of a given molecule. For example, the Hopp and Woods technique assigns each amino acid a numerical value of hydrophilicity and then repeatably averages these values along the peptide chain. The points of highest average, local hydrophilicities are indicative of antigenic portions of the molecule. By "immunological carrier" is meant any molecule that, when associated with a GnRH immunogen of interest, imparts immunogenicity to that molecule, or better to the immunogenicity of the molecule. Examples of suitable carriers include large, slowly metabolized macromolecules, such as: proteins, polysaccharides, such as sepharose, agarose, cellulose, cellulose beads and the like, polymeric amino acids such as polyglutamic acid, polylysine and the like; amino acid copolymers; inactive virus particles; bacterial toxins such as diphtheria toxoid, tetanus, cholera, leukotoxin molecules and the like. The carriers are described in further detail later. A GnRH immunogen is "linked" to a specific carrier molecule when the immunogen is chemically coupled or associated with the carrier, or when the immunogen is expressed from a chimeric DNA molecule encoding the immunogen and the carrier of interest . An "immunoconjugate" is a GnRH immunogen such as a GnRH peptide or multimer that binds to a carrier molecule, as defined above. The term "leukotoxin polypeptide" or "LKT polypeptide" is proposed for a polypeptide that is derived from a protein that corresponds to the family of molecules characterized by the consensus amino acid sequence of the carboxy terminus Gly-Gly-X- Gly-X-Asp (Highlander et al., (1989) DNA 8: 15-28), wherein X is Lys, Asp, Val or Asn. These proteins include, among others, leukotoxins derived from P. haemolytica and Actinobacillus pleuropneumoniae, as well as alpha-hemolysin from E. coli (Strathdee et al. (1987) Infect. Immun. 55: 3233-3236; Lo (1990) Can J. Vet. Res. 54: S33-S35; Welch (1991) Mol. Microbiol. 5: 521-528). This family of toxins is known as the "RTX" family of toxins (Lo (1990) Can, J. Vet, Res. 54: S33-S35). In addition, the term "leukotoxin polypeptide" refers to a leukotoxin polypeptide that is chemically synthesized, isolated from an organism expressing the same, or produced recombinantly. Additionally, the term is proposed for an immunogenic protein having an amino acid sequence substantially homologous to a continuous amino acid sequence found in the native, particular, leukotoxin molecule. In this way, the term includes both full-length and partial sequences, as well as the like. Although full-length, native leukotoxins exhibit cytotoxic activity, the term "leukotoxin" is also proposed for molecules that remain immunogenic even in the lack of cytotoxic character of native leukotoxins. The nucleotide sequences and the corresponding amino acid sequences for various leukotoxins are known. See, for example, U.S. Patent Nos. 4,957,739 and 5,055,400; Lo et al. (1985) Infect. Immun. 50: 667-67; Lo et al. (1987) Infect. Immun. 55: 1987-1996; Strathdee et al. (1987) Infect. Immun. 55: 3233-3236; Highlander et al. (1989) DNA 8: 15-28; and W Welch (1991) Mol. Microbiol. 5: 521-528. In preferred embodiments of the invention, leukotoxin chimeras having a selected leukotoxin polypeptide sequence that impart enhanced immunogenicity to one or more GnRH multimers fused thereto are provided. Particular examples of leukotoxin polypeptides, immunogenic for use in the present invention are truncated leukotoxin molecules described in U.S. Patent Nos. 5,476,657 and 5,837,268. These truncated molecules include LKT 352, LKT 111 and LKT 114. LKT 352 is derived from the lktA gene present in plasmid pAA352 (Accession No. ATCC 68283). The nucleotide sequence and the corresponding amino acid sequence of this gene are described in U.S. Patent No. 5,476,657. The gene codes for a truncated leukotoxin having 914 amino acids and an estimated molecular weight of approximately 99 kDa. LKT 111 is a leukotoxin polypeptide derived from the lktA gene present in plasmid pCBlll (Accession No. ATCC 69748). The nucleotide sequence of this gene and the corresponding amino acid sequence are described in U.S. Patent No. 5,837,268. The gene codes for a shortened version of leukotoxin that developed from the recombinant leukotoxin gene present in plasmid pAA352 (Accession No. ATCC 68283) by removing an internal DNA fragment of approximately 1300 bp in length. The LKT 111 polypeptide has an estimated molecular weight of 52 kDa (compared to the LKT 352 polypeptide of 99 kDa), but retains portions of the N-terminus of LKT 352 that contain epitopes of T cells that are necessary for sufficient immunogenicity of T cells, and portions of the C-terminus of LKT 352 that contain suitable restriction sites for use in the production of fusion proteins for use in the present invention. LKT 114 is derived from the gene present in plasmid pAA114 (described in U.S. Patent No. 5,837,268). LKT 114 differs from LKT 111 by virtue of additional amino acid suppression of the inner portion of the molecule. The term "immunological adjuvant" refers to an agent that acts in a non-specific manner to increase an immune response to a particular antigen, thereby reducing the amount of antigen needed from any given vaccine, and / or the frequency of injection required in order to generate an adequate immune response to the antigen of interest. See, for example, A.C. Allison J. Reticuloendothel. Soc. (1979) 26: 619-630. "Native" proteins, polypeptides or peptides are proteins, polypeptides or peptides isolated from the source in which the proteins naturally occur. "Recombinant" polypeptides refers to polypeptides produced by recombinant DNA techniques; that is, produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide. "Synthetic" polypeptides are those prepared by chemical synthesis. By "polynucleotide" is meant a nucleotide sequence that includes, but is not limited to, RNA such as mRNA, cDNA, genomic DNA sequences and even synthetic DNA sequences. The term also encompasses sequences that include any of the known base analogs of DNA and RNA. The term "derivative of", as used herein, denotes a real or theoretical source or origin of the molecule or immunogen of interest. For example, an immunogen that is "derived from" a particular GnRH molecule will carry a close sequence similarity to a relevant portion of the reference molecule. In this way, an immunogen that is "derived from" a particular GnRH molecule can include the entire wild-type GnRH sequence, or can be altered by insertion, deletion or substitution of amino acid residues, while the derived sequence provides an immunogen that corresponds to the GnRH molecule sought. Immunogens derived from an implicit molecule will contain at least one epitope specific to the implied molecule. By "vertebrate subject" is meant any member of the subphylum cordata, including without limitation, mammals such as sheep, bovine, porcine, equine, cervinos, including cattle, sheep, pigs, goats, horses, deer and humans; domestic animals such as domestic dogs and cats (dogs and cats); and birds, including domestic, wild and hunting birds such as roosters and chickens including chickens, turkeys and other gallinaceous birds, fish, rodents such as hamsters, guinea pigs, deer, gophers, marmots, lagomorphs, rabbits, urones, squirrels and reptilian subjects . The term does not denote a particular age or gender. In this way, it is proposed that both male and female adults and newborn animals, as well as fetuses and eggs, be covered. By "suppression" of reproductive behavior and / or fertility in a vertebrate subject is meant a significant reduction in mating behavior and / or fertility in an animal treated according to the invention, compared to mating behavior and / or fertility normally expected from a post-pubertal animal of the same species. In this way, a suppression in reproductive behavior and / or fertility may result in a reduction in the number of matings attempted, or a reduction in successful matings, such as a reduction in the number of conceptions, in the case of a female, or fertilizations, in the case of a male, compared to an untreated animal, typical of the same species. The suppression may result in complete sterility of the animal in question.

Without being bound by a particular theory, the effect may be the result of either suppression in the pituitary or hypothalamic suppression at the gonadal level, such as a suppression of testicular development or testicular function in the male and ovarian development or function in the female . By "long-term suppression" or "prolonged" reproductive behavior and / or fertility is meant a suppression of reproductive behavior and / or fertility, as described above, which persists after the GnRH antibody titers have been decreased. , or in the case of a GnRH analogue, such as a GnRH agonist or antagonists, which persists after the circulating blood level of the substance has declined below a pharmacologically active level. This effect may be permanent, or persistent for 6 months to a year or more after the GnRH antibody titers 0 the circulating blood level of the GnRH analogue has declined, preferably from 8 to 10 months to 1 year or more, up to the animal's life span. This prolonged effect can easily be terminated by measuring the levels of GnRH antibodies with blood levels of the analog using methods well known in the art, such as those described in the following examples. By "pre-pubertal" is meant any time before the onset of puberty. The term "pre-pubertal" also includes the peripubertal period. According to the present invention, the onset of puberty is indicated by the ability of an animal in question to conceive, in the case of a female, for example, the onset of ovulation, or to fertilize, for example, the ability to to produce sperm, in the case of a male. The typical timing for the onset of puberty for a given species is well known in the art. The onset of puberty is accompanied by the presence of several circulating hormones in the subject of interest. For example, clinical studies of puberty may be preceded by the increased pulsatile secretion of GnRH produced in the hypothalamus, followed by the increased sensitivity of the pituitary to GnRH that causes the release of LH and FSH from the pituitary gland. This, in turn, controls the production of testosterone in males and estrogen and progesterone in females. The secretion of gonadotropin, particularly LH, increases progressively during the peripubertal period resulting in gonadal stimulation, the secretion of sex hormones and progressive physical maturation. The levels of these hormones associated with puberty are known in the art and will vary from species to species. For a detailed description of puberty and the hormonal changes associated with puberty, see, for example, Apter D., Ann. NY Acad. Sci. (1997) 816: 9-21. Hormone levels, such as LH and FSH levels, can be measured using standard techniques such as radioimmunoassays (see, for example, Lee et al., J. Reproduc. Fertil. (1976) 46: 1-6; Bremner et al. al., Endocrin. (1980) 106: 329-336), as well as highly sensitive immunofluorometric assays (see, for example, Apter et al., J. Clin. Endocrinol. Metab. (1989) 68: 53-57; Dunkel et al. al., Pediatr Res. (1990) 27: 215-219; Gon et al., Pediatr Res. (1992) 31: 535-539; Wu et al., J. Clin. Endocrinol. Metab. (1991) 72: 1229-1237, and the commercially available assay, IFMA, Delfia, Wallac, Turku, Finland) and immunochemiluminometric assays (see, for example, Neely et al., J. Pediatr. (1995) 127: 40-46; Pandian et al., Clin. Chem. (1993) 39: 1815-1819). Similarly, in females, estradiol secretion can be measured using several well-known assays such as cell bioassays (see, for example, Klein, et al., J. Clin. Invest. (1994) 94: 2475-2480 k ). 2. General Methods Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or process parameters since these may vary of course. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting. Although various compositions and methods similar or equivalent to those described herein can be used, in the practice of the present invention, the preferred materials and methods are described herein. The central point of the present invention is the discovery of a method for reducing the circulating levels of GnRH, thereby achieving long-term suppression of reproductive performance and / or fertility, such as the suppression of testicular development or function in the male, or ovarian development or function in the female. The method can be achieved by passively or actively immunizing against endogenous GnRH and / or by using agonists or GnRH antagonists. Although GnRH is generally recognized as "self" and therefore not "immunogenic", the compositions described herein surprisingly provide a means to produce a long-term immune response in a subject immunized therewith. If immunization is used, the method involves one or more primary immunizations before the onset of puberty, optionally followed by one or more boosters, with the same or different GnRH composition, to cause long-term suppression as described above. Reinforcements can be given before, or after the onset of puberty. The timing of vaccinations depends on the animal in question which is generally a mammal such as a cat, dog, horse or deer. For example, in these species, a primary vaccination will generally be administered at about 0 to about 35 weeks of age, more preferably about 3 to about 15 weeks, preferably about 5 to about 12 weeks of age, and even more preferably at about 6 to about 10 weeks of age. One or more booster treatments may be given, before puberty, at an appropriate time after the primary immunization, generally at about 2 to about 10 weeks after the primary immunization, preferably at about 3 to about 4 weeks. after primary immunization. Additionally, additional immunizations can be given at regular intervals in order to ensure a prolonged effect on reproductive performance and / or fertility. For example, in one embodiment, additional immunizations are given at least 6 months to 1 year after the second immunization and can be administered annually thereafter. If multiple immunizations are used, for example, at least two vaccinations, the animal can be immunized post-pubertally in order to achieve a prolonged effect. The vaccine compositions of the present invention are administered directly to the animal in question (active immunization) or laboratory animals in order to produce antibodies which in turn can be used to immunize the animal in question (passive immunization). Additionally, passive immunization can be achieved using monoclonal antibodies, monospecific antisera, as well as preparations that include hybrid antibodies, altered antibodies, F (ab ') 2 fragments, F (ab) fragments, Fv fragments, domain antibodies. individual, chimeric antibodies, humanized antibodies and functional fragments thereof, described in greater detail below.

