MXPA00010869A - Methods of raising animals for meat production - Google Patents

Abstract

Methods for raising uncastrated male animals for meat production are disclosed. The methods use compositions which include GnRH immunogens. The methods are useful for producing cuts of meat with enhanced organoleptic qualities.

Description

METHODS FOR RAISING ANIMALS FOR MEAT PRODUCTION TECHNICAL FIELD The present invention relates generally to methods for raising animals for the production of meat. More particularly, the invention is directed to methods for immunizing animals with a primary vaccination of a GnRH immunogen that causes a reduction in the circulating levels of gonadal steroids, followed by revaccination with a GnRH immunogen in a short time before of killing to substantially reduce the level of one or more androgenic and / or non-androgenic steroids.

BACKGROUND OF THE INVENTION Typically, the males of cattle, pigs and sheep are more heavily muscled and have a mature body size larger than the females. The conventional explanation is that as male animals reach sexual maturity, the secretion of testosterone, the anabolic steroid produced by the testes, results in a deposit Increased muscle protein and decreased fat content. Also, numerous studies have established that males use feed more efficiently than females during the growth period (Field, R.A., J. Animal Sci. (1971) 32: 849-958). These differences are more obvious around the time of puberty and testosterone has an important role in regulating these changes. In males, androgenic steroids, testosterone and androsterone, are involved in the regulation of two different biological processes. One effect is physiological and results in increased muscle deposition, reduced fat synthesis and increased efficiency of feed utilization. Testosterone has similar direct effects on the growth of the sex glands such as seminal vesicles, the prostate gland and testes. These actions include the direct interaction of androgen with receptors in target tissues. The second general effect of androgens is to provoke different sexual and behavioral changes, typical of males. These effects were measured through the central nervous system.

It is possible that due to these, several effects are presented in different tissues by different molecular mechanisms, some of them can be maintained at low androgen plasmatic concentrations while other effects may require high levels. Although, it is generally assumed that androgens are required early in life to maintain optimal growth, muscle deposition, and feed efficiency, an assumption that can not be true even though median amounts of androgen are present. Alternatively, androgen-sensitive tissues, such as muscle, may be much more sensitive to lower levels of androgen early in life than at puberty or later. Allrich et al., J. Animal Sci. (1982) 55: 1139-1146, compared rates of gain in body weight and weights of testosterone-sensitive tissues in pigs as a function of age and testosterone concentration. In particular, a comparison of the growth rate of different tissues at different ages showed that tissue sensitivity to testosterone, as well as concentration of testosterone, changed with age. For example, the authors showed that from day 40 to 100, while serum testosterone increased 3.1 times, body weight increased 3.2 times, testicles 3.6 and seminal vesicles 4.0 times. These data also show that pre-pubertal testosterone levels were low but easily detectable and that body weight increased at approximately the same rate as other tissues in the presence of low testosterone levels. The pigs begin to reach sexual maturity at approximately 130 to 150 days of age (65 to 85 kg of body weight) and the serum concentrations of testosterone increase significantly at that time. From day 100 to 190, whereas serum testosterone is increased 4.0 times, body weight, testicular weight and weight of the seminal vesicles increases 2.6 times, 13.5 times and 9.2 times, respectively. Additionally, Knudson et al., J. Animal, Sci. (1985) 61: 789-796, showed that castrated male pigs gained weight at a similar speed compared to intact males until approximately 90 days of age, but beyond that age intact males grow more efficiently than castrated males. This characteristic is particularly important for the immunostealization of herd animals, and particularly where it is desired to immunize male piglets to prevent the "boar taint" that is produced by the synthesis of sex steroids in testicles normally functioning of male piglets. See, for example Meloen et al., Vaccine (1994) 12 (8): 741-746. A large number of studies have been done in pigs and cattle to explore the use of GnRH immunization as a method to improve growth rate and feed efficiency in animals. See, for example, Adams and Adams, J. Animal Sci. (1992), 70: 1691-1698; Caraty and Bonneau, C.R. Acad. Sc. Paris (1986) 303: 673-676; Chaffaux et al., Recueil de Medecine Veterinaire (1985) 161: 133-145; Finnerty et al., J. Repro. Fertile. (1994) 101: 333-343. The goal of many of these studies has been to allow the animals to grow as intact males until the approach of the end of the fattening period and then to castrate them immunologically. For achieve immunological castration towards the end of the fattening period and just before slaughter, the animals are vaccinated one or more times early in life to prime the immune system so that it will respond strongly to the revaccination given towards the end of the fattening period. The first vaccination is designed to prime the immune system with the GnRH antigen but to prevent the induction of high titers of anti-GnRH antibodies that will lower serum testosterone levels or prevent them from increasing as the animals reach puberty. This was based on the belief that the reduction of serum testosterone will also reduce the rate of growth or feed efficiency in young animals. For example Meloen et al., Vaccine (1994) 12: 741-756, describes the use of a GnRH tandem vaccine, administered in two doses, in order to reduce boar taint in pigs. There was no reduction in the size of the testicles until after the second immunization. See, also, Publication International No. WO 90/11298, published October 4, 1990. Falvo et al., J. Anim. Sci. (1986 63: 986-994 reports the use of GnRH vaccines to study various effects on pigs, including the presence of boar taint and characteristics of the dead animal. The authors report that plasmatic levels of testosterone were significantly reduced two weeks after the first booster injection. Similarly, U.S. Patent No. 5,573,767, relates to a method for improving the organoleptic qualities of meat using GnRH immunization. The method involves two immunizations, one immunization designed to have no effect on the secretion of gonadal steroids and a second immunization before slaughter in order to suppress the action of the androgenic and non-androgenic spheroids. Additionally, previous attempts at immunosorption have not yielded uniform results due to insufficient immunogenicity of the GnRH peptides and / or related carrier systems, and the resulting inability of several previous GnRH-based vaccines to induce sufficient immune responses to the GnRH. Endogenous GnRH. See, for example Robertson, Vet., Record (1981) 108: 381-382.

Therefore, reliable methods for the immunization of food-producing animals would be desirable.

DESCRIPTION OF THE INVENTION The present invention is based on a reproducible, reliable method for raising a male animal producing food for the production of meat. In particular, contrary to the prevailing belief, based on the trials described herein, it seems that avoiding a substantial reduction in testosterone early in life is not necessary in order to produce commercially acceptable amounts of meat and that immunization of primary GnRH that induces antibodies that have a measurable effect on the secretion of gonadal spheroids during the fattening period can be achieved without loss significant growth rate or food efficiency. Primary immunization can be followed later in life with a secondary immunization that suppresses the action of the androgenic and / or non androgenic spheroids. Therefore, in one embodiment, the invention is directed to a method for raising a food producing animal, male, not castrated for the production of meat, which comprises the vaccination of the animal with a first vaccine composition comprising a GnRH immunogen before or during fattening employment of the animal to cause a reduction of the levels circulating testosterone and vaccinating the animal with a second vaccine composition comprising the GnRH immunogen in about 2 to about 8 days prior to slaughter of the animal to substantially reduce the level of one or more androgenic and / or non-androgenic steroids . In particularly preferred embodiments, the first and / or second vaccine compositions comprise an immunological adjuvant such as an adjuvant comprising an oil and dimethyldioctadecylammonium bromide. Additionally, the GnRH immunogen in the first and / or second vaccine composition can be a GnRH multimer comprising the General Formula (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 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 certain embodiments, administration of the first vaccine composition results in the production of antibodies that cross-react with the endogenous GnRH of the animal and the second composition is administered after that have declined antibody levels. In another embodiment, the invention is directed to a method for breeding a bovine, ovine or porcine, male, non-castrated animal for the production of meat comprising the vaccination of the animal with a first vaccine composition comprising a GnRH immunogen before or after during the fattening period of the animal to cause a reduction in circulating levels of testosterone and vaccination of the animal with a second vaccine composition comprising a GnRH immunogen at about 2 to about 8 weeks prior to slaughter of the animal, for Substantially reduce the level of one or more androgenic and / or non-androgenic steroids. The first and / or Second vaccine compositions may additionally comprise an immunological adjuvant. In yet another embodiment, the invention is directed to a method for breeding a bovine, ovine or porcine, male, non-castrated animal for meat production, comprising: (a) vaccinating the animal with a first vaccine composition comprising a immunological adjuvant and a GnRH multimer comprising the General Formula (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 leucotoxin polypeptide 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, wherein the first vaccine composition is administered before or during the fattening period of the animal to cause a reduction in testosterone circulation levels; and (b) vaccinating the animal with a second vaccine composition comprising a mummological adjuvant and a GnRH multimer comprising the General Formula (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 l; and y is an integer greater than or equal to 1, wherein the second vaccine composition is administered approximately 2 to approximately 8 weeks prior to slaughter of the animal, to substantially reduce the level of one or more androgenic steroids and / or not androgenic In still another embodiment, the invention is directed to a method for breeding a bovine, ovine or porcine animal, male, non-castrated for meat production, comprising: (a) vaccination of the animal with a first vaccine composition comprising an immunological adjuvant and a GnRH multimer comprising the amino acid sequence depicted in Figures 3A-F (SEQ ID Nos. 12 and 13), an amino acid sequence that is at least about 75% identical in sequence to it, in where the first vaccine composition is administered before or during the fattening period of the animal for cause a reduction in circulating levels of testosterone; (b) vaccinate the animal with a second vaccine composition comprising an immunological adjuvant and a GnRH multimer comprising the amino acid sequence depicted in Figures 3A-3F (SEQ ID Nos. 12 and 13) or an amino acid sequence with at least about 75% sequence identity thereto, wherein the second vaccine composition is administered about 2 to about 8 weeks prior to slaughter of the animal, to substantially reduce the level of one or more androgenic and / or non androgenic spheroids The adjuvant in the first and / or second vaccine composition may comprise a light mineral oil and dimethyldioctadecylammonium bromide. 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 represents the relationship between antibody titers before vaccination of reinforcement on day 35 of the trial when the pigs were 63 days of age and 14 days after the booster injection on day 49 of the test, when the animals were 77 days old, as described in the examples. Figures 2A (SEQ ID Nos. 8 and 9) and 2B (SEQ ID Nos. 10 and 11) show the nucleotide sequences and amino acid sequences of the GnRH constructs used in the gene fusions of leukotoxin-GnRH gone polypeptide , chimerical, in the present. Figure 2A (SEQ ID Nos. 8 and 9) represent an individual copy of a GnRH decapeptide. Figure 2B (SEQ ID Nos. 10 and 11) represents a molecule with four copies of a decapeptide of GnRH when n = 1 and eight copies of GnRH when n = 2, etc. Figures 3A through 3F (SEQ ID Nos. 12 and 13) show the nucleotide sequence and predicted amino acid sequence of the chimeric LKT-GnRH protein of plasmids pCB122 and pCB130. Figure 4 shows body weight as a function of age in pigs treated with GnRH vaccines according to the invention (immunocastrated), compared to male pigs castrated (tumults) and unneutered male pigs (boars).