In a GnRH conjugate As explained above, GnRH is an endogenous molecule and as such, it may be desirable to further increase the immunogenicity of the GnRH polypeptides (or multimers described below) by linking them to carriers to form GnRH immunoconjugates. This is especially necessary if the GnRH immunogen will be administered to the same species from which it is derived. In general, suitable carriers are polypeptides that include antigenic regions of a protein derived from an infectious material such as a viral surface protein or a carrier peptide sequence. These carriers serve to non-specifically stimulate the activity of the T helper cells and to help direct an immunogen of interest to the cells presenting the antigen (APC) for processing and presentation on the cell surface in association with molecules of the main histocompatibility complex (MHC). Several carrier systems have been developed for this purpose. See, for example, often the small peptide haptens are coupled to protein carriers such as the limpet hemocyanin (Bittle et al (1982) Nature 298: 3033), bacterial toxins such as tetanus toxoid (Muller et al. (1982) Proc. Nati, Acad. Sci. USA 79: 569573), ovalbumin, leukotoxin polypeptides, and whale sperm myoglobin, to produce the immune response. These coupling reactions typically result in the incorporation of several moles of the peptide hapten per mole of the carrier protein. Other suitable carriers for use with the present invention include rotavirus VP6 polypeptides, or functional fragments thereof, as described in U.S. Patent No. 5,071,651. Also useful is a fusion product of a viral protein and one or more epitopes from GnRH, fusion products that are made by 'methods described in U.S. Patent No. 4,722,840. Still other suitable carriers include cells, such as lymphocytes, since the presentation of this form mimics the natural mode of presentation in the subject, which leads to the immunized state. Alternatively, the GnRH immunogens can be coupled to erythrocytes, preferably the erythrocytes of the subject. Methods for coupling peptides to proteins or cells are known to those skilled in the art. Distribution systems useful in the practice of the present invention can also utilize carriers in the form of particles. For example, preformed particles have been used as a platform on which immunogens can be coupled and incorporated. Protensome-based systems (Lowell et al (1988) Science 240: 800-802) and immunostimulatory complexes (Morein et al (1984) Nature 308: 457-460), are also known in the art. Carrier systems using recombinantly produced chimeric proteins that self-assemble into particles can also be used with the present invention. For example, the yeast retrotransposon, Ty, codes for a series of proteins that are mounted on virus-like particles (Ty-VLPs, Kingsman et al. (1988) Vaccines 6: 304-306). In this manner, a gene, or a fragment thereof, which codes for the GnRH immunogen of interest can be inserted into the TyA gene which is expressed in yeast as a fusion protein. The fusion protein retains the ability to self-assemble into particles of uniform size.

Other useful virus-like carrier systems are based on HBsAg, (Valenzuela et al (1985) Bio / Technol.3: 323-326; U.S. Patent No. 4,722,840; Delpeyroux et al. (1986) Science 233: 472- 475), hepatitis B core antigen (Clarke et al. (1988) Vaccines 88 (Ed. H. Gmsberg, et al.) Pp. 127-131), Poliovirus (Burke et al. (1988) Nature 332: 81 -82), and tobacco mosaic virus (Haynes et al. (1986) Bio / Technol. 4: 637-641). Especially preferred carriers include serum albumins, lapam emotion, ovalbumin, whale sperm myoglobin, leukotoxin molecules as described above, and other proteins well known to those skilled in the art. For example, chimeric systems using a leukotoxin polypeptide, as defined above, such as a leukotoxin polypeptide (LKT) from Pasteurella haemolytica fused to the antigen of interest, may also be used herein. In this regard, the nucleotide sequences and the corresponding amino acid sequences for various leukotoxin carriers are known. See, for example, U.S. Patent Nos. 5,422,110, 5,708,155, 5,723,129 and International Publications Nos. WO 98/06848 and WO 96/24675. Particular examples of leukotoxin polypeptides, immunogenic for use herein include LKT 342, LKT 352, LKT 111, LKT 326 and LKT 101 which are described in the patents and publications cited above. Particularly preferred are LKT 111 and LKT 114. The gene encoding LKT 111 was developed from the recombinant leukotoxin gene present in plasmid pAA352 (Accession No. ATCC 68283) by removal of an internal DNA fragment of approximately 1300 bp. of length. The LKT 111 polypeptide has an estimated molecular weight of 52 kDa (compared to the LKT 352 polypeptide of 99 kDa), but retains portions of the N-terminus of LKT 352 containing T-cell epitopes that are necessary for sufficient immunogenicity of T cells. , and portions of the C-terminus of LKT 352 containing convenient restriction sites for use in the production of fusion proteins of the present invention. LKT 114 differs from LKT 111 by virtue of an additional amino acid expression of the inner portion of the molecule. See, for example, U.S. Patent No. 5,837,268 and International Publications Nos. WO 98/06848 and WO 96/24675 for descriptions of these molecules. Protein carriers can be used in their native form or their content of functional groups can be modified for example by succinylation of lysine residues or reaction with Cys-thiolactone. A sulfhydryl group can also be incorporated into the carrier (or antigen) for example by reaction of the amino functions with 2-iminothiolane or the N-hydroxysuccinimide ester of 3- (4-dithiopyridyl) -propinonate. Suitable carriers can also be modified to incorporate spacer arms (such as hexamethylene diamine or other bifunctional molecules of similar size) for the binding of peptide immunogens. The carriers can be physically conjugated to the GnRH immunogen of interest, using normal coupling reactions. Alternatively, chimeric molecules can be prepared recombinantly for use in the present invention, such as by fusing a gene encoding a suitable polypeptide carrier to one or more copies of a gene, or fragment thereof, which codes for a selected RH gene immunogen. The GnRH portion can be fused either 5 'or 3' of the carrier portion of the molecule, or the GnRH portion can be located at sites internal to the carrier molecule. GnRH immunogens can also be administered via a carrier virus that expresses the same. Carrier viruses that found use in the present include, but are not limited to, vaccinia and pox viruses, adenoviruses and herpes viruses. By way of example, the vaccinia virus recombinants expressing the proteins can be constructed as follows. The DNA encoding a particular protein is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking the vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells that are simultaneously infected with vaccinia. The homologous recombination serves to insert the vaccinia promoter plus the gene encoding the desired immunogen in the viral genome. The recombinant TK, resulting can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and collecting the viral plaques resistant to it.

GnRH Multimers Also, the immunogenicity of GnRH immunogens can be increased significantly by producing immunogenic forms of the molecules comprising multiple copies of the selected epitopes. In this way, endogenous GnRH can become an effective autoantigen. Accordingly, in one aspect of the invention, vaccine compositions containing multimers of the GnRH immunogen are provided in either the nucleic acid or peptide form. The GnRH multimer will have more than one copy of the selected GnRH immunogens, peptides or epitopes, as described above, or multiple tandem repeats of a selected GnRH immunogen, peptide or epitope. In this manner, the GnRH multimers may comprise either multiple or tandem repeats of the selected GnRH sequences, multiple or tandem repeats of the selected GnRH epitopes, or any conceivable combination thereof. The GnRH epitopes can be identified using techniques as described in detail above. For example, the GnRH multimer may correspond to a molecule with repeat units of the General Formula (GnRH-X-GnRH), wherein GnRH is a GnRH immunogen, X is selected from the group consisting of a peptide bond, a amino acid spacer group, a carrier molecule and [GnRH] n, where n is an integer greater than or equal to 1; and is an integer greater than or equal to l, and wherein additionally "GnRH" can comprise any GnRH immunogen. In this manner, the GnRH multimer can contain from 2 to 64 or more GnRH immunogens, preferably 2-34 or 2-16 GnRH immunogens. Additionally, the selected sequences of GnRH immunogens may be all or the same, or may correspond to different GnRH derivatives, analogs, variants or epitopes as long as they maintain the ability to produce an immune response. Additionally, if the GnRH immunogens are linked either chemically or recombinantly to a carrier, the GnRH immunogens can be linked to either the 5 'end, the 3' end or they can flank the carrier in question. Additionally, the GnRH multimer can be located at sites internal to the bearer. A particular carrier for use with the present GnRH multimers is a leukotoxin polypeptide as described above. As explained above, the spacer sequences may be present between the GnRH portions. For example, Ser-Gly-Ser trimers and Gly-Ser dimers are present in the GnRH multimers exemplified herein that provide spacers between the repeat sequences of the GnRH immunogens. See, for example, Figure 5B. The strategy placement of the various spacer sequences among the selected GnRH immunogens can be used to confer increased immunogenicity in the present constructs. Accordingly, according to the invention, a selected spacer sequence can encode a wide variety of portions such as an individual amino acid linker or a sequence of two or more amino acids. The selected spacer groups can preferably provide enzyme cleavage sites so that the expressed multimer can be processed by proteolytic enzymes in vi (by APC, or the like) to produce various peptides, each of which contains at least one T cell epitope derived from the carrier portion, and which are preferentially fused to a substantially complete GnRH polypeptide sequence. The spacer groups can be constructed such that the binding region between the selected GnRH portions comprises a sequence clearly foreign to the immunized subject, thereby conferring enhanced immunogenicity on the associated GnRH immunogens. Additionally, the separator sequences can be constructed to provide T-cell antigenicity, such as those sequences encoding antiphatic and / or a-helical peptide sequences that are generally recognized in the art to provide immunogenic epitopes of helper T cells. The choice of epitopes of the particular T cells to be delivered by these spacer sequences may vary depending on the particular vertebrate species to be vaccinated. Although particular GnRH portions are exemplified as including spacer sequences, it is also an object of the invention to provide one or more GnRH multimers comprising directly adjacent GnRH sequences (without intervening splicing sequences). The multimeric GnRH sequence produced in this manner returns to a highly immunogenic GnRH antigen for use in the compositions of the invention. Polypeptides, immunoconjugates and GnRH multimers can be produced using the methods described below and used for nucleic acid immunization, gene therapy, protein-based immunization methods and the like.

Nucleic Acid-based Immunization Methods In general, nucleic acid-based vaccines for use with the present invention will include relevant regions coding for a GnRH immunogen, appropriate control consequences, and optionally, therapeutic, auxiliary nucleotide sequences. Nucleic acid molecules are prepared in the form of vectors that include the elements necessary to direct transcription and translation in a recipient cell. In order to increase an immune response in an immunized subject, the nucleic acid molecules can be administered in conjunction with auxiliary substances, such as pharmacological agents, adjuvants or in conjunction with the distribution of vectors encoding biological response modifiers such as cytokine and the like. Other auxiliary substances include, but are not limited to, substances for increasing weight gain, body mass or muscular resistance, such as growth hormones, or growth-promoting agents, β-antagonists, dividing agents and antibiotics.

The nucleotide sequences selected for use in the present invention can be derived from known sources, for example, by isolating them from cells or tissue containing a desired gene or nucleotide sequence using standard techniques, or by using recombinant or synthetic techniques. Once coding sequences for the immunogens have been prepared and isolated. of GnRH, these sequences can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those skilled in the art, and the selection of an appropriate testing vector is a matter of choice. Ligations to other sequences, for example, auxiliary molecules or carrier molecules, are performed using standard procedures known in the art. One or more portions of GnRH immunogen from the chimera can be fused 5 'and / or 3' to a desired helper sequence or carrier molecule. Alternatively, one or more portions of GnRH immunogen can be located at sites internal to the carrier molecule, or these portions can be placed at both terminal and internal locations on the chimera. Alternatively, the DNA sequences encoding the GnRH immunogens of interest, optionally linked to the carrier molecules, can be synthetically prepared instead of being cloned. The DNA sequences can be digested with codons appropriate for the particular sequence. The complete sequence of the immunogen is then assembled from overlapping oligonucleotides prepared by normal methods and assembled and assembled in a complete coding sequence. See, for example, Edge (1981) Nature 292: 756; Nambair et al. (1984) Science 223: 1299; and Jay et al. (1984) J. Biol. Chem. 259: 6311. The coding sequence is then placed under the control of suitable control elements for expression in suitable host tissue, in vi vo. The choice of control elements will depend on the subject and the type of preparation used. Thus, if the endogenous transcription and translation machinery of the subject is used to express the immunogens, the control elements compatible with the particular subject will be used. In this regard, several promoters are known in the art for use in mammalian systems. For example, typical promoters for expression of mammalian cells include the SV40 early promoter, a CMV promoter such as the CVM immediate early promoter, the LTR promoter of the mouse mammary tumor virus, the late major promoter of adenovirus (Ad MLP), and the herpes simplex virus promoter, among others. Other non-viral promoters such as a promoter derived from the murine metallothionein gene also found use for expression in mammals. Typically, the transcription termination and polyadenylation sequences will also be present, located 3 'to the translation finalizing codon. Preferably, a sequence for the optimization of translation initiation, located 5 'to the coding sequence, is also present. Examples of transcription / polyadenylation terminator signals include those derived from CV40, as described in Sambrook et al., Supra, as well as the bovine growth hormone terminator sequence. Introns, which contain the splice donor and acceptor sites, are also designated in the constructions for use with the present invention. Intensifying elements can also be used in the present to increase the expression levels of the constructions. Examples include the SV40 early gene enhancer (Dijkema et al (1985) EMBO J. 4: 761), the enhancer / promoter derived from the long terminal repeat (LTR) of Rous Sarcoma virus (Gorman et al. al. (1982) Proc. Nati, Acad. Sci. USA 79: 6777) and elements derived from human CMV (Boshart et al., (1985) Cell 41: 521), such as elements included in the intron sequence. A of CMV. Once prepared, the nucleic acid vaccine compositions can be delivered to the subject using known methods. In this regard, several techniques have been described for immunization with DNA encoding the antigen. See, for example, U.S. Patent No. 5,589,466 to Felgner et al.; Tang et al. (1992) Nature 358: 152; Davis et al. (1993) Hum. Molec. Genet 2: 1847; Ulmer et al. (1993) Science 258: 1745; Wang et al. (1993) Proc. Nati Acad. Sci. USA 90: 4156; Eisenbraun et al. (1993) DNA Cell Biol. 12: 791; Fynan et al. (1993) Proc. Nati Acad. Sci. USA 90: 12476; Fuller et al. (1994) AIDS Res. Human Retrovir. 10: 1433; and Raz et al. (1994) Proc. Nati Acad. Sci. USA 91: 9519. General methods for the delivery of nucleic acid molecules to cells in vitro may also be used for subsequent reintroduction of the host, such as liposome-mediated gene transfer. See for example Hazinski et al. (1991) Am. J. Respir. Cell Mol. Biol. 4: 206-209; Brigham et al. (1989) Am. J. Med. Sci. 298: 278-281; Canonical et al. (1991) Clin. Res. 39: 219A; and Nabel et al. (1990) Science 249: 1285-1288. In this manner, the nucleic acid vaccine compositions can also be delivered in either the liquid or particulate form using a variety of known techniques. Typical vaccine compositions are described more fully below.

Protein-based distribution methods Protein-based compositions can also be produced using a variety of methods known to those skilled in the art. In particular, GnRH polypeptides can be isolated directly from native sources, using normal purification techniques. Alternatively, the polypeptides can be produced recombinantly using nucleic acid expression systems, well known in the art and described for example in Sambrook et al., Supra. GnRH polypeptides can also be synthesized using chemical polymer synthesis such as solid phase peptide synthesis. These methods are known to those skilled in the art. See for example J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, IL (1984) and G. Barany and R.B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, Academic Press, New York, (1980), pp. 3-254, for solid phase peptide synthesis techniques. GnRH polypeptides for use in the compositions described herein may also be produced by cloning the coding sequences therefor into any suitable expression vector or replicon. Numerous cloning vectors are known to those skilled in the art and the choice of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning, and host cells that can be transformed, include bacteriophage lambda (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFRl (gram-negative bacteria), pME290 (gram-negative bacteria not from E. coli), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pIJßl (Streptomyces), pUC6 ( Streptomyces), YIp5 (Saccharomyces), YCpl9 (Saccharomyces) and bovine papilloma virus (mammalian cells). See in general, DNA Cloning: Vols. I & II, supra; Sambrook et al., Supra; B. Perbal, supra. For example, the coding sequences for porcine, bovine and ovine GnRH have been determined (Murad et al (1980) Hormones and Hormone Antagonists, in The Pharmacological Basis of Therapeutics, Sixth Edition), and the cDNA for human GnRH is has cloned so that its sequence has been well established (Seeburg et al. (1984) Nature 311: 666-668). Additional GnRH polypeptides of known sequences have been described, such as the GnRH molecule is presented in salmon and chickens (International Publication No. WO 86/07383, published December 18, 1986). The particular GnRH coding sequences for use with the present invention are shown in Figures 5A and 5B herein. The GnRH coding sequence is highly conserved in vertebrates, particularly in mammals, and the porcine, bovine, ovine and human GnRH sequences are identical to each other. Portions of these sequences encoding the desired GnRH polypeptides, and optionally, a sequence encoding a carrier protein, can be cloned, isolated and ligated together using recombinant techniques generally known in the art. See, for example, Sambrook et al. , supra. The gene can be placed under the control of a promoter, the ribosome binding site (for bacterial expression) and optionally an operator, so that the DNA sequence of interest is transcribed into RNA by a suitable transformant. The coding sequence may or may not contain a signal peptide or a leader sequence, the polypeptides may be expressed using for example the tac promoter of E. coli or the promoter of the protein A gene (spa) and the signal sequence. The guide sequences can be removed by the bacterial host in post-transductional processing. See, for example, U.S. Patent Nos. 4,431,739; 4,425,437; 4,338, 397. Auxiliary sequences may also be present, such as those described above. In addition to the control sequences, it may be desirable to add regulatory sequences that allow the regulation of the expression of the polypeptide sequences relative to the growth of the host cell. Regulatory sequences are known to those skilled in the art and examples include those which cause the expression of a gene to be activated or deactivated in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences. An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the placement and orientation of the coding sequence with respect to the control sequences which is such that the coding sequence is transcribed under the "control" of the control sequence (ie, the RNA polymerase that binds to the DNA molecule in the control sequences transcribes the coding sequence). Modification of the sequences encoding the particular GnRH polypeptide may be desirable to achieve this purpose. For example, in some cases it may be necessary to modify the sequence so that the control sequences can be attached in the appropriate orientation; that is, to maintain the reading frame. The control sequences and other regulatory sequences can be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site. In some cases, it may be desirable to add sequences that cause secretion of the polypeptide from the host organism, with subsequent cleavage of the secretory signal sequence. It may also be desirable to produce mutants or analogs of the polypeptide. Mutants or analogs can be prepared by deleting a portion of the sequence encoding the reference polypeptide, or if present, a portion of the sequence encoding the desired carrier molecule, by inserting a sequence, and / or by substitution of one or more nucleotides within the sequence. Techniques for modifying the nucleotide sequence, such as sequence directed mutagenesis, and the like, are well known to those skilled in the art. See, for example, Sambrook et al., Supra; DNA Cloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra; Kunkel, T.A. Proc. Nati Acad. Sci. USA (1985) 82: 448; Geisselsoder et al. BioTechniques (1987) 5: 786; Zoller and Smith, Methods Enzymol. (1983) 100: 468; Dalbie-McFarland et al. Proc. Nati Acad. Sci USA (1982) 79: 6409.