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 Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratoy Manual; DNA Cloning, Vols. I and II (D.N. Glover ed.); Oligonucleotide Synthesis (M.J. Gait ed.); Nucleic acid Hybridi zation (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. Black ell 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 these may vary, for their Market Stall. 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 luteinizing 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 mammals including human, bovine, ovine and porcine GnRH (formerly designated LHRH) has the amino acid sequence pyroGlu-Kis-Trp-Ser-Tyr-Gly-Leu-Arg-Pro -Gly-NH2 (SEQ ID NO: 1) (Murad et al., Hormones and Hormone Antagonists, in The Pharmacological 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. 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, according to 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 the amino acid in place of His (see Figures 2A (SEQ ID Nos. 8 and 9) and 2B (SEQ ID Nos. 10 and 11), an analogous of GnRH 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) 7: 5: 2609-2619; Brown et al., J. Reproduct. Fertil. (1994) 101: 15- twenty-one); the GnRH analogue (D-Trp6-Pro9-ethyl-amide) GnRH (see, for example, Tilbrook et al., Hormones and Behavior (1993) 22: 5-28) or (D-Trp 6) GnRH, (see example, Chaffaux et al., Recueil de Medecine Veterinaire (1985) 161: 133-145); GnRH analogs with the first, sixth and / or tenth amino acid that occurs normally replaced by Cys and / or where the N-terminus is acetylated and / or the C-terminus is amidated (see, for example, US Pat. United States Nos. 4,608,251 and 4,975,420); the GnRH analog pyroGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Gly-YZ (SEQ ID NO: 2), where X is Gly or a D-amino acid, and Y is one or more residues of amino acids which may be the same or different, preferably 1-3 Gly residues, and Z is Cys or Tyr (see, UK Patent Publication No. GB 2196969); the GnRH analogs described in U.S. Patent No. 5,588,506, including the GnRH analogue Cy-Pro-Pro-Pro-Pro Ser-Ser-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly (SEQ ID NO: 3), pyroGlu-Hi s -Trp- Ser-Tyr-G ly-Leu- Arg-Pro -Gly-Ser-Ser-Pro-Pro-Pro-Pro-Cys (SEQ ID NO: 4), pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Arg-Pro-Pro- Pro-Pro-Cys (SEQ ID NO: 5); 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 to produce the formation of antibodies that cross-react with naturally occurring GnRH. Thus, the term "GnRH polypeptide" includes a GnRH molecule different from the reference sequence by having one or more substitutions, deletions and / or additions of amino acids and having at least about 50% amino acid identity a the reference molecule, more preferably about 75-85% identity and more preferably about 90-95% identity or more, 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 approximately 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; aspartate and glutamate; (2) basic.- lisma, arginma, histidma; (3) non-polar.- alamna, valina, leucma, ísoleucma, proline, phenylalanine, metionma, tpptofan; and (4) uncharged polar. - glycine, asparagma, glutamine, cyst, serma, tremon, tyrosma. Phenylalanma, tryptophan and tyrosine are sometimes classified as aromatic amino acids. For example, one can reasonably predict that an isolated replacement of leucma with ísoleucma or valma, or vice versa, an aspartate with a glutamate or vice versa; a trionmma with a serma 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. Proteins that have substantially the same amino acid sequence as the reference molecule, but that have minor amino acid substitutions that retain the desired activity, are therefore within the definition of a GnRH polypeptide. A "GnRH polypeptide" also includes peptide fragments of the reference GnRH molecule, while the molecule retains the desired activity. GnRH epitopes are also captured by definition. Particularly contemplated herein are the 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, optionally including spacer sequences, such as those described in International Publication Nos. WO 98/06848 and WO 96/24675 and shown in Figure 2B (SEQ ID Nos. 10 and 11) 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 subjects, bovine, porcine, ovine, canine, 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 GnRH peptides including 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). By "GnRH multimer" is meant a molecule having more than one copy of a selected GnRH polypeptide, GnRH immunogen, GnRH peptide or epitope, or multiple tandem repeats of a GnRH polypeptide selected, GnRH immunogen, GnRH peptide or epitope. The GnRH multimer may correspond to a molecule with repeat units of the General Formula (GnRH-X-GnRH) and, where GnRH is a GnRH polypeptide, 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 is an integer greater than or equal to 1 and further wherein "GnRH" can comprise any GnRH polypeptide. And therefore it can define 1-40 or more repeating units in a preferential manner, 1-30 repeating units and more preferably, 1-20 repeating units. Additionally, the selected GnRH sequences may all be the same, or may correspond to different GnRH derivatives, analogs, variants or epitopes, while retaining the ability to produce an immune response. Additionally, if the GnRH units are linked either 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 is can locate the carrier in internal sites. The GnRH multimers are discussed in further detail in a subsequent manner. The term "GnRH mmunogen" refers to GnRH polypeptides, as described above, that produce a mmunological response without a carrier, adjuvant or immunological, associated, immunobemulant, as well as GnRH polypeptides capable of being immunogenic, or more immunogenic. , by means of the 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 a 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 mmunogen will produce antibodies that cross-react with endogenous GnRH, which occurs naturally in the vertebrate species to which this mmunogen is derived. The term ? GnRH mmunogen "also refers to molecules of nucleic acid, such as DNA and ARS molecules that encode GnRH polypeptides that are capable of expression in vi when administered using nucleic acid distribution techniques described hereinafter. "Homology" refers to percent identity between two portions of polynucleotides or two polypeptides. Two DNA or two polypeptide sequences are "substantially homologous" to each other when the sequences are exhibited at least about 75% -85%, preferably at least about 90% and more preferably at least about 95% -98% of sequence identity according to 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 the two molecules by aligning the sequences, counting the exact number of correspondences between the two sequences aligned, dividing by the length of the shortest sequence and multiplying the result by 100. Easily available computer programs could be used to aid in the analysis, such as ALING, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl. 5Y353-358, 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. The programs to determine the identity of nucleotide sequences 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, which also depend on the Smith algorithm. and Waterman. These programs are easily used with the default parameters recommended by the manufacturer described in the 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 homology algorithm of Smith and Waterman with a score table per omission and a separation penalty of six nucleotide positions. 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 almost the entire length of a protein sequence, or even a fusion protein comprising one or more epitopes of a protein in question. Because GnRH is a very small molecule, identification of the epitopes thereof that will be capable of producing an antibody response will be readily achieved using techniques well known in the art. For example, 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 Z8 (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, the peptides corresponding to portions of the protein molecule and reacting the peptides with antibodies while the peptides are bound to the supports. These techniques are known in the art and are 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. 2_3_: 709-715. Similarly, conformational epitopes are easily identified when 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 of the amino acid sequence of the protein, which utilize 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; Y Hopp and Woods, Proc. Nati Acad. Sci. USA (1981) 7_8_: 3824 -3828, could also be used to determine the antigenic portions of a given molecule. For example, the Hopp and Woods sequence assigns each amino acid a numerical value of hydrophilicity and then repeatably averages these values along the peptide chain. The highest local average hydrophilicity points 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 this molecule or enhances the immunogenicity of the molecule. Examples of suitable carriers include large, slowly metabolized macromolecules, such as: proteins; polysaccharides, such as safarose, agarose, cellulose, cellulose beads and the like; polymeric amino acids such as iglutamic pol acid, polylysine and the like; amino acid copolymers; inactive virus particles, bacterial toxins such as diphtheria toxoid, tetanus, cholera, leukotinin molecules and the like. The carriers are described in detail later . A GnRH immunogen is "linked" to a specified 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 amino acid sequence of carboxy terminus Gly-Gly-X- Gly-X- Asp SEQ ID NO: 14) (Highlander et al., (1989) DAN 8? L5-28), where X is Lys, Asp, Val or Asn. These proteins include, among others, leukotoxins derived from P. has emolyt i ca and ñc t in oba ci l l us pl and uropn eumoni a e, as well as alf a-haemolysin from E. col i (Strathdee et al., (1987) Infect. Immun. 55: 3233-3236; Lo (1990) Can. J. Vet. Res. 54: 533- 535, Welch (1991) Mol. Microbiol. 5: 521-528). This toxin family is known as the "RTX" family of toxins (Lo (1990) Can, J. Vet, Res. 54 533-535). In addition, the term "leukotoxin polypeptide" refers to a leukotoxin polypeptide that is chemically synthesized, isolated from an organism that expresses it, or is produced recombinantly. Additionally, the term is proposed for a mmunogenic protein having an amino acid sequence substantially homogeneous to a contiguous 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 "leukotox" is also proposed for molecules that remain immunogenic even though they lack the cytotoxic character of native leukotoxmas. The nucleotide sequences and the corresponding amino acid sequences for several leukotoxes are known. See, for example, U.S. Patent Nos. 4,957,739 and ,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 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 immunogenic leukotoxin polypeptides for use in the present invention are truncated leukotoxin molecules described in U.S. Pat., 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 (ATCC Accession No. 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, which has 914 amino acids and an estimated molecular weight of approximately 99 kDa. The LKT 111 is a leukotoxin polypeptide derived from the IktA gene present in the plasmid pCBlll (ATCC Accession Number 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 was developed from the recombinant leukotoxin gene present in the plasmid pAA352 (ATCC Accession No. 68283) by removal of an internal DNA fragment of approximately 1300 bp in length. The LKT 111 polypeptide has an estimated molecular weight of 52 kDa (as compared to the LKT 352 polypeptide at 99 kDa, but retains the N-terminus portions of LKT 352 which contains the T cell epitopes that are necessary for sufficient immunogenicity of T cells and portions of the C-terminus of LKT 352 containing 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 the plasmid pAA114 (described in U.S. Patent No. 5,837,268.) LKT 114 differs from LKT 111 by virtue of an additional amino acid deletion of , r the inner portion of the molecule. By "immunological adjuvants" is meant an agent that acts in a non-specific manner to increase an immune response to a particular antigen, thereby reducing the amount of the antigen needed in any given vaccine and / or the frequency of injection necessary in order to generate an adequate immune response to the antigen of interest- See, for example, AC Allison J. Ret iculoendothe 1. Soc. (1979) 26: 619-630. "Native" proteins, polypeptides or peptides are isolated proteins, polypeptides or peptides. from the source in which the proteins are presented naturally, "recombinant" polypeptides refer to polypeptides produced by recombinant DNA techniques, ie, produced from cells transformed by an exogenous DNA construct that encodes The "synthetic" polypeptides are those prepared by chemical synthesis, by "polynucleotides" it is meant a nucleotide sequence including, but not limited to, RNA such as mRNA, cDNA, sequences Genomic DNA and even synthetic DNA sequences. The term also captures sequences that include any of the known base analogs of DNA and RNA. The term "derived from" as used herein, denotes a real or theoretical source or origin of the molecule or immunogen of interest. For example, a mmunogen that is "derived from" a particular GnRH molecule will have close sequence similarity with a relevant production of the reference molecule. In this manner, a mRNA which is "derived from" a particular GnRH molecule may include the entire wild-type GnRH sequence, or may be altered by insertion, deletion or substitution of amino acid residues, while the derived sequence provides a munogen that corresponds to the GnRH molecule sought. The mmunogens derived from a designated molecule will contain at least one epitope specific to the designated molecule. By "animal producing food" is meant an animal proposed for consumption by humans or domestic pets such as cats and dogs. These animals include, without limitation, mammals such as ovine, bovine, porcine and cervine subjects, including sheep, cattle, pigs and deer. By "improvement of the organoleptic qualities of the meat" is meant the improvement of the smell, taste and / or tenderness of the meat of an animal treated under the invention in comparison to the meat of a non-castrated member, typical of the same species that has not been treated. The meat of uncastrated males suffers in general from several disadvantages. In this regard, meat derived from unneutered male pigs and sheep frequently has an unpleasant taste and odor. For example, "boar taint" refers to a urine-like odor found in the cooked meat of uncastrated pigs. The boar taint is produced by steroids stored in tissues in male piglets with testicles that function normally. See, for example, Brooks et al., J. Anim. Sci. (1986) 62Y1279. The presence of androsterone in the dead boar animal is a measure of the boar's stain and is considered a measure of the production of gonadal steroids. Additionally, skatole can contribute to the boar's stain. See, for example, Mortensen and Sorensen, Proc. 30tb European Meeting of Meat Research Workers, Ghent, Belgium (1986), pp. 394-396 for a test method for skatole in fat. Similarly, unneutered cattle males often produce lean, but harder, meat by virtue of increased muscle mass. In this way, meat with improved organoleptic properties is meat with an odor, tenderness and / or taste. By "circulating testosterone reduction" is meant a statistically significant reduction in serum testosterone levels as measured using a normal assay, such as an RIA, as described herein, as compared to serum testosterone levels expected in an untreated male, not neutered, typical of the same age and species. The "androgenic" steroids include andros tenona, androstenedione, androsine diol and / or testosterone. Androgenic steroids can be measured using well-known techniques. For example, testosterone and the other androgenic steroids can be measured using ELISA and RIA well known in the art. Particularly convenient measures can be made using commercially available test equipment, for example, the Total Testosterone Kit Coat-A-Count ™ (Diagnostic Products Corporation, Los Angeles, CA). This equipment is a RIA of solid phase designed for the quantitative measurement of testosterone in serum, based on the specific antibody of testosterone immobilized to the wall of a polypropylene tube. See, also Schanbacher and D'Occhio, J. Andrology (1982) 3: 45-51, for a description of a direct RIA for determining testosterone levels. ELISAs for determining androstenone levels are described, for example, in Abouzied et al., J. Agri. Food Chem. (1990) 38: 331-335. See, also Meloen et al., Vaccine (1994) 1_2_ (8): 741-746; and Booth et al., Anim. Prod. (1986) 4 ^: 145-152 which describes the ELISAs elaborated in androstenone extracted from fat. The RIAs for determining androstenone levels are also known. See, for example, Andersen, Q. r Minutes. Vet. Scand. (1979) 20: 343-350. "Non-androgenic" steroids include those derived from 16-andros dye, including 5o-androstenone (5a-andros-l 6-en-3-one). Non-androgenic steroids can be measured using techniques well known in the art, such as by ELISA and RIA. See, for example, Claus et al., Archiv fuer Lebensmi ttelhygiene (1988) 39: 87-90. By a "substantially reduced" level of one or more androgenic and / or non-androgenic steroids is meant that the level of at least one androgenic or non-androgenic steroid is at least about 50% less than expected in an untreated male, not castrated, typical of the same age and species, preferably at least about 75% less, more preferably at least about 80% to 90% or less. The term "fattening period" is proposed for the period from weaning to slaughter and thus includes the pre-, peri- and post-pubertal periods. A typical fattening period will vary from species to species and even within a species, depending on the preference of the food producer and the country where the animals are bred. In this way, the fattening period is widely a matter of choice and one skilled in the art can easily determine the appropriate fattening period for a given animal. 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 in the practice of the present invention, various compositions and methods similar or equivalent to those described herein can be used., preferred materials and methods are described herein. The central point of the present invention is the discovery of a method to improve the quality of the meat by modulating the secretion of gonadal steroids. The method includes one or more primary immunizations before or during the fattening period of the animal with a GnRH formulation designed to cause a measurable reduction in circulating levels of testosterone, but generally does not result in complete immunocasting. Primary vaccination is followed with a reinforcement with the same or different composition of GnRH in a short time before slaughter, to substantially reduce the level of one or more androgenic and / or non-androgenic steroids. Although GnRH is generally recognized as "auto" and therefore non-immunogenic, the compositions described herein surprisingly provide a means to produce an adequate immune response in a subject immunized therewith. • Timing of vaccinations depends on the animal in question which is generally a sheep, cow or pig, as well as the preference of the food producer. However, the first vaccination will be given before or during the fattening period of the animal. For example, in pigs and sheep, primary immunization will generally occur at a time between the birth of the animal and about 15 weeks of age, preferably at a time between the birth of the animal and about 10 weeks of age. In cows, primary immunization will generally occur at a time between birth and approximately 48 weeks of age. One or more reinforcement treatments are given before the killing. The reinforcement timing will also be dependent on the animal in question. For example, in pigs and sheep ,. the reinforcement will generally be about one to about 12 weeks before slaughter, preferably about 2 to about 8 weeks before slaughter and more preferably about 4 to about 6 weeks before slaughter and still 2 to about 3 weeks. weeks before the killing. In cows, it may be preferable to administer the second vaccine composition several months prior to killing. In certain embodiments, the subsequent immunization (s) is (are) after the GnRH antibodies, formulated against the primary immunization, have declined, i.e., at a level at least about 50% by below the maximum levels of antibodies detected, preferably decreased to at least approximately 75% below the maximum levels detected. The vaccine compositions of the present invention employ GnRH polypeptides, as defined above, optionally linked to mol e cu l s s po rtadora s to f i n to improve the m emunogeni city of the m os s.