GnRH polypeptides can be expressed in a wide variety of systems, including insect, mammalian, bacterial, viral and yeast expression systems, all well known in the art. For example, insect cell expression systems, such as baculovirus systems, are known to those skilled in the art and are described in, for example, Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus / insect cell expression systems are commercially available in the form of equipment from, inter alia, Invitrogen, San Diego, CA ("MaxBac" equipment). Similarly, bacterial and mammalian cell expression systems are well known in the art and are described in, for example, Sambrook et al., Supra. Yeast expression systems are also known in the art and are described for example in Yeast Genetic Engineering (Barr et al., Eds., 1989) Butterworths, London. Various host cells suitable for use with the above systems are also known. For example, mammalian cell lines are known in the art and include mortalized cell lines available from the American Collection of Species Collection (ATCC-American Type Culture Collection), such as, but not limited to, Chinese hamster ovary (CHO), HeLa cells, infant hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g. Hep G2), Madin bovine kidney cells -Darby ("MDBK"), as well as others. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., Found use with the present expression constructs. Yeast hosts useful in the present invention include inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltose, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa californica, Bo byx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni. Depending on the expression system, and the selected host, the GnRH polypeptides are produced with growing host cells transformed by an expression vector described immunogens include polyclonal and monoclonal antibody preparations, monospecific antisera, as well as preparations including hybrid antibodies, antibodies altered, F (ab ') 2 fragments, F (ab) fragments, Fb fragments, individual domain antibodies, chimeric antibodies, humanized antibodies and functional fragments thereof, which retain specificity for the target molecule in question. For example, an antibody can include variable regions or fragments of variable regions, which retain specificity for the molecule in question. The rest of the antibody can be derived from the species in which the antibody will be used. In this way, if the antibody is to be used in a human, the antibody can be "humanized" in order to reduce the immunogenicity while still retaining the activity. For a description of the chimeric antibodies, see for example, Winter, G. and Milstein, 'C. (1991) Nature 349: 293-299; Jones, P.T. et al. (1986) Nature 321: 522-525; Riechmann, L. et al. (1988) 332: 323-327; and Cárter, P. et al. (1992) Proc. Nati Acad. Sci. USA 89: 4285-4289. These chimeric antibodies can contain not only combination sites for the target molecule, but also binding sites for other proteins. In this way, bifunctional reagents with targeted specificity to both external and internal antigens can be generated. If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunized with the desired antigen, or its fragment, or a mutated antigen, as described above. Prior to immunization, it may be desirable to further increase the immunogenicity of a particular immunogen. This can be accomplished in any of several ways known to those skilled in the art. For example, immunization for the production of antibodies is generally carried out by mixing or emulsifying the protein in a suitable excipient, such as saline, preferably in an adjuvant such as Freund's complete adjuvant, or any of the described adjuvants. subsequently, indicate the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). The animal is generally strengthened 2-6 weeks later with one or more infections of the protein in saline, preferably using incomplete Freund's adjuvant or the like. Antibodies can also be generated by in vitro immunization, using methods known in the art. Polyclonal antisera are then obtained from the immunized animal and treated according to known procedures. See for example Jurgens et al. (1985) J. Chrom. 348: 363-370. If the serum containing polyclonal antibodies is used, the polyclonal antibodies can be purified by immunogenicity chromatography, using known procedures. Monoclonal antibodies are generally prepared using the method of Kohler and Milstein, Nature (1975) 256: 495-96, or a modification thereof. Typically, a mouse or rat is immunized as described above. However, instead of bleeding the animal to extract the serum, the spleen is removed (and optionally several large lymph n) and disassociated into individual cells. If desired, the spleen cells can be selected (after removal of specifically nonadherent cells) when applying a cell suspension to a plate or cavity coated with the protein antigen. B cells, which express the immunoglobulin bound to the membrane specific for the antigen, will bind to the plate, and will not rinse with the rest of the suspension. The resulting B cells, or all of the dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, then cultured in a selective medium (eg, hypoxanthine, aminopterin, thymidine, "HAT" medium). The resulting hybridomas are plated by limiting the reduction and titrated for the production of antibodies that specifically bind to the immunizing antigen (and which do not bind to the unrelated antigens). Hybridomas secreted by the selected monoclonal antibodies are then cultured either in vitro (for example in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice). See, for example, M. Schreier et al., Hybridoma Techniques (1980); Hammerling et al., Monoclonal Antibodies and T-cell Hybridomas (1981); Kennett et al., Monoclonal Antibodies (1980); see also United States Patents Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,452,570; 4,466,917; 4,472,500, 4,491,632; and 4,493,890. The panels of monoclonal antibodies raised against the GnRH immunogen of interest, or fragment thereof, can be selected for various properties, i.e., for isotype, epitope, affinity, etc. Functional fragments of the antibodies can also be made against the GnRH immunogen of interest and can be produced by cleaving a constant region, not responsible for the antigen binding, of the antibody molecule, using for example pexin, to produce the fragments of F (ab ') 2. These fragments will contain two antigen-binding sites, but lack a portion of the constant region of each of the heavy chains. Similarly, if desired, the Fab fragments, which comprise an individual antigen binding site, can be produced, for example, by digestion of the polyclonal and monoclonal antibodies with papain. Functional fragments, which include only the variable regions of the heavy and light chains, can also be produced, using standard techniques. These fragments are known as Fv. Humanized chimeric antibodies can also be produced using the present immunogens. These antibodies can be designed to minimize unwanted immunological reactions attributable to variable regions of species specific structure and heterologous constants, typically present in monoclonal and polyclonal antibodies. For example, if antibodies are to be used in human subjects, they can be additionally later. The compositions of the present invention may also include auxiliary substances, such as pharmacological agents, cytokines or other biological response modifiers. As explained above, the vaccine compositions of the present invention may include adjuvants to further increase the immunogenicity of the GnRH immunogen. Adjuvants may include for example, emulsifiers, muramyl dipeptides, avridine, aqueous adjuvants such as aluminum hydroxide, chitosan-based adjuvants, and any of the various saponins, oils and other substances known in the art. For example, compounds that can serve as emulsifiers herein include natural and synthetic emulsifying agents, as well as anionic, cationic and nonionic compounds. Among the synthetic compositions, the anionic emulsifying agents include, for example, the potassium, sodium and ammonium salts of lauric and oleic acid, the calcium, magnesium and aluminum salts of fatty acids (ie, metallic soaps), and organic sulfonates such as sodium lauryl sulfate. Synthetic cationic agents include, for example, cetyltrimethylammonium bromide, while the synthetic nonionic agents are exemplified by glyceryl esters (e.g., glyceryl monostearate), polyoxyethylene glycol esters and ethers, and sorbitan fatty acid esters (e.g., sorbitan monopalmitate) and their polyoxyethylene derivatives (e.g. polyoxyethylene sorbitan monopalmitate). Natural emulsifying agents include acacia gum, gelatin, lecithin and cholesterol. Other suitable adjuvants can be formed with an oil component, such as an individual oil, or a mixture of oils, a water-in-oil emulsion, or an oil-in-water emulsion. The oil can be a mineral oil, a vegetable oil, or an animal oil. Mineral oil, or oil-in-water emulsions in which the mineral oil component is most preferred. In this regard, a "mineral oil" is defined herein as a mixture of liquid hydrocarbons obtained from petrolatum via a distillation technique; the term is synonymous with "liquid paraffin", "liquid petrolatum" and "white mineral oil". The term is also proposed to include "light mineral oil", that is, an oil that is obtained in a similar manner by distillation of petrolatum, but which has a specific weight 77 slightly smaller than the white mineral oil. See, for example, Remington's Pharmaceutical Sciences, supra. A particularly preferred oil component is the oil-in-water emulsion sold under the trade name EMULSIGEN PULUS ™ (comprising a light mineral oil as well as 0.05% formalin, and 3 mcg / ml gentamicin as preservatives), available from MVP Laboratories, Ralston , Neb'raska. Suitable animal oils include, for example, cod liver oil, halibut oil, shad oil, rough orange oil and shark liver oil, all of which are commercially available. Suitable vegetable oils include, without limitation, canola oil, almond oil, cottonseed oil, corn oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil, and Similar. Alternatively, several aliphatic nitrogen bases can be used as adjuvants with the vaccine formulations. For example, known immunological adjuvants include amines, quaternary ammonium compounds, guanidines, benzamidines and thiourunia (Gall, D. (1966) Immunology 11: 369-386). Specific compounds include dimethyldioctadecylammonium bromide (DDA) (available from Kodak), and N, N-dioctadecyl-N, N-bis (2-hydroxyethyl) propanediamine ("avridine"). The use of DDA as an immunological adjuvant has been described; see for example; the Kodak Laboratory Chemicals Bulletin 56 (1): 1-5 (1986); Adv. Drug Deliv. Rev. 5 (3): 163-187 (1990); J. Controlled Reléase 7: 123-132 (1988); Clin. Exp. Immunol. 78 (2): 256-262 (1989); J. Immunol. Methods 97 (2): 159-164 (1987); Immunology 58 (2): 245-250 (1986); and Int. Arch. Allergy Appl. Immunol. 68 (3): 201-208 (1982). Avridine is also a well-known adjuvant. See, for example, U.S. Patent No. 4,310,550 to Wolff, III et al., Which discloses the use of N, N-higher alkyl-N ', N' -bis (2-hydroxyethyl) propane diamines in general and the avridine in particular, as vaccine adjuvants. U.S. Patent No. 5,151,267 to Babiuk, and Babiuk et al. (1986) Virology 159: 57-66, also refers to the use of avridine as a vaccine adjuvant. Particularly preferred for use herein is a known adjuvant, "VSA-3" which is a modified form of the adjuvant EMULSIGEN PLUS ™ including DDA (see, United States Patent Application, allowed, Serial No. 08 / 463,837). ). Current methods for preparing these dosage forms are known, or will be evident, for those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 18th edition, 1990. The composition or formulation to be administered will contain an amount of the appropriate GnRH polypeptide to achieve the desired state in the subject being treated. The compositions of the present invention are usually prepared as injectables, either as liquid solutions or suspensions, or as solid forms which are suitable for solution or suspension in liquid carriers prior to injection. The preparation can also be emulsified by the active ingredient encapsulated in liposome vehicles or other carriers in particulate form is used. The compositions can also be prepared in solid form. For example, solid particle formulations can be prepared for distribution from commercially available needleless injection devices. Alternatively, solid dose implants for implantation in a subject can be provided. Sustained controlled release formulations can also be used and elaborated by incorporating the GnRH polypeptides into carriers or vehicles such as liposomes, non-resorbable waterproof polymers such as Liposomes. such as ethylene-vinyl acetate copolymers and Hytrel® copolymers, inflatable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures. Additionally, the polypeptides can be formulated into compositions either in natural or saline form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acids such as for example hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric acids , mandélico and similars. Salts formed from free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and these organic bases such as isopropylamine, trimethylamine, 2-ethylamino-ethanol , histidine, procaine and the like. The composition is formulated to contain an effective amount of GnRH polypeptide, the exact amount which is readily determined by one skilled in the art, wherein the amount depends on the animal being treated, in the case of a vaccine composition, the capacity of the animal's immune system to synthesize antibodies, and the degree of immunoneutralization of the desired GnRH. For purposes of the present invention, formulations include about 1 μg to about 2 mg, more generally about 5 μg to about 800 μg, most particularly from 10 μg to about 400 μg of GnRH polypeptide per mL of injected solution should be adequate to elicit an immune response when administered. If a peptide-carrier chimera is used, the ratio of the immunogen to the carrier to the vaccine formulation will depend on the particular carrier and the selected immunogen to construct these molecules. For example, if a leucotoxin-GnRH chimera is used, the ratio of GnRH to leukotoxin in the vaccine formulation will vary based on the particular portions of leukotoxin and the GnRH polypeptide selected to construct these molecules. A preferred vaccine composition contains a leukotoxin-GnRH chimera having about 1 to 90% GnRH, preferably about 3 to 80% and more preferably about 10 to 10% of the GnRH polypeptide per molecule of fusion. The 82 Increases in the percentage of GnRH present in the LKT-GnRH fusions reduce the amount of the total antigen that must be administered to a subject in order to produce a sufficient immune response to GnRH. The subject is administered with one of the compositions described above, for example, in a primary immunization, before puberty, in at least one dose, and optionally, two or more doses. The primary administration (s) is followed by one or more reinforcers with the same or different GnRH composition and either pre-pubertal, post-pubertal, or both, in order to result in a deletion prolonged development or testicular function in males or ovarian development or function in females. Any means of pharmaceutical distribution suitable for distributing the compositions to the vertebrate subject can be employed. For example, syringes with conventional needles, spring or compressed gas injectors (air) (U.S. Patent Nos. 1,605,763 to Smoot; 3,788,315 to Laurens; 3,853,125 to Clark et al; 4,596,556 to Morrow et al .; and 5,062,830 to Dunlap), liquid jet injectors (U.S. Patent Nos. 2,754,818 to Scherer, 3,330,276 to Gordon, and 4,518,385 to Lindmayer et al.), and particle injectors (Patents 83 from the United States Nos. 5,149,655 to McCabe et al. and 5,204,253 to Sanford et al.) are all suitable for the distribution of the compositions. Preferably, the composition is administered intramuscularly, subcutaneous, intravenous, subdermal, intradermal, transdermal or transmucosal to the subject. If a jet injector is used, an individual jet of the liquid vaccine composition is ejected under high pressure and velocity, for example 1200-1400 PSI, thereby creating an opening in the skin penetrating to depths suitable for immunization. Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. 3. Experimental part Example 1 Construction of pCB! 30 Plasmid pCB130 was used to produce a GnRH fusion protein for use in the examples described below. This construction of GnRH 84 contains 8 tandem repeats of the GnRH sequence fused to both the 5 'and 3' ends of a DNA sequence encoding a leukotoxin carrier-polypeptide. Each alternating GnRH sequence has a change in the fourth base in the guanoasin cytokine sequence. This results in an individual change of amino acid in the second amino acid of the GnRH molecule from His to Asp. See, Figures 5A and 5B. The leukotoxin portion of the construct codes for a shortened version of leukotoxin that was developed from the recombinant leukotoxin gene present in plasmid pAA352 (Accession No. ATCC 68283 and described in U.S. Patent No. 5,476,657) by Removal of an internal DNA fragment of approximately 1300 bp in length. The leukotoxin polypeptide has an estimated molecular weight of 52 kDa and contains convenient restriction sites for use in the protection of fusion proteins of the present invention. The chimeric construct is under the control of the Tac promoter and the emulsion is controlled through the use of Lac I. The GnRH-leukotoxin fusion protein produced by the plasmid pCB130 is shown in Figures 6A through 6F. Plasmid pCB130 was prepared as follows. The leukotoxin gene was isolated as described in U.S. Patent Nos. 5,476,657 and 5,837,268. In particular, to isolate the leukotoxin gene, gene libraries of P. haemolytica Al (strain B122) were constructed using standard techniques. See, Lo et al., Infect. Immun., Supra; DNA CLONING: Vols. I and II, supra; and Sambrook et al., supra. A genomic library was constructed in the pUC13 plasmid vector and a DNA library constructed in the bacteriophage lambda gtll. The resulting clones were used to transform E. coli and individual colonies were mixed and selected for the reaction with serum from a calf that has survived P. haemolytica infection and that has been boosted with a concentrated culture supernatant of P. haemolytica. to increase the levels of anti-leukotoxin antibody. Positive colonies were selected for their ability to produce leukotoxin by incubating cell lysates with bovine neutrophils and subsequently measuring the release of lactate dehydrogenase from the latter. Several positive colonies were identified and these recombinants were analyzed by correlation with restriction endonucleases. One clone appeared to be identical to a previously cloned leukotoxin gene. 86 See, Lo et al., Infect. Immun., Supra. To confirm this, small fragments were cloned and restriction maps were compared. It was determined that approximately 4 kilobases of DNA bases have been cloned. Larger clones were progressively isolated by carrying out a chromosomal walk (in the 5 'to 3' direction) in order to isolate the full-length recombinants that were approximately 8 kb in length. The final construction was called pAA114. This construct contained the sequence of the leukotoxin gene, complete. lktA, a restriction endonuclease fragment of Mael from pAA114 that contained the complete leukotoxin gene, was treated with the Klenow fragment of DNA polymerase I plus nucleotide triphosphates and ligated into the Smal site of the pUC13 cloning vector. This plasmid was called pAAl79. From this, two expression constructs were made in the vector pGH432 based on ptac: lacl digested with Smal. One, pAA342, consisted of the 5'-AhaIII fragment of the lktA gene while the other, pAA345, contained the complete Mael fragment described above. Clone pAA342 expressed a truncated leukotoxin peptide at high levels while pAA345 expressed full-length leukotoxin at 87 very low levels. Therefore, the 3 'end of the lktA gene (Styl-BamHI fragment from pAA345) was ligated to pAA342 digested with Styl BamHI, yielding the plasmid pAA352. The leukotoxin of P. haemolytica produced from the construction pAA352 is subsequently referred to herein as LKT 352. The plasmid pAA352 was then used to prepare a shortened version of the recombinant leukotoxin polypeptide. The shortened LKT gene was produced by deleting an internal DNA fragment about 1300 bp in length from the recombinant LKT gene as follows. Plasmid pCB113 (ATCC Accession No. 69749 and described in U.S. Patent No. 5,837,268) including the LKT 352 polypeptide, was digested with the restriction enzyme BstBl (New England Biolabs). The resulting linearized plasmid was then digested with bean nuclease (Pharmacia) to remove the individual strand leaving term produced by digestion with BstBl. The blunt-ended DNA was then digested with the restriction enzyme Nael (New England Biolabs), and the digested DNA was loaded onto a 1% agarose gel where the DNA fragments were separated by electrophoresis. A large DNA fragment of approximately 6190 bp was isolated and puxified from the agarose gel using a Gene Clean Kit (Bio 101), and the purified fragment was allowed to bind itself using bacteriophage T4-DNA ligase (Pharmacia). The resulting ligation mixture was used to transform competent E. coli JM105 cells, and positive clones were identified for their ability to produce an aggregate protein having an appropriate molecular weight. The recombinant plasmid formed in this manner was designated pCBlll, (Accession No. ATCC69748), and produces a shortened leukotoxin polypeptide (referred to below as LKT111) fused to four copies of the GnRH polypeptide. The plasmid to pCB114 has the multiple copy GnRH sequence (corresponding to the oligomer of Figure 5B) was inserted twice. Both of these plasmids are described in U.S. Patent No. 5,837,268 and produce shortened leukotoxin polypeptides called LKT 111 and LKT 114, respectively. A recombinant LKT-GnRH fusion molecule having two GnRH multimers of 8 copies, one arranged in the N '-terminus of LKT 114 and the other arranged in the C-terminus of LKT 114, was constructed from the sequence of LKT-GnRH fusion obtained from plasmid pCB114 by ligating the multiple copy GnRH sequence (corresponding to oligomer 89 of Figure 5B) twice to 5 'end of the coding sequence LKT114. A synthetic nucleic acid molecule having the following nucleotide sequence: 5 'ATGGCTACTGTTATAGATCGATCT-3' (SEQ ID NO:) was ligated at the 5 'end of the multiple copy GnRH sequences. The synthetic nucleic acid molecule codes for a sequence of 8 amino acids (Met-Ala-Thr-Val-Ile-Asp-Arg-Ser) (SEQ ID NO).