Immunoconjugates of GnRH 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 binding them to carriers to form immunoconjugates of GnRH. This is especially unnecessary if the GnRH munogen is administered to the same species from which it is derived. Suitable carriers are generally polypeptides that include antigenated 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 T-helper cells and to help direct an immunogen of antigens to antigen presenting cells (APC) for processing and presentation on the cell surface in association with complex molecules. main histocompatibility (MHC).

Several carrier systems have been developed for this purpose. For example, small peptide haptens are often coupled to protein carriers such as keyhole limpet hemocyanin (Bittle et al., (1982) Nature 298: 30-33), bacterial toxins such as tetanus toxoid (Muller et al., (1982). Proc. Nati, Acad. Sci. USA 7_9: 569-573), ovalbumin, leukotoxin polypeptides, and whale sperm myoglobin, to produce an immune response. These coupling reactions typically result in the incorporation of several moles of peptide hapten per mole of carrier protein. Other carriers suitable for use with the present invention include rotavirus VP6 polypeptide, 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 GnRH epitopes, fusion products that are made by the methods described in U.S. Patent No. 4,722,840. Still other suitable carriers include cells, such as lymphocytes, since the presentation in this form mimics the natural mode of presentation in the subject, which gives rise to the immunized state. Alternatively, the GnRH immunogens can be coupled to erythrocytes, preferably to the erythrocytes of the subject. Methods for coupling the 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, the pre-formed particles have been used as platforms on which the immunogens can be coupled and incorporated. Proteosome-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 rotavransposon, Ty, codes for a series of proteins that mount virus-like particles (Ty-VLPs, Kingsman et al. (1988) Vaccines 6 ^: 304-306). In this way, a gene, or fragment thereof, that codes for the GnRH immunogen of interest can be inserted into the TyA gene and 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.2: 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. Ginsberg, 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, limpet hemocyanin, or albumin, whale sperm myoglobin, leukotoxin molecules as described above and other peptides 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) of Pasteurella h a emolyt i ca fused to the antigen of interest, may also be used herein. To this respect, 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 WO 98/06848 and WO 96/24675. Particular examples of immunogenic leukotoxin polypeptides 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 pAA323 (ATCC Accession Number 68283) by removal of an internal DNA fragment of approximately 1300 bp. length. The LKT 111 polypeptide has an estimated molecular weight of 52 kDa (in comparison to the LKT 352 polypeptide of 99 kDa), but retains portions of the N-terminus of LKT 352 which contains T-cell epitopes that are necessary for sufficient immunogenicity of T cells and portions of the C-terminus of LKT 352 containing suitable restriction sites for use in the . production of the fusion proteins of the present invention. LKT 114 differs from LKT 111 by virtue of an additional amino acid suppression 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 functional group content it can be modified for example by succinylation of lysine residues or reaction with Cys-1-iolactone. A sulfonyl group can also be incorporated into the carrier (or antigen), for example by the reaction of animo function with 2-aminothiolane or the N-hydroxysuccinimide ester of 3- (-ditopyridyl-propionate.) Suitable carriers can also be modify to incorporate spacer arms (such as hexamethylene-diamma or other bifunctional molecules of similar size) for the binding of peptide mmunogens. The carriers can be physically conjugated to the immunogenic GnRH mRNA., using normal coupling reactions. From Alternatively, the 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 GnRH immunogen. The GnRH portion can be fused either 5 'or 3' to the carrier portion of the molecule, a portion of GnRH 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 will find use herein include, without limitation, vaccinia and other viruses of pox, adenovirus and herpes virus. By way of example, recombinant vaccinia viruses 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 vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells that are infected simultaneously with vaccinia.

Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the desired immunogen in the viral genome. The resulting recombinant TK can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and collecting viral plaques resistant thereto.

GnRH Multimers The immunogenicity of GnRH immunogens can also be increased significantly by producing immunogenic forms of the molecules comprising multiple copies of selected epitopes. In this way, endogenous GnRH can become an effective autoantigen.

Accordingly, in one aspect of the invention, vaccine compositions containing multimers of GnRH immunogen are provided, either in the form of nucleic acid or peptide. 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 indozogen, peptide or epitope. In this way, the GnRH multimers can already comprise either multiple repetitions or tandem repeats of the selected GnRH sequences, multiple repetitions or tandem repeats of 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 can correspond to a molecule with repeat units of the General Formula (GnRH-X-GnRH) and where 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] ", where n is an integer greater than or equal to 1, and is an integer greater than or equal to 1 and additionally wherein" GnRH "can comprise any immunogen of GnRH. In this manner, the GnRH multimer can contain 2-64 or more GnRH immunogens, more preferably 2-32 or 2-16 GnRH immunogens. Additionally, the selected GnRH immunogen sequences may all be the same, or may correspond to different GnRH derivatives, analogs, variants or epitopes. as long as they retain 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 that can be passed through 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 separating 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 2B (SEQ ID Nos. 10 and 11). The strategic placement of several spacer sequences among the selected GnRH immunogens can be used to confer increased homogeneity in the present constructs. Accordingly, according to the invention, a separating sequence selected it can code for a wide variety of portions such as an individual amino acid linker or a sequence of two to several amino acids. The selected spacer groups can preferably provide enzyme cleavage sites so that the expressed multimer can be processed by proteolytic enzymes in vivo (by APC or the like) to produce various peptides, each of which contains at least one epitope of T cells derived from the carrier portion and that are preferably 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 spacer sequences can be constructed to provide antigenicity of T cells, such as those sequences that encode amphipathic and / or alpha-helical peptide sequences that are generally recognized in the art as providing epitopes of T helper, immunogenic cells. The choice of particular T cell epitopes to be delivered by these spacer sequences may vary depending on the particular vertebrate species to be vaccinated. Although the particular GnRH portions are exemplified as including spacer sequences, it is also an object of the present invention to provide one or more GnRH multimers comprising directly adjacent GnRH sequences (without intervening spacer sequences.) The multimeric GnRH sequence produced from this way it returns to a highly immunogenic GnRH antigen for use in the compositions of the invention.GnRH, immunocognate polypeptides and multimers can be produced using the methods described below and used for nucleic acid immunization, gene therapy, immunization methods based on protein and similar.

Immunization Methods Based on Nucleic Acid In general, nucleic acid-based vaccines for use with the present invention they will include relevant regions that code for a GnRH immunogen, with suitable control sequences and optionally, nucleotide, therapeutic, ancillary 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 cytokines. and similar. Other auxiliary substances include, but are not limited to, substances to increase weight gain, muscle mass or muscular resistance, such as growth hormones, growth promoting agents, ß-antagonists, dividing agents and antibiotics.

The nucleotide sequences selected for use in the present invention can be derived from known sources, by way of example, by isolating the same from cells or tissue containing a desired gene or nucleotide sequence using standard techniques, or by the use of recombinant or synthetic techniques. Once the coding sequences have been prepared or isolated for the GnRH immunogens, these sequences can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those skilled in the art and selection of an appropriate cloning vector is a matter of choice. Ligations to other sequences, eg, auxiliary molecules or carrier molecules, are performed using standard procedures, known in the art. One or more portions of GnRH immunogens of the chimera can be fused 5 'and / or 3' to an auxiliary sequence or desired carrier molecule. Alternatively, one or more portions of the GnRH immunogen can be located at sites internal to the carrier molecule, or these portions can be placed at both terminal and internal locations in the chimera. Alternatively, the DNA sequences encoding the GnRH immunogens of interest, optionally linked to carrier molecules, can be synthetically prepared instead of being cloned. The DNA sequences can be designed with appropriate codons for the particular sequence. The entire sequence of the immunogen is then assembled or assembled from the overlap oligonucleotides prepared by normal methods and assembled into a complete coding sequence. See, e.g., 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 control of suitable control elements for expression in the appropriate host tissue in vi vo. The choice of control elements will depend on the subject and the type of preparation used. In this way, if the endogenous transcription and the translation machinery of the subject will be used to express the immunogens, control elements compatible with the particular subject will be used. In this regard, several promoters for use in mammalian systems are known in the art. For example, typical promoters for the expression of mammalian cells include the SV40 early promoter, a CMV promoter such as the early promoter. of CMV, the LTR promoter of the mouse mammary tumor virus, the adenovirus major late promoter (Ad MLP) and the herpes simplex virus promoter, among others. Other non-viral promoters, such as a promoter derived from the metallothione a mur gene, will also find use for expression in mammals. Typically, the transcription termination and polyadenylation sequences will also be present, located 3 'to the translation coding codon. Preferably, a sequence for the optimization of translation initiation, located 5' to the coding sequence, also is present Examples of transcription / polyadenylation terminator signals include those derived from CV40, as described in Sambrook et al., Supra, as well as a bovine growth hormone terminator sequence. Trones, which contain splice donors and acceptor sites, can also be designed in constructions for use with the present invention. Stressing elements can also be used herein to increase the expression levels of the constructions. The examples include the CV40 early gene enhancer (Dijkema et al., (1985) EMBO J. 4? 761), the enhancer / promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma virus (Gorman et al., (1982) Proc. Nati, Acad. Sci. USA 7_9: 6777) and elements derived from human CMV (Boshart et al., (1985) Cell 4_1_: 521), such as elements included in the sequence of CMV intron A. Once prepared, the nucleic acid vaccine compositions can be delivered to the subject using known methods. In this regard, several techniques for immunization with DNA encoding the antigen have been described. 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. Gener 2: 1847; Ulmer et al., (1993) Science 258: 1745; Wang et al., (1993) Proc. Nati- Acad. ? ci. USA 90_: 4156; Eisenbraun et al., (1993) DNA Cell. Biol. 12Y791; Fynan et al., (1993) Proc. Nati Acad. Sci. USA 9 ^ 0: 12476; Fuller et al, (1994) AIDS Res. Human Retrovir. 1_0_: 1433; and Raz et al., (1994) Proc. Nati Acad. Sci. USA 91_: 9519. The general methods for the distribution of acid nucleic cells in vi tro, for subsequent reintroduction into the host can also be used, such as liposome-mediated gene transfer. See, for example, Hazinski et al., (1991) Am. J. Respir. Cell Mol. Biol. 4_: 204-209; Brigham et al., (1989) Am. J. med. Sci. 298: 278-281; Canónico et al., (1991) Clin. Res. 3_9: 219A; and Nabel et al., (1990) Science 249: 1285-1288. In this manner, the nucleic acid vaccine compositions can be delivered in either liquid or particulate form using a variety of known techniques. Typical vaccine compositions are more fully described later.

Protein-based Distribution Methods Protein-based compositions can also be produced using a wide 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, polypeptides can be produced recombinantly using nucleic acid expression systems, either known in the art and described for example in Sambrook et al., supra. GnRH polypeptides can also be synthesized using chemical synthesis of polymers such as synthesis of solid phase peptides. 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. Merpfield, The Peptides. Analysis, Synthesis, Biology, Editors E. Groos and J Meienhofer, Vol. 2, Academic Press, New York, (1980), pp. 3-254, for peptide synthesis techniques in solid phase. GnRH polypeptides for use in the compositions described herein may also be produced by cloning the coding sequences therefor into any suitable expression or replicon vector. The numerous cloning vectors are known to those skilled in the art and selection 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.Coil), pKT230 (gram-negative bacteria a), pGV1106 (gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290 (gram-negative bacteria not E. coli), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus ), pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), YCpl9 (Saccharomyces) and bovine papillomavirus (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 adn Hormone Antagonists, in The Pharmacological Basis of Therapeutics, sixth edition), and the cDNA for human GnRH have been cloned so that the sequence has been well established (Seeburg et al., (1984) Nature 311: 666-668). Additional GnRH polypeptides of the known sequences have been described, such as the GnRH molecule that occurs 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 2A (SEQ ID Nos. 8 and 9) and 2B SEQ ID Nos. 10 and 11) in the I presented. The GnRH coding sequence is highly conserved in invertebrates, particularly in mammals, and the porcine, bovine, ovine and human GnRH sequences are identical to each other. Portions of these sequences encoding 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, 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 sequences may or may not contain a signal peptide or a leader sequence. The polypeptides can be expressed using for example, the E. coli tac promoter or the protein A gene promoter (spa) and the signal sequence. The guide sequences can be removed by the bacterial host in post-transductional processing. See, for example, the Patents of the States United 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 that elicit the expression of a gene that is to be turned on or off 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 sequences are 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 transcribe under the "control" of the control sequence (ie, 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 joined in the proper 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. It may also be desirable to produce mutants or analogs of the polypeptide. The mutants or analogues 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 replacing one or more nucleotides within the sequence. Techniques for modifying the nucleotide sequence, such as site-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 Hybr idization, supra; Kunkel, T.A. Proc. Nati Acad. Sic. 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 example, Summers and Smith. Texas Agpcultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for the baculovirus / insect cell expression subject are commercially available in the form of a kit from, inter alia, Invitrogen, San Diego CA ("MaxBac" kit). Similarly, bacterial and mammalian cell expression systems are well known in the art and are described for example in Sambrook et al., Supra. Yeast expression systems are also known in the art and are described in for example 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 known in the art include immortalized cell lines available from the American Species Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary cells ( CHO), HeLa cells, neonatal hamster kidney cells (BHK), monkey kidney cells (COS), hepatocellular carcinoma cells human (for example, Hep G2), Madin-Darby bovine kidney cells ("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 ali, Aedes aegypti, autographa californica, Bombyx mori, Drosophila melanogas ter, Spodoptera frugiperda, and Trichoplusia ni. Depending on the expression system and the selected host, the GnRH polypeptides are produced by culturing host cells transformed by an expression vector described above under expression conditions by which the polypeptide is expressed. The expressed polypeptide is then isolated from host cells and purified. If the system expression secretes the polypeptide in the growth medium, the product can be purified directly from the medium. If it is not segregated, it can be isolated from cell lysates. The selection of appropriate growth conditions and methods of recovery are known to those skilled in the art. Once obtained, GnRH polypeptides, with or without an associated carrier, can be formulated into compositions, such as in vaccine compositions as described further and subsequently, in order to elicit the production of antibodies.