The resulting recombinant molecule thus contains in the order given in the 5 'to 3' direction: the synthetic nucleic acid molecule; a nucleotide sequence encoding a first GnRH multimer of 8 copies; a nucleotide sequence encoding the shortened LKT polypeptide (LKT 114); and a nucleotide sequence encoding a second GnRH multimer of 8 copies. The recombinant molecule was circularized, and the resulting molecule was used to transform competent E. coli JM105 cells. Positive clones were identified by their ability to produce an aggregated protein having a molecular weight of approximately 74 kb. The recombinant plasmid formed in this manner was designated pCB130 which produces the LKT 114 polypeptide fused to 16 copies of the GnRH polypeptide. The nucleotide sequence of the 90 Recombinant LKT-GnRH fusion of pCB130 is shown in Figures 6A through 6F.

Example 2 Purification of LKT-antigen fusions The recombinant LKT-GnRH fusion of Example 1 was purified using the following procedure. Five to ten colonies of transformed E. coli strains were inoculated in 10 ml of TB broth supplemented with 100 μg / ml of ampicillin and incubated at 37 ° C for 6 hours on a G10 shaker220 rpm. 4 mL of this culture was diluted in each of the two Fernbach flasks with diverters containing 400 mL of TB broth plus ampicillin and incubated overnight as described above. Cells were trimmed by centrifugation for 10 minutes at 4,000 rpm in polypropylene bottles, with a volume of 500 mL, using a Sorvall GS rotor. The pellet was redispersed in an equal volume of TB broth containing ampicillin that has been preheated to 37 ° C (i.e., 2 x 400 ml), and the cells were incubated for 2 hours as described above. 3.2 mL of isopropyl-B, D-thiogalactopyranoside (IPTG, Gibco / BRL), 500 Mm in water, (final concentration = 4 mM), were added to each culture at the end of each culture. induce the synthesis of recombinant fusion proteins. The cultures were incubated for two hours. Cells were harvested by centrifugation as described above, redispersed in 30 mL of 50 mM Tris-hydrochloride, 25% (w / v) sucrose, pH 8.0 and frozen at -70 ° C. The frozen cells were thawed at room temperature after 60 minutes at -70 ° C and 5 ml of lysozyme (Sigma, 20 mg / mL in 250 mM Tris-HCl, pH 8.0) was added. The mixture was subjected to vertex at high speed for 10 seconds and then placed on ice for 15 minutes. The cells were then added to 500 mL of lysis buffer in a 1000 mL beaker and mixed by shaking with a 12 mL pipette. The beaker containing the suspension of used cells was placed on ice and treated with sound for a total of 2.5 minutes (bursts of 5-30 seconds with 1 minute of cooling between each) with a Braun sounding device, long probe , adjusted to a power of 100 watts. Equal volumes of the solution were placed in Teflon SS34 centrifuge tube and centrifuged for 20 minutes at 10,000 rpm in a Sorvall SS34 rotor. The sediments were redispersed in a total of 100 mL of doubly sterile water when subjected to 92 vortex at high speed and the centrifugation step was repeated. Supernatants were rescued and the pellets were combined in 20 mL of 10 mM Tris-HCl, 150 M NaCl, pH 8.0 (saline buffered with Tris) and the suspension was frozen overnight at -20 ° C. The recombinant suspension was thawed at room temperature and added to 100 mL of 8 M guanidine-HCl (Sigma) in Tris buffered saline and mixed vigorously. A magnetic stir bar was placed in the bottle and the solubilized sample was mixed at room temperature for 30 minutes. The solution was transferred to a 2000 mL Erlenmeyes flask and 1200 mL of Tris-buffered saline was added rapidly. This mixture was stirred at room temperature for an additional 2 hours. Aliquots of 500 mL were placed in dialysis bags (Spectrum, diameter of 63.7 mm, cut of PM 6,000-8,000, # 132670, Fisher Scientific) and these were placed in 4,000 mL beakers containing between 3,500 of the solution saline buffered with Tris + guanidine -HCl 0.5 M. The beakers were placed in a room at 4 ° C on a magnetic stirrer overnight after which the dialysis buffer was replaced with saline buffered with Tris + 93 guanidine-HCl O.lM and dialysis was continued for 12 hours. The buffer was then replaced with saline buffered with Tris + guanidine-HCl 0.05M and the dialysis was continued overnight. The buffer was replaced with saline buffered with Tris (no guanidine) and dialysis was continued for 12 hours .. This was repeated three more times. The final solution was poured into a 2000 mL plastic bottle (Corning) and 13 mL of 100 mM PMSF (in ethanol) was added to inhibit the protease activity. The solution was stored at -20 ° C in 100 mL aliquots. To confirm that the fusion protein has been isolated, aliquots of each preparation were diluted 20 times in double distilled water, mixed with an equal volume of SDS-PAGE sample buffer, placed in a boiling water bath for 5 minutes and they were run through 12% polyacrylamide gels. Controls of recombinant leukotoxin were also run. The fusion protein was expressed at high levels as inclusion antibodies.

Example 3 Immunization of mature cats by GnRH vaccination The following experiment uses an immunological approach to demonstrate the use of GnRH vaccines in order to achieve prolonged immunological castration. Five mature male cats were immunized twice, 67 days apart, with a GnRH vaccine. In particular, vaccines derived from the cultures containing the pCB130 plasmid described above were formulated to contain 50 μg of GnRH-multimer fusion molecules (30 μg total of GnRH) in a final volume of 0.5 mL of adjuvant VSA-3 (an adjuvant EMULSIGEN PLUSMR modified containing a light mineral oil and dimethyldioctadecyl ammonium bromide, see, United States Patent Application, allowed, Serial No. 08 / 463,837). After immunization, the GnRH titers were measured as% inhibition at a dilution of 1: 5000 and the testicular size was determined. As can be seen in Figure 1, immunization of mature male cats with the GnRH immunogen induced GnRH antibody titers, which resulted in a decline in serum testosterone concentration to undetectable levels. A significant reduction in testicular size compared to pretreatment values was also observed. Biologically active GnRH titers, that is, the level of the titer that results in the significant suppression of Serum testosterone was achieved in the space of 35 days of vaccination and has a duration of effect of at least 300 days after primary immunization.