Production of Antibodies The present GnRH immunogens can be used to generate antibodies for use in passive immunization methods. Typically, polypeptides useful for producing antibodies will usually be amino acids of about 3-5 amino acids in length, preferably 7-10 amino acids in length. Antibodies against the present immunogens include polyclonal and monoclonal antibody preparations, antisera monospecies, as well as preparations including hybrid antibodies, altered antibodies, fragments of F (ab ') 2, fragments of F (ab), fragments of F ", antibodies of individual domain, chimeric antibodies, humanized antibodies and functional fragments of the they retain the specificity for the target molecule in question. For example, an antibody may include variable regions or fragments of variable regions, which retain the specificity of for the molecule in question. The rest of the antibody can be derived from the species from which the antibody will be used. Thus, if the antibody is to be used in a human, the antibody can be "humanized" in order to reduce immunogenicity even while retaining the activity. For a description of 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 8_9: 4285-4289. These chimeric antibodies can contain not only combination sites for the target molecule, but also binding sites for other proteins. This Thus, bifunctional reagents can be generated with specificity sought in both external and internal antigens. 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 mounted antigen, as described above. Prior to immunization, it may be desirable to further increase the immunogenicity of a particular immunogen. This can be achieved in various 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 adjuvants described below. inject the mixture or emulsion parenterai (generally subcutaneously or intramuscularly). The animal is generally strengthened 2-6 weeks later with one or more injections of the protein in saline, preferably using incomplete Freund's adjuvant, or the like. The antibodies are also can be generated by in vitro immunization, using methods well known in the art. The 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 serum containing polyclonal antibodies is used, the polyclonal antibodies can be purified by immunoaffinity 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 (and optionally several large lymph nodes) is removed and dissociated into individual cells. If desired, the spleen cells can be selected (after removal of non-specifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen. B cells, which express the membrane-bound immunoglobulin, specific for the antigen, will bind to the plate and do 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, and are cultured in a selective medium (e.g., hypoxanthine, aminoopterin, thymidine, "HAT"). The resulting hybridomas are plaqueed by limiting the dilution and assayed for the production of antibodies that specifically bind to the immunizing antigen (and that do not bind to the unrelated antigens). Hybridomas secreting the monoclonal antibody, selected then, are cultured either in vitro (for example, in tissue culture bottles or hollow fiber reactors), or in vivo (in 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 monoclonal antibody panels produced by the GnRH immunogen of interest, fragment thereof, can be selected for several properties, say, for the isotype, epitope, affinity, etc. Functional fragments of the antibodies can also be made against the GnRH immunogens of interest and can be produced by cleaving a constant region, not responsible for antigen binding, of the antibody molecule, using a pepsin, to produce F fragments. (ab ') 2. These fragments will contain two antigen-binding sites, but lack a portion of the constant region for each of the heavy chains. Similarly, if desired, Fab fragments can be produced, comprising an individual antigen-binding site, for example, by digestion of polyclonal or monoclonal antibodies with papain. Functional fragments, which include only the variable regions of the heavy and light chains, can also be produced, using normal techniques. These fragments are known as F ". Chimeric or humanized antibodies can also produce the present immunogens. These antibodies can be designed to minimize the unwanted immunological reactions attributable to variable regions of structure, specific to the species and constants, heterologous, typically present in monoclonal and polyclonal antibodies. For example, if the antibodies are to be used in human subjects, chimeric antibodies can be created by replacing non-human constant regions, either heavy and light chains, or both, with human constant regions, using techniques well known in the art. The technique. See, for example Winter, G. and Milstein, C. (1991) Nature 349: 293-299; Jones, P.T. et al., (1986 Nature 321: 522-525; Riechamnn, L. et al., (1988) 332: 323-327; and Carter, P. et al., (1992) Proc. Nati. Acad. Sci. USA 89: 4285-4289.

GnRH Compositions Once the above GnRH polypeptides or antibodies are produced, they are formulated into compositions for distribution to a vertebrate subject. The relevant GnRH molecule is administered alone, or mixed with a pharmaceutically acceptable excipient carrier. Suitable carriers are for example water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, the vehicle may contain minor amounts of substances auxiliaries such as wetting or emulsifying agents, pH buffering agents, or adjuvants in the case of vaccine compositions, which enhance the effectiveness of the vaccine. Suitable adjuvants are described further below. The compositions of the present invention may also include adjuvants, 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, and muramyl peptides, avridine, aqueous adjuvants, such as aluminum hydroxide and any of the various saponins, chitosan-based adjuvants, oils and other substances known in the art. For example, compounds that can serve as emulsifiers include natural and synthetic emulsifiers, as well as anionic, cationic and nonionic compounds. Among the synthetic compounds, the anionic emulsifying agents include, for example, the potassium, sodium and ammonium salts of laupco and oleic acid, the calcium, magnesium and aluminum salts and fatty acids (ie, metallic soaps) and organic sulfonates such as sodium laupl sulfate. Synthetic cationic agents include, for example, cetyl methylmethyl bromide, while synthetic nonionic agents are exemplified by glyceryl esters (eg, glyceryl monostearate), polyoxyethylene glycol esters and ethers, and sorbitan fatty acid esters ( for example, sorbitan monopalmi- tate) and its polyoxyethylene derivatives (for example, polyoxyethylene sorbitan monopalmi- tate). Natural emulsifying agents include acacia, gelatin, lecithin and cholesterol. Other suitable adjuvants can be formed with an oily component, such as an individual oil, a mixture of oils, or 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 oily component is mineral oil are preferred. In this regard, a "mineral oil" it is defined herein as a mixture of liquid hydrocarbons obtained from petrolatum via a distillation technique; the term is synonymous with "paraf a liquida", "liquid petrolatum" and "white mineral oil". The term is also proposed to include "light mineral oil", that is, an oil obtained similarly by petrolatum distillation, but having a specific weight slightly lower than white mineral oil. see, for example, Ramington'n Pharmaceutical Sciences, supra. A particularly preferred oil component is the oil-in-water emulsion sold under the trade name EMULSIGEN PLUS ™ comprising a light mineral oil as well as 0.05% formam and 30 mcg / mL gentamicm as preservatives), available from Laboratories, Ralston, Nebraska. Suitable animal oils include, for example, cod liver oil, halibut oil, shad oil, rough orange oil and shark liver oil, all of which are available commercially. Suitable vegetable oils include, without limitation, canola oil, almond oil, cottonseed oil, corn oil, olive oil, and oil.

Similar . Alternatively, several aliphatic nitrogenous bases can be used as adjuvants with the vaccine formulations. For example, known immunological adjuvants include amines, quaternary ammonium compounds, guanidines, benzamidines and tiouroniums (Gall, D. (1966) Immunology 1: 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 Kodak Laboratory Chemicals Bulletin 5_6 (l): l-5 (1986); Adv. Drug. Deliv. Rev. 5 3): 163-187 (1990); J. Controlled Reléase 1_: 12 3 - 1 32 (1988); Clin. Exp. Immunol. 7j3_: (2): 256-262 (1989); J. Immunol. Methods 97_ (2): 159-164 (1987); Immunology 5JM2): 245-250 (1986); and Int. Arch. Allergy Appl. Immunol. 6_8 (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) p diamines in general and avridine in particular, as vaccine adjuvants. U.S. Patent Nos. 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 an adjuvant known as "VSA-3" which is a modified form of the adjuvant EMULSIGEN PLUS ™ R which includes DDA (see, application of the US Pat. 463,837). Current methods for preparing these dosage forms are known, or will be apparent to 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 injectable compositions, 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 is also it can be emulsified or the active ingredient encapsulated in liposome vehicles or other particulate carriers, used. The compositions can also be prepared in solid form. For example, formulations in solid particle form can be prepared for distribution from commercially available needleless injection devices. Alternatively, solid dose implants may be provided for implantation in a subject. Sustained controlled release formulations can also be used and are made by incorporating the GnRH polypeptides into carriers or vehicles such as liposomes, non-resorbable waterproof polymers such as ethylene vinyl acetate copolymers and Hytrel® copolymers, swellable polymers such as hydrogels or polymers. resorbable such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures. Additionally, the polypeptides can be formulated into compositions in either neutral or salt forms. 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, mandelic and the like. 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 organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine. , procaine and the like. The composition is formulated to contain an effective amount of the GnRH polypeptide, the exact amount that is easily determined by a person skilled in the art, wherein the amount depends on the animal being treated, in the case of a vaccine composition, the ability 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 from about 1 μg to about 2 mg, more generally from about 5 μg to about 800 μg, and more particularly from about 10 μg to about 400 μg of the 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 immunogen to carrier, in the vaccine formulation will vary based on the particular carrier and immunogen selected 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 leukotoxin and selected GnRH polypeptide portions to construct these molecules. A preferred vaccine composition contains a leukotoxin-GnRH chimera having from about 1 to about 90% GnRH, preferably from about 3 to about 80% and most preferably from about 10 to about 70% of GnRH polypeptide per molecule of fusion. Increases in the percentage of GnRH present in LKT-GnRH fusions reduce the amount of total antigen that must be administered to the subject in order to produce a sufficient immune response to GnRH. The subject is administered one of the compositions described above, for example, in a primary immunization, during the fattening period, in at least one dose and optionally two or more doses. The primary administration (s) is followed by one or more reinforcements with the same or different GnRH composition in a short time before slaughter, in order to substantially reduce the circulating level of one or more androgenic and / or non-androgenic steroids. Any suitable pharmaceutical delivery means can be employed to distribute the compositions to the vertebrate subject. For example, syringes with conventional needles, spring or compressed air (air) injectors (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 (U.S. Patent Nos. 5,149,655 to MaCabe et al. al., and 5,204,253 to Sanford et al) are all suitable for the distribution of the compositions. Preferably, the composition is administered intramuscularly, subcutaneously, intravenously, subdermally, mderdermally, transdermally or transmucosally to the subject. If a jet injector is used, an individual jet of the liquid vaccine composition is expelled under high pressure and velocity, for example 1200-1400 PSI, thereby creating an opening to the skin and penetrating to depths suitable for immunization. . Below are examples of specific modalities 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 EXAMPLE 1 Construction of pCB122 and pCB130 Plasmids pCB12 and pCB130 were used to produce a GnRH fusion protein for use in the examples described below. Both plasmids produce a protein with the same amino acid sequence. The construction of GnRH in both plasmids contains 8 tandem repeats of the GnRH sequence fused to both the 3 'and 5' ends of a DNA sequence encoding a carrier leukotoxin polypeptide. Each alternating GnRH sequence has a change in the fourth sequence base from cytokine to guanosine. This results in an individual change of amino acid in the second amino acid of the GnRH molecule from His to Asp. See, Figures 2A (SEQ ID Nos. 8 and 9) and 2B (SEQ ID Nos. 10 and 11). 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 (ATCC Accession No. 68283) and is described in U.S. Patent No. 5, 476,657) by removal of 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 production of the fusion proteins of the present invention. The chimeric construct is under the control of the Tac promoter and the induction is controlled through the use of Lac I. The GnRH-leukotoxin fusion protein produced by the plasmids pCB122 and pCB130 is shown in Figures 3A through 3F (SEQ.

ID Nos. 12 and 13). The pCB122 plasmid 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, the 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 plasmid vector pUC13 and a DNA library was constructed in the bacteriophage lambda gtll. The resulting clones were used to transform E. coli and individual colonies were mixed and selected for the serum reaction from a calf that has survived an infection of P. haemolytica and that has been reinforced with a concentrated culture supernatant of P. haemolytica to increase the levels of anti-leukotoxin antibodies. The positive colonies were selected for their ability to produce leukotoxin by incubating Cells 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. See, Lo et al., Infect. Immun. , supra. To confirm this, smaller fragments were re-cloned and restriction maps were compared. It was determined that approximately 4 kilobases of DNA bases have been cloned. The progressively larger clones were isolated by carrying out a chromosomal walk (5 'or 3' direction) in order to isolate full-length recombinants that were approximately 8 kb in length. The final construction was called pAA114. This construct contained the complete leukotoxin gene sequence. LktA, a restriction endonuclease fragment of Mael from pAA114 which contained the complete leukotoxin gene, was treated with the Klenow fragment of DNA polymerase I plus nucleotide triphosphates which was ligated into the Smal site of the cloning vector pUC13 . This plasmid was named pAA179. From this, two expression constructs were elaborated in the vector pGH432: lacI based on ptac 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 polypeptide at high levels while pAA345 expressed full-length leukotoxin at very low levels. Therefore, the 3 'end of the lktA gene (BamHI fragment of Styl from pAA345) was ligated to pAA342 digested with StylU BamHI-haemolytica, yielding the plasmid pAA352. The haemolytic leukotoxin produced from the construction of pAA352 is hereinafter referred to as LKT 352. Plasmid pAA352 was then used to prepare a shortened version of the recombinant leukotoxin polypeptide. The shortened LKT gene was produced by deleting an approximately 1300 bp long internal DNA fragment of the recombinant LKT gene as follows. Plasmid pCB113, (ATCC, Accession No. 69749 and described in U.S. Patent No. 5,837,268) which includes the LKT 352 polypeptide, was digested with the restriction enzyme BstBl (New England Biolabs) - The linearized plasmid The resultant was then digested with bean nuclease (Pharmacia) to remove the outgoing individual strand terms produced by the BstBl digestion. The blunt-ended end DNA was then digested with the restriction enzyme Nael (New England biolabs), and the digested DNA was loaded on a 1% agarose gel where the DNA fragments were separated by electrophoresis. The large DNA fragment of approximately 6190 bp was isolated and purified from the agarose gel using gene cleansing equipment (Bio 101), and the purified fragment was allowed to bind itself using bacteriophage T4 -ADN-ligase (Pharmacia) . The resulting ligation mixture was used to transform competent E. coli JM105 cells and positive clones were identified by their ability to produce an aggregated protein having the appropriate molecular weight. The recombinant plasmid formed in this manner was designated pCBll, (ATCC, Accession No. 69748) and produces a shortened leukotoxin polypeptide (hereinafter referred to as LKT111) fused to four copies of the GnRH polypeptide. Plasmid pCB114 has the multiple copy GnRH sequence (corresponding to the oligomer of Figure 2B (SEQ ID Nos. 10 and 11) was inserted twice. these plasmids are described in U.S. Patent No. 5,836,268 and produce shortened leukotoxin polypeptides called LKT 111 and LKT 114, respectively. A fusion molecule of recombinant LKT-GnRH having two GnRH multimers of 8 copies, one arranged in the N '-terminus of LKT 114 and the other arranged in the C'-termmo LKT 114 was constructed from the sequence of fusion of LKT-GnRH obtained from plasmid pCB114 by ligating the GnRH sequence of multiple copies (corresponding to the oligomer of Figure 2B (SEQ ID Nos. 10 and 11) twice at the 5 'end of the coding sequence of LKT 114. A synthetic nucleic acid molecule having the following nucleotide sequence (5'-ATGGCTACTGTTATAGATCGATCT-3 '(SEQ ID NO: 6) was ligated at the 5' end of the multi-copy GnRH sequence. of synthetic nucleic acid encodes a sequence of 8 amino acids (Met-Ala-Thr-Val-Ile-Asp-Arg-Ser) (SEQ ID No. 7). The resulting recombinant molecule thus contains in the order given in 5 'to 3' direction: the synthetic nucleic acid molecule, a nucleotide sequence what codes for a first GnRH multimer of 8 copies, a nucleotide sequence encoding the shortened LKT peptide (LKT 114); and a nucleotide sequence encoding a second GnRH multimer of 8 copies. The recombinant molecule was circulated, 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 kDa. The recombinant plasmid formed in this manner was designated pCB122 which produces the LKT 114 polypeptide fused to 16 copies of the GnRH polypeptide. For plasmid pCB130, the ampr or pCB122 gene was replaced with the tetr gene. In this way, the plasmid is under selections of tetracycline. The nucleotide sequence of the recombinant LKT-GnRH fusion of plasmids pCB122 and pCB130 are shown in Figures 3A to 3F (SEQ ID Nos. 12 and 13).

Example 2 Purification of LKT-antigen Fusions The fusion of recombinant LKT-GnRH of the Example 1 was purified using the following procedure. Five to ten colonies of transformed E. coli strains were inoculated into 10 ml of TB broth supplemented with 100 fruit juice at 100 μg / ml ampicillin and incubated at 37 ° C for 6 hours on a G10 shaker, 220 rpm . Four ml of this culture was diluted in each of the two Fernbach flasks with reflectors containing 400 ml of the TB + ampicillin broth and incubated overnight as described above. The cells were harvested by centrifugation for 10 minutes at 4,000 rpm in polypropylene bottles, volume 500 ml, using a Sorvall GS3 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), was added to each culture in order to induce synthesis of the recombinant fusion proteins. The cultures were incubated for two hours. Cells were harvested by centrifugation as described previously, redispersed in 30 ml of 50 mM 3-hydrochloride, 25% sucrose (w / v), pH 8.0 and frozen at -70 ° C. The frozen cells were thawed at room temperature after and 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 vortexed 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 2 ml pipette. The beaker containing the used cell suspension was placed on ice and treated with sound for a total of 2.5 minutes (bursts of 5-30 minutes with cooling of 1 minute between each) with a Braun sonicator., large probe, adjusted to a power of 100 watts. Equal volumes of the solution were placed in Teflon centrifuge tubes SS34 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 distilled water by vortexing at high speed and the centrifugation step was repeated. The supernatants are they were removed and the pellets were combined in 20 ml of 10 mm Tris-HCl, 150 mM 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 Erlenmeyer flask and 1200 ml of Tris-buffered saline were added rapidly. This mixture was stirred at room temperature for an additional 2 hours. 50O ml aliquots were placed in dialysis bags (Spectrum, 63.7 mm in diameter, cut from PM 6,000-8,000, # 132670, Fisher Scientific) and placed in 4,000 ml beakers containing 3,500 ml of this buffered saline solution of Iris + 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 solution saline buffered with 0.1 M Tris + Guanidine-HCl and dialysis was continued for 12 hours. The buffer was then replaced with saline buffered with 0.05 M Tris + Guanidine-HCl and the dialysis was continued overnight. The buffer was replaced with saline buffered with Tris (no guanidine) and the dialysis was continued for 12 hours. This was repeated three more times. The final solution was poured into a 2,000 ml rotating 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 was 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 five 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 bodies.