Example 4 Immunization of pre-pubertal cats by vaccination of GnRH at 8, 12 and 16 weeks of age A vaccine derived from cultures containing plasmid pCB130 described above was formulated to contain 100 μg of GnRH multimer fusion molecules (72 total μg of GnRH) in a final volume of 0.25 mL adjuvant VSA-3. Seven kittens (six females and one male) from two groups of bait were immunized with the vaccine at 8, 12 and 16 weeks of age. Each bait group included an untreated control kitten coupled in age and sex. The immunological activity of the GnRH fusion protein vaccine was assessed by measuring anti-GnRH antibody titers using a normal radioimmunoassay procedure at a serum dilution of 1: 5000. As can be seen in Figure 2, the titers of anti-GnRH antibodies increased dramatically in the immunized animals and remained at levels significantly in excess of the minimum amount 96 required to produce a biological effect (approximately 30% binding in Figure 2). The biological activity of the GnRH fusion protein vaccine was also assessed by measuring the serum testosterone concentration in the males and the serum estradiol concentration in the females using normal radioimmunoassay processing. In particular, testosterone was measured using a Total Coat-A-Count Testosterone Ki 'tMR device (Diagnostic Products Corporation, Los Angeles, CA). The equipment is a solid phase RIA designed for the quantitative measurement of testosterone in serum, based on the specific antibody of testosterone and mobilized to the wall of a polypropylene tube. Testosterone labeled with 125 I was competed for 3 hours at 37 ° C with testosterone) in the test sample for the antibody sites. The tube was decanted to separate the united from the free, and counted in a gamma counter. The amount of testosterone present in the test sample was determined from a calibration curve. The results are shown in Figure 7. As can be seen, serum testosterone was almost absent in the immunized group compared to the control group. 97 Serum estradiol was measured using an estradiol antibody kit of Double Antibody Estradiol Kit ™ (Diagnostic Products Corporation, Los Angeles, CA). The test is a sequential radioimmunoassay in which the test sample was pre-incubated with anti-estradiol antibodies. After incubation with estradiol labeled with 125 I for 3 hours, separation of the free was achieved by the accelerated antibody method with PEG. The antibody-bound fraction was precipitated and counted and the concentrations of the test sample were read from the calibration curve. The antibodies were highly specific for estradiol. The results are shown in Figure 8. As can be seen, there was no significant difference between the control and immunized animals. This is probably due to the fact that the levels of estrogen in animals of the tested age are below those levels at maturity and are low so that no difference can be detected between the levels and the control and immunized animals. Estrogen-dependent mating behavior was assessed by exposing male kittens to a lady in estrus at approximately 9 weeks of age, 98 for which the males of age will normally be sexually active. Reproductive function in the cells was assessed by exposing the female kittens to an intact male cat at approximately 10 months of age by which females would normally be sexually active. Sexual behavior was not evident in male 9-month-old kittens when exposed to a lady in estrus. The sensitivity of the pituitary to exogenous GnRH was assessed by a GnRH stimulation test performed when the immunized kittens were 10 months old. Milk concentrations were measured by a radioimmunoassay procedure. The purpose of the GnRH stimulation test was to determine if the pituitary gland in immunized kittens was sensitive to exogenous GnRH. In the normal animal, the exogenous GnRH analogue, leuprolide acetate, binds to the GnRH receptors in the pituitary and results in an immediate increase in serum LH concentrations. If GnRH receptors are downregulated or other post-translational effects occur, the pituitary will not respond to exogenous GnRH and serum LH concentrations will remain constant. In vitro studies have shown that 99 leuprolide has minimal cross-sensitivity with GnRH antibodies so that any reduction in the action of leuprolide, demonstrated by any change in LH concentrations, is likely at the receptor or post-translational level. Four immunized kittens (3 females, designated cats 1-3 and 1 male, designated cat 4), a non-immunized female bait mate and a non-immunized adult female were tested. A catheter was inserted into the cephalic vein of each animal. Leuprolide (1 mg / ml) was diluted to 100 mg / ml with 0.9% NaCl. The animals were subcutaneously administered the leuprolide solution at a dose of 10 mg / kg body weight at T = 0. Blood was drawn from the catheter immediately before administration of the test material, and at T = 30, 60, 90 and 150 minutes. 1.5 ml of blood was obtained from each cat at each sample point. The catheters were flooded with heparinized saline after each sample point. The blood was centrifuged, extracted in serum and frozen at -20 ° C until the analysis of the sample. LH concentrations were analyzed using a sequential radioimmunoassay as follows. The radioactive ligand was bovine LH iodinated with I 125 and the antibody was formulated in rabbits using bovine LH as 100 the immunogen. The antibody did not cross-react with growth hormone, FSH or prolactin. The highly purified bovine LH, provided by the National Institutes of Health, was used as the reference standard. The test sample was preincubated with bovine LH antibodies. After incubation with LH labeled with I125 for 3 hours, separation of the bound from the free was achieved by the double antibody method accelerated with PEG. The fraction bound to the antibody was precipitated and the concentrations of the test sample were counted and read from a calibration curve. All samples were run in an individual assay that had a 15% assay coefficient. The animals were also monitored on a daily basis in the morning for signs of estrus behavior (vocalization, lordosis, winding, etc.), up to 10 days after T = 0. As can be seen in Figure 3, the LH levels in the immunized animals did not increase significantly with respect to the pretreatment values. The levels of LH in non-immunized animals, on the other hand, increased significantly within 30 minutes afterwards. 101 of administration of leuprolide and remained elevated for at least 2.5 hours. The adult control female showed signs of estrus in the space of 5 days after the test. None of the kittens showed signs of estrus and the immunized male kitten showed no signs of sexual behavior. The lack of sexual behavior seen in these kittens at an age when the onset of sexual function is expected can be attributed to high circulating, continuous levels of GnRH antibodies that bind to endogenous GnRH and prevent receptor activation of GnRH from the pituitary. Low levels of endogenous GnRH are also required for the maintenance of normal concentrations of GnRH receptors of the pituitary. Removal of GnRH by circulating antibodies can result in decelerating regulation in the pituitary receptors, with the subsequent inability of the pituitary to respond to exogenous GnRH. A prolonged reduction in the number of GnRH receptors of the pituitary may have a permanent effect on the maturation of the hypothalamic-pituitary-gonadal axis and leads to long-term sterilization. For valorax if the decelerated regularization of the 102 is permanent receptors, this test can be repeated again when the antibody titers have declined. Since the pre-pubertal gonadal development seems to be more sensitive than the post-pubertal gonadal at withdrawal of gonadotropin stimulation, immunization of the pre-pubertal or peripubertal animal, followed by a second immunization one or two months later, should give as a result a long-term sterilization in effect. He also obtains vaginal cytology to assess the reproductive cycle.

Example 5 In a pre-pubertal cat castration by GnRH vaccination at 6 and 10 weeks of age The GnRH vaccine described above was used to immunize two kittens (one female and one male) from a group of bait at 6 and 10 o'clock. weeks of age The bait group also included an untreated control kit matched in sex and age. Serum antibody titers to GnRH, serum testosterone concentrations, serum estradiol concentrations, and LH concentrations in response to the exogenous GnRH stimulation test were measured as described above. Additionally, the 103 Pituitary sensitivity to exogenous GnRH is assessed by a GnRH stimulation test, also as described above. Estrogen-dependent behavior and mating is assessed by exposing male kittens to a lady in estrus at approximately 9 months of age, the males of age for which males would normally be sexually active. Reproductive function in females is assessed by exposing female kittens to an intact male cat at approximately 10 months of age by which females will normally be sexually active. Vaginal cytology is also obtained to assess the reproductive sign. As can be seen in Figure 4, the anti-GnRH antibody titers were dramatically increased in the immunized animals and remained at levels significantly in excess of the minimum amount required to produce a biological effect (approximately 30% binding in Figure 4). ).

Deposits of strains useful in the practice of the invention A deposit of biologically pure cultures of the following strains was made with the American Type Culture Collection (ATCC), 10801 University Boulevard, 5 Manassas, VA. The indicated access number was assigned after the successful viability test and the necessary cups were separated. The deposits were made under the conditions of the Budapest treaty in the International Recognition of Deposits of Microorganisms for the purpose of patent procedure and the regulations according to this one (Budapest Treaty). This ensures the maintenance of viable crops for a period of thirty (30) years from the date of deposit and at least five (5) years after the most recent request for the submission of a deposit sample by the depositary. The agencies will be made available by the ATCC in accordance with the terms of the Budapest Treaty, which ensures the permanent and unrestricted availability of crops to one determined by the North American Patent and Trademark Commissioner to be titled to it in accordance with 35 USC §122 and the rules of the commissioner according to this (including) 35 U.S.C. §122). In the granting of a patent, all restrictions and availability to the public of the crops deposited will be irrevocably removed. 105 These deposits are provided only as a convenience to those skilled in the art and are not an admission that a deposit is required in accordance with 35 U.S.C. §122. The nucleic acid sequences of these plasmids, as well as the amino acid sequences of the polypeptides encoded by them, are incorporated herein by reference and are in control in the case of any conflict with the description herein. A license to do, how to use, or sell the deposited materials may be required, and this license is not granted in this way.

No. of stocks ATCC deposit date PAA352 in W1485 of E. coli March 30, 1990 68283 PCB113 in JM105 of E. coli February 1, 1995 29749 PCB111 in JM105 of E. coli February 1, 1995 69748 Therefore, new methods to reduce GnRH levels in pre-pubertal vertebrates have been described. Although preferred embodiments of the present invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and scope of the invention as defined by the appended claims. 106 LIST OF SEQUENCES < 110 > Biostar Inc. < 120 > METHODS TO DEPRIVE REPRODUCTIVE BEHAVIOR IN ANIMALS < 130 > 08-883615WO < 140 > < 141 > < 150 > US 60/088, 024 < 151 > 1998-06-04 < 160 > 14 < p? > Patentln See 2 0 < 210 > 1 < 211 > 10 < 212 > PRT < 213 > Artificial Sequence < 220 > < 221 > MOD_RES < 222 > (1) < 223 > Xaa is pyroglutamic acid < 220 > < 223 > Definition of Artificial Sequence The Sequence defines analogous GnRH < 400 > 1 Xaa His Trp Ser Tyr Gly Leu Arg Pro Gly 1 5 10 < 210 > 2 < 211 > 12 < 212 > FRT • 213 > Artificial Sequence < 220- > < 221 > MOD_RES < 222 > (6) < ? 23 > Xaa is Gly or a D-amino acid < 220 > < 221 > MOD_ ES < 222 > (11) < 223 > Xaa is one or more amino acid residues which may be the same or different, preferably 1-3 Gly < -220 ^ < 221 > MOD_RES < 222 > (12. <223> Xaa is Cys or Tyr <220> <221> MOD RES <222> (I) - 107 < 223 > Xaa is pyroglutamic acid < 220 > < 223 > Description of Artificial Sequence: The Sequence defines analogous GnRH < 400 > 2 Xaa His Trp Ser Tyr Xaa Leu Arg Pro Gly Xaa Xaa 1 5 10 < 210 > 3 < 211 > 17 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: The Sequence defines analogous GnRH < 400 > 3 Cys Pro Pro Pro Ser Ser Glu His Trp Ser Tyr Gly Leu Arg Pro 1 5 10 15 Gly < 210 > 4 < 211 > 17 < 212 > PRT < 213 > Artificial Sequence < 220 > < 221 > MOD_RES < 222 > (1) < 223 > Xaa is pyroglutatic acid < 220"< 223 > Description of Artificial Sequence - The Sequence Defines Analog GnRH <400> 4 Xaa His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Pro Pro Pro Pro 1 5 10 15 Cys < 210 > 5 < 211 > 16 < 212 > PRT < 213 > Artificial Sequence < 220 > < 221 > M0D_R? S < 222 > (1) < 223 > Xaa is pyroglutamic acid < 220 > < 223 > Description of Artificial Sequence: The Sequence Defines Analog GnRH 108 < 400 > 5 Xaa His Trp Ser Tyr Gly Leu Arg Pro Gly Arg Pro Pro Pro Cys 1 5 10 15 < 210 > 6 < 2 U > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: The Sequence defines synthetic nucleic acid linker < 400 > 6 atggctactg ttatagatcg atct 24 < 210 > 7 < 211 > 8 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: The Sequence defines synthetic amino acid linker < 400 > 7 Met Wing Thr Val lie Asp Arg Ser 1 5 < 210 > 8 < 211 > 30 < 212 > DNA ^ 213 > Artificial Sequence < 220 > < 222 > (1) .. (30) < 220 > < 223 > Artificial Sequence Discovery: The Sequence Defines GnRH < 400 > 8 cag cat tgg age tac ggc ctg cgc cct ggc 30 Gln His Trp Ser Tyr Gly Leu Arg Pro Gly 1 5 10 < 210 > 9 < 211 > 10 < 212 > PRT < 213 > Artificial Sequence < 400 > 9 Gln His Trp Ser Tyr Gly Leu Arg Pro Gly 1 5 10 < 210 > 10 < 2il > 147 < 212 > DNA 109 < 213 > Artificial Sequence < 220 > < 221 > CDS < 222 > (1) .. (147) < 220 > < 223 > Description of Artificial Sequence: The Sequence defines a GnRH last < 400 > 10 cag cat tgg age tac ggc ctg cgc cct ggc age ggt tct ca gt tgg 48 Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp 1 5 10 15 age tac ggc ctg cgt ceg ggt ggc tct age cag cat tgg age tac ggc 96 Ser Tyr Gly Leu Arg Pro Gly Gly Ser Ser Gln His Trp Be Tyr Gly 20 25 30 ctc cgc cct ggc age ggt age ca gt gat age gcc tac ggc cgt ceg 144 Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr Gly Leu Arg Pro 35 40 45 ggt 147 Gly < 210 > 11 < 211 > 49 < 212 > PRT < 213 > Artificial Sequence < 400 > 11 Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp 1 5 10 15 Ser Tyr Gly Leu Arg Pro Gly Gly Ser Ser Gln His Trp Ser Tyr Gly 20 25 30 Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Being Tyr Gly Leu Arg Pro 35 40 45 Gly < 210 > 12 < 211 > 2088 < 212 > DNA < 213 > Artificial Sequence < 220 > < 221 > CDS < 222 > (1) .. (2088) < 220 > < 223 > Describing Artificial Sequence: The Sequence defines a GnRH leucotoxam chimera < 400 > 12 atg gct act gtt ata gat cga tct cag cat tgg age tac ggc ctg cgc 4 E Met Ala Thr Val He Asp Arg Ser Gln His Trp Ser Tyr Gly Leu Arg 1 5 10 15 110 cct ggc age ggt tct ca gt ggc ggc cgt ggt ggc cgt ggt ggt Pro Gly Ser Gly Ser Gly Gp Tp Gly Gly Leg Arg Pro Gly Gly 20 25 30 tct age cag cat tgg age tac ggc cg cgc gcg ggc age ggc age caa 144 Being Ser Gln His Trp Being Tyr Gly Leu Arg Pro Gly Being Gly Being Gln 35 40 45 gat tgg age tac ggc ctg cgt ceg ggt gga tct cag cat tgg age tac 192 Asp Trp Ser Tyr Gly Leu Arg Pro Gly Gly Ser Gln His Trp Ser Tyr 50 55 60 ggc ctg cgc cct ggc age ggt tct ca gt tgg age tac ggc ctg cgt 240 Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr Gly Leu Arg 65 70 75 80 ceg ggt ggc tct age cag cat tgg age tac ggc ctg cgc cct ggc age 288 Pro Gly Gly Ser Ser Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser 85 90 95 ggt age ca gt tgg age tac ggc ctg cgt ceg ggt gga tct age ttc 336 Gly Ser Gln Asp Trp Ser Tyr Gly Leu Arg Pro Gly Gly Ser Ser Phe 100 105 110 cea aaa act ggg gca aaa aaa att atc ctc tat att ccc caat aat tac 384 Pro Lys Thr Gly Ala Lys Lys He He Leu Tyr He Pro Gln Asn Tyr 115 120 12 5 ca g tat tat gaa ca ggt aat ggt tta cag gat tta gtc aaa gcg 432 Gln Tyr Asp Thr Glu Gln Gly Asn Gly Leu Gln Asp Leu Val Lys Wing 130 135 140 gcc gaa gag ttg ggg att gag gta caa aga gaa gaa cgc aat aat att 480 Wing Giu Glu Leu Gly He Glu Val Gln Arg Glu Glu Arg Asn Asn He 145 150 155 160 gca here gct ca g acc agt tta gc acg att g gc tta 528 Ala Thr Ala Gln Thr Ser Leu Gly Thr He Gln Thr Wing He Gly Leu 165 170 175 act gag cgt ggc att gtg tta tcc gct cea caa att gat aaa ttg cta 576 Thr Glu Arg Gly He Val Leu Ser Wing Pro Gln He Asp Lys Leu Leu 180 185 190 cag aaa act aaa gca ggc ca gca gta tta ggt tct gcc gaa age att gta 624 Gln Lys Thr Lys Wing Gly Gln Wing Leu Gly Ser Wing Glu Ser He Val 195 200 205 caat aat gca aat aaa gcc aaa act gta tta tct ggc att caa tct att 672 Gln Asn Wing Asn Lys Wing Lys Thr Val Leu Ser Gly He Gln Ser He 210 215 220 tta ggc tea gta ttg gct gga atg gat tta gat gag gcc tta "cag aat 720 Leu Gly Ser Val Leu Wing Gly Met Asp Leu Asp Glu Wing Leu G ln Asn 225 230 235 240 aac age aac ca ght cat gct ett gct aaa gct ggc ttg gag cta here aat 768 Asn Ser Asn Gln His Ala Leu Ala Lys Ala Gly Leu Glu Leu Thr Asn 245 250 255 tea tta att gaa aat att gct aat tea gta aaa here ett gac gaa ttt 816 Ser Leu He Glu Asn He Wing Asn Ser Val Lys Thr Leu Asp Glu Phe 260 265 270 111 ggt gag cag att agt caa ttt ggt tea aaa cta caat aat atc aaa ggc 864 Gly Glu Gln He Ser Gln Phe Gly Ser Lys Leu Gln Asn He Lys Gly 275 280 285 tta ggg act tta gga gac aaa ctc aaa aat atc ggt gga et gat aaa 912 Leu Gly Thr Leu Gly Asp Lys Leu Lys Asn He Gly Gly Leu Asp Lys 290 295 300 gct ggc ett ggt tta gat gtt atc tea ggg cta tta teg ggc gca here 960 Wing Gly Leu Gly Leu Asp Val He Ser Gly Leu Leu Ser Gly Ala Thr 305 310 315 320 gct gca ett gta ett gca gat aaa aat gct tea here gct aaa aaa gtg 1008 Ala Ala Leu Val Leu Ala Asp Lys Asn Ala Ser Thr Ala Lys Lys Val 325 330 335 ggt gcg ggt ttt gaa ttg gca aac ca gtt gtt ggt aat att acc aaa 1056 Gly Ala Gly Phe Glu Leu.Ala Asn Gln Val Val Gly Asn He Thr Lys 340 345 350 gcc gtt tct tct tac att tta gcc ca gt c gt gca gca ggt tta tct 1104 Ala Val Ser Ser Tyr He Leu Wing Gln Arg Val Wing Wing Gly Leu Ser 355 360 365 tea act ggg cct gtg gct gct tta att gct tct act gtt tct ett gcg 1152 Ser Thr Gly Pro Val Wing Wing Leu He Wing Being Thr Val Ser Leu Ala 370 375 380 att age cea tta gca ttt gcc ggt att gcc gat aaa ttt aat cat gca 1200 He Ser Pro Leu Wing Phe Wing Gly He Wing Asp Lys Phe Asn His Wing 385 390 395 400 aaa agt tta gag agt tat gcc gaa cgc ttt aaa aaa tta ggc tat gac 1248 Lys Ser Leu Glu Ser Tyr Ala Glu Arg Phe Lys Lys Leu Gly Tyr Asp 405 410 415 gga gat aat tta tta gca gaa tat cag cgg gga here ggg act att gat 1296 Gly Asp Asn Leu Leu Wing Glu Tyr Gln Arg Gly Tnr Gly Thr He Aso 420 425"430 gca teg gtt act gca att aat acc gca ttg gcc gct att gct ggt ggt L344 Wing Ser Val Thr Wing He Asn Thr Ala Leu Wing Wing He Wing Gly Gly 435 440 445 gtg tct gct gct gca gcc gat tta here ttt gaa aaa gtt aaa cat aat 1392 Val Ser Ala Ala Ala Ala Asp Leu Thr Phe Glu Lys Val Lys His Asn 450 455 460 ett gtc atc acg aat age aaa aaa gag aaa gtg acc ata caac aac tgg L440 Leu Val He Thr Asn Ser Lys Lys Glu Lys Val Thr He Gln Asn Trp 465 470 475 480 ttc cga gag gct gat ttt gct aaa gaa gtg cct aat tat aaa'gca act 1488 Pne Arg Glu Wing Asp Phe Wing Lys Glu Val Pro Asn Tyr Lys Wing Thr 485 490 495 aaa gat gag aaa atc gaa gaa atc atc ggt caat aat ggc gag cgg atc 1536 Lys Asp Glu Lys He Glu Glu He He Gly Gln Asn Gly Glu Arg He 500 505 510 ac tea aag ca gtt gat gat ett atc gca aaa ggt aac ggc aaa att 1584 Thr Ser Lys Gln Val Asp As p Leu He Ala Lys Gly Asn Gly Lys He 515 520 525 112 acc cat gat gag cta tea aaa gtt gtt gat aac tat gaa ttg ctc aaa 1632 Thr Gln Asp Glu Leu Ser Lys Val Val Asp Asn Tyr Glu Leu Leu Lys 530 535 540 cat age aaa aat gtg here aac age tta gat aag tta atc tea tct gta 1680 His Ser Lys Asn Val Thr Asn Ser Leu Asp Lys Leu He Ser Ser Val 545 550 555 560 agt gca ttt acc teg tct aat gat teg aga aat gta tta gtg gct cea 1728 Being Wing Phe Thr Ser Being Asn Asp Being Arg Asn Val Leu Val Wing Pro 565 570 575 act at t tg g t tg t tg t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t tc t t Thr Ser Met Leu Asp Gln Ser Leu Ser Ser Leu Gln Phe Wing Arg Gly 580 585 590 tct cag cat tgg age tac ggc etg cgc cct ggc age ggt tct caa gat 1824 Ser Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp 595 600 605 tgg age tac ggc ctg cgt ceg ggt ggc tct age cag cat tgg age tac 1872 Trp Ser Tyr Gly Leu Arg Pro Gly Gly Ser Ser Gln His Trp Ser Tyr 610 615 620 ggc ctg cgc cct ggc age ggt age ca gt gat age gc tac gcc ctg 1920 Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr Gly Leu Arg 625 630 635 640 ceg ggt gga tct cag cat tgg age tac ggc ctg cgc cct ggc age ggt 1968 Pro Gly Gly Ser Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly 645 650 655 tct tac at tgg age tac ggc ctg cgt ceg ggt ggc tct age cag cat 2016 Ser Gln Ásp Trp Ser Tyr GÍy Leu Arg Pro Gly Gly Ser Ser Gln His 660 '665 670 tgg age tac ggc ctg cgc cct ggc age ggt age ca gt gat tgg age tac 2064 Tro Ser Tyr Glv Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tvr 675"'680 685: tg cgt zz zz zs tcc tsg 2088 590 '6S5 < 210 > 13 < 211 > 695 < 212 > PRT < 213 > Artificial Sequence < 400 > 13 Met Wing Thr Val He Asp Arg Ser Gln His Trp Ser Tyr Gly Leu Arg 1 5 10 * 15 Pro Gly Ser Glv Ser Gln Asp Trp Ser Tyr Gly Leu Arg Pro Gly Gly 20 25 30 Ser Ser Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly Ser Gln 35 40 45 Asp Tm Ser Tyr Gly Leu Arg Pro Gly Gly Ser Gln His Trp Ser Tyr 50 55 60 Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr Gly Leu Arg 113 65 70 75 80 Pro Gly Gly Ser Ser Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser 85 90 95 Gly Ser Gln-Asp Trp Ser Tyr Gly Leu Arg Pro Gly Gly Ser Ser Phe 100 105 110 Pro Lys Thr Gly Wing Lys Lys He He Leu Tyr He Pro Gln Asn Tyr 115 120 125 Gln Tyr Asp Thr Glu Gln Gly Asn Glv Leu Gln Asp Leu Val Lys Wing 130 135 140 Wing Glu Glu Leu Gly He Glu Val Gln Arg Glu Glu Arg Asn Asn He 145 150 155 160 Ala Thr Ala Gln Thr Ser Leu Gly Thr He Gln Thr Ala He Gly Leu 165 170 175 Thr Glu Arg Gly He Val Leu Ser Ala Pro Gln He Asp Lys Leu Leu 180 185 190 Gln Lys Thr Lys Ala Gly Gln Ala Leu Gly Ser Ala Glu Ser He Val 195 200 205 Gln Asn Ala Asn Lys Ala Lys Thr Val Leu Ser Gly He Gln Ser He 210 215 220 Leu Gly Ser Val Leu Wing Gly Met Asp Leu Asp Glu Wing Leu Gln Asn 225 230 235 240 Asn Ser Asn Gln His Wing Leu Wing Lys Wing Gly Leu Glu Leu Thr Asn 245 250 255 Ser Leu He Glu Asn He Wing Asn Ser Val Lys Thr Leu Asp Glu Phe 260 265 270 Gly Glu Gln He Ser Gin Phe Glv Ser Lys Leu Gln Asn He Lys Gly 275 260 285 Leu Gly Tnr Leu Lys Leu Lys Asn He Gly Gly Leu Asp Lys 290 295 300 Wing Giy Leu Gly Leu Asp Val He Ser Gly Leu Leu Ser Gly Wing Thr 305 310 315 320 Ala Ala Leu Val Leu Ala Asp Lys Asn Ala Ser Thr Ala Lys Lys Val 325 330 335 Gly Ala Gly Phe Glu Leu Ala Asn Gln Val Val Gly Asn He Thr Lys 340 345 350 ft Wing Val Ser Ser Tyr He Leu Wing Gln Arg Val Wing Wing Gly Leu Ser 355 360 365 Ser Thr Gly Pro Val Wing Wing Leu Wing Wing Ser Thr Val Ser Leu Wing 370 375 380 He Ser Pro Leu Wing Phe Wing Gly He Wing Asp Lys Phe Asn His Ala 385 390 395 400 Lys Ser Leu Glu Ser Tyr Wing Glu Arg Phe Lys Lys Leu Gly Tyr Asp 405 410 415 114 Gly Asp Asn Leu Leu Wing Glu Tyr Gln Arg Gly Thr Gly Thr He Asp 420 425 430 Wing Ser Val Thr Wing He Asn Thr Wing Leu Wing Wing He Wing Gly Gly 435 440 445 Val Wing Wing Wing Wing Wing Asp Leu Thr Phe Glu Lys Val Lys His Asn 450 455 460 Leu Val He Thr Asn Ser Lys Lys Glu Lys Val Thr He Gln Asn Trp 465 470 475 480 Phe Arg Glu Wing Asp Phe Wing Lys Glu Val Pro Asn Tyr Lys Wing Thr 485 490 495 Lys Asp Giu Lvs He Glu Glu He He Gly Gln Asn Gly Glu Hendr G He 500 505 510 Thr Ser Lys Gln Val Asp Asp Leu He Wing Lys Gly Asn Gly Lys He 515 520 525 Thr Gln Asp Glu Leu Ser Lys Val Val Asp Asn Tyr Glu Leu Leu Lys 530 535 540 His Ser Lys Asn Val Thr Asn Ser Leu Asp Lys Leu He Ser Ser Val 545 550 555 560 Being Wing Phe Thr Ser Being Asn Asp Being Arg Asn Val Leu Val Wing Pro 565 570 575 Thr Ser Met Leu Asp Gln Ser Leu Ser Ser Leu Gln Phe Ala Arg Gly 580 585 590 Ser Gln His Trp Ser Tvr Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp 595 600 605 Trp Ser Tyr Gly Leu Arg Pro Gly Gly Ser Ser Gln His Trp Ser Tyr 610 615 620 Gly Leu Arg be iji, Ser Gln Asp Trp r Gly Leu Arg 625 635 Pro Glv Gly Ser Gln His. p Ser Tyr Gly Leu Arg Pro Glv Ser Gly 645 650"655 Ser Gln Asp Trp Ser Tyr Gly Leu Arg Pro Gly Gly Ser Ser Gln His 660 665 670 Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr 675 680 685 Gly Leu Arg Pro Gly Gly Ser 690 695 < 210 > 14 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: The Sequence defines a consensus amino acid sequence of RTX 115 < 220 > < 221 > MOD_RES < 222 > (3) < 223 > Xaa is Lys, Asp, Val or Asn < 220 > < 221 > MOD_RES < 222 > (5) < 223 > Xaa is Lys, Asp, Val or Asn < 400 > 14 Gly Gly Xaa Gly Xaa Asp 1 5

Claims (36)

116 CLAIMS 1. The use of a GnRH immunogen, a GnRH analog or antibody that cross-reacts with the endogenous GnRH of a vertebrate subject, in the preparation of a first composition for pre-pubertal administration to the vertebrate subject to result prolonged suppression of reproductive behavior and / or fertility in the vertebrate subject.

2. The use according to claim 1, wherein the first composition comprises an effective amount of a GnRH immunogen.

3. The use according to claim 2, wherein the first composition further comprises an immunological adjuvant.

4. The use according to claim 3, wherein the immunological adjuvant comprises an oil and dimethyldiocatadecylammonium bromide.

The use according to claim 3, wherein the immunological GnRH is a multimer of GnRH comprising the general formula (GnRH-X-GnRH) wherein: GnRH is a GnRH immunogen; X is one or more molecules selected from the group consisting of a peptide bond, an amino acid spacer group, a carrier molecule and [GnRH] n, where n is an integer greater than or equal to l, and 117 and is an integer greater than or equal to 1.

The use according to claim 5, wherein the carrier molecule is a leukotoxin polypeptide.

The use according to claim 2, wherein the GnRH immunogen is a nucleic acid molecule.

8. The use according to claim 1, wherein the GnRH analog is a GnRH agonist or antagonist.

9. The use according to claim 1, wherein the antibodies are polyclonal.

10. The use according to claim 1, wherein the antibodies are monoclonal.

The use according to claim 1, further comprising administering to the vertebrate subject a second composition comprising an effective amount of a GnRH immunogen, a GnRH analog or antibodies that cross-react with endogenous GnRH from vertebrate subject.

The use according to claim 2, further comprising administering to the vertebrate subject a second composition comprising an effective amount of a GnRH immunogen.

The use according to claim 12, wherein the first and second compositions comprise the same GnRH immunogen.

14. Use according to any of the 118 claims 1-13, wherein the vertebrate is selected from the group consisting of a feline subject,. a canine subject, or an equine subject and a cervino subject.

15. The use according to claim 14, wherein the first composition is administered to the vertebrate subject at about 3 to about 15 weeks of age.

The use according to claim 15, wherein the second composition is administered to the vertebrate subject from about 2 to about 10 weeks after administration of the first composition.

The use according to claim 1, wherein the pre-pubertal administration results in a long-term, prolonged suppression of testicular development and / or function in males, or a long-term, prolonged suppression of development and / or function ovarian in females.

18. Use the use of a first and a second GnRH multimer for the preparation of a first and a second vaccine composition for the use of a method for prolonged suppression of reproductive behavior and / or fertility in a vertebrate subject, the first and second vaccine compositions comprising an adjuvant, the first and second GnRH multimers comprising the general formula (GnRH-X-GnRH) wherein: GnRH is a GnRH immunogen; 119 X is one or more molecules selected from the group consisting of a peptide bond, an amino acid spacer group, a lipotoxin polypeptide [GnRH] n, where n is an integer greater than or equal to 1, and y is a whole number greater than or equal to, wherein the first composition is administered to the subject, pre-pubertally at about 3 to about 15 weeks of age, and the second vaccine composition is administered to a subject around week 2 to about week 10 after administration of the first vaccine composition.

The use according to claim 18, wherein the first and second vaccine compositions comprise the same GnRH multimer.

The use according to claim 19, wherein the GnRH multimer comprises the amino acid sequence depicted in Figures 6A-6F (SEQ IN NO:), or an amino acid sequence with at least about 75% sequence identity to the same.

The use according to claim 19, wherein the GnRH multimer comprises the amino acid sequence depicted in Figures 6A-6F (SEQ IN NO:).

22. The use of a first and second rarimonimers of GnRH in the preparation of a first and second vaccine compositions for the use of a method for 120 prolonged suppression of reproductive behavior and / or fertility of a feline subject, the first and second vaccine compositions comprising an immunological adjuvant comprising a light mineral oil and dimethyl octadecyl ammonium bromide, the first and second GnRH multimers comprising the amino acid sequence depicted in Figures 6A-6F (SEQ ID NO:), or an amino acid sequence with at least about 75% sequence identity thereto, wherein the first composition is administered to the subject, pre-pubertally at about 5 to about 12 weeks of age and the The second vaccine composition is administered to the subject in about 2 to about 10 weeks after administration of the first vaccine composition.

23. The use according to claim 22, wherein the GnRH multimer in the first and second vaccine compositions comprises the amino acid sequence depicted in Figures 6A-6F (SEQ IN NO:).

24. The use of a first GnRH immunogen in the preparation of a first vaccine composition for use in a method for suppressing reproductive performance and / or fertility in a feline, canine, equine or cervino subject for at least 10 months .

25. The use according to claim 24, which 121 further comprises administering to the subject a second vaccine composition comprising an effective amount of a GnRH immunogen.

26. The use according to any of claims 24 or 25, wherein the first and / or second vaccine composition is administered pre-pubertally.

27. The use according to any of claims 24 or 25, wherein the first and / or second vaccine composition is administered post-pubertally.

The use according to claim 25, wherein the first vaccine composition is administered pre-pubertal and the second vaccine composition is administered post-pubertal.

29. The use according to claim 25, wherein the first and second vaccine compositions are the same.

30. The use according to claim 25, wherein the GnRH immunogen in the first and second vaccine compositions are different.

31. The use according to any of claims 24 or 25, wherein the first and / or vaccine composition additionally comprises an immunological adjuvant.

32. The use according to claim 31, wherein the immunological adjuvant and / or second vaccine composition 122 it comprises an oil and dimethyldiocatadecylammonium bromide.

The use according to claim 24, further comprising administering additional vaccine compositions comprising an effective amount of a GnRH immunogen of at least about 6 up to about 12 months subsequent to the previous administration.

34. The use according to claim 33, wherein the additional vaccine compositions are administered at approximately annual intervals.

35. The use according to claim 24, wherein the immunological GnRH in the first vaccine composition is a nucleic acid molecule.

36. The use according to claim 25, wherein the immunological GnRH in the first and second vaccine compositions is a nucleic acid molecule.