EXAMPLE 3 Titers of Antibodies Following Immunization of GnRH in Pigs This trial was designed to evaluate the variables including volume, site of the second injection in relation to the first and number (one against two) injections for primary vaccination. For the study, 160 pigs, 28 days old and weighing 3 to 4 kg, were assigned to each of the eight treatment groups (see Table 1). There were 10 females and 10 castrated male pigs in each group. The animals were housed 10 per pen and cared for to use the normal operating procedures developed by the Prairie S ine Center, Inc., an experimental facility affiliated with the University of Saskatchewan and the Canadian Animal Care study was inspected. For groups 1 to 7, GnRH vaccines were made using the GnRH immunogen from plasmid pCB122, described above. In particular, the GnRH immunogen was dissolved at a concentration of 20 mg / ml in 8 M urea. The adjuvant used to formulate the GnRH vaccines was VSA-3, a modified form of the adjuvant.

EMULSIGEN PLUSMR including DDA (see also United States Patent Application Permit No. Series 08 / 463,837). GnRH vaccines are prepared by combining the concentrated solution of the GnRH mmunogen with phosphate buffered saline and mixed with VSA-3 at a ratio of 1: 1 (v / v) to form a stable emulsion. The GnRH immunogen dose for groups 1, 2, 3, 4 and 6 was 40 μg, however the volume differed in some of the formulations. Table 1 provides details for each treatment group. The GnRH vaccines for groups 5 and 7 contained 30 μg of the GnRH immunogen / 0.25 ml while group 8 received 40 μg of the GnRH immunogen of plasmid pCB130 in 0.4 ml of adjuvant. In all cases, the ratio of the adjuvant VSA-3 to the aqueous phase (saline buffered with phosphate) remained at 1: 1 v / v). Adjustments were made by altering the volume of the concentrated immunogen solution.

TABLE 1 Dose, site and volume of GnRH vaccine administered in the first and second injections * L and R refer to left and right ears ** The indicated volume is given in each of these ears. Therefore, groups 5, 6 and 7 received a total of 0.5, 0.3 and 0.5 mL, respectively, in primary injection.

The vaccines ** were all administered with a Biojector 2000 needleless injection device manufactured by Bioject Inc., Portland, Oregon, USA. This device uses a gas cylinder to inject the vaccine under high pressure through a small opening. The vaccine penetrates through the skin and is deposited subcutaneously. In each treatment group, the first injection occurs when the pigs are 28 days old and the second is given 35 days later. Injections are given on the outer surface of the pinna except for the second injection of group 7 that occurs in the dorsal midline 10-15 cm behind the head. Blood was collected by perforation of the jugular vein on days 35, 49 and 63 of the trial (in relation to the beginning of the study (day 0)). The blood is allowed to clot at room temperature and then centrifuged to collect serum that was stored at -20 ° C until analyzed for GnRH antibody titers. The GnRH antibody titers were determined by a modified or modified radioimmunoassay procedure. Synthetic GnRH (Bachem, Inc.) was helped with I (Amersham, Oakville, Ontario). HE ~ «, ^ - *.,.; &&; -«. made dilutions of serum to tet tubes followed by a normal amount of 125 I-labeled GnRH to give a final incubation volume of 0.7 ml. A suspension of carbon in assay buffer was added at the end of a 24 hour incubation at 2-6 ° C to absorb the non-antibody bound GnRH-I 125. After centrifugation, the radioactivity in the carbon fraction was measured. The data is presented as a numerical value that is the percent of a normal dose (approximately 12,000 cpm) of GnRH-I125 bound to a specific serum dilution. Descriptive statistics, analysis of variance and "t" tests were done using the Student version of Statistix, Version 1, Copyright 1996. Volumes of 0.15, 0.25 and 0.35 given at the individual injection site were evaluated in the groups 1, 2 and 3, respectively. Figure 1 shows the relationship between the antibody titer before the booster vaccination on day 35 of the test, when the animals were 63 days of age and 14 days after the booster injection, on day 49 of the test when the animals were 77 days old. The animals that have titles greater than 10% binding at 1: 5000 on day 35 gave a better response to booster vaccination than animals that had a weaker response to the primary injection. Based on other experiments, it is known that the binding of approximately 20% at a dilution of 1: 5000 will give partial suppression of testosterone secretion. These results indicate the usefulness of a strong response to primary immunization by saying that there is no effect on the growth or efficiency of feed utilization.

E xemple 4 Effects of Immunization of GnRH on Testosterone Levels The following experiment uses an immunological approach to demonstrate the lack of effect of the reduction of testosterone concentration in pre-pubertal animals. Sixty intact male pigs were divided into 3 treatment groups. Group 1 was surgically castrated at birth and groups 2 and 3 were left intact. Approximately on day 21 of age, group 3 was immunized with a GnRH immunogen comprising 8 copies of GnRH linked to an internally suppressed leukotoxin molecule comprising amino acids 38-378 and 815-951 of native leukotoxin. The GnRH immunogen was formulated in the adjuvant VSA-3 as described above. The immunization resulted in an increase in antibody production sufficient to cause a detectable decrease in testosterone secretions. Group 2 was left intact throughout the experiment and was not immunized. Previous studies have shown that in animals immunized with this adjuvant will have a sustained increase, moderate in GnRH antibody titers that reduces testosterone concentrations to low but detectable levels. The feed intake of the composition of the dead animal was measured during the experiment to compare these parameters in the various stages. The animals treated with this vaccine GnRH (immunocharged) performed similarly to castrated males (tumult) and non-castrated males (boars) until approximately 90 days of age. Additionally, as shown in Figure 4, the three groups have a large of similar body weight.

Example 5 Immunocastration of Sexually Mature Pigs by GnRH Vaccination The objects of this study were to determine whether GnRH vaccination decreased serum testosterone and androstenone fat concentrations in sexually mature male pigs at values equivalent to those seen in surgically castrated pigs and for determine the kinetics of the GnRH antibody response, serum testosterone concentrations and fat androstenone levels after a primary and secondary immunization. 24 intact male pigs were randomly assigned before day 0 to one of the three treatment groups (Groups 1, 2 and 3) as shown in Table 2. Six pigs coupled by age and litter that have been surgically castrated at of 1 week of age were assigned to the fourth treatment group (group 4, castrated early). The pigs were housed 10 animals per pen until they were approximately 60 kg of weight at which time They lodged 2 animals per pen. The pigs were given free access to food and water and care was taken to use the normal operating procedures documented by the Prairie Swine Center, an animal facility affiliated with the University of Saskatchewan and inspected by the Canadian Council on Animal Care. GnRH vaccines were washed using the GnRH immunogen from the pCB122 plasmid, dissolved at a concentration of 28 mg / ml in 4 M guanidine-HCL. The adjuvant used to formulate the GnRH vaccine was VSA-3. The vaccine was prepared by combining the GnRH immunogen with phosphate buffered saline and mixing with VSA-3 at a ratio of 1: 1 (v / v) to form a stable emulsion. The vaccine contained 40 μg of GnRH immunogen per 0.5 ml dose and IM was administered. The placebo contained saline buffered with phosphate and VSA-3. The pigs were given two IM injections of vaccine or placebo in the neck. The first injection occurred on day 0 of the experiment at which time the pigs were 21 days old. The second injection was given when the pigs had reached sexual maturity time in the which were approximately 100 kg of body weight (day 110 - day 120) (Table 2). The pigs in group 2 (castrated late) were surgically castrated when they reached sexual maturity, which was strongly influenced by body weight and presented at approximately 110 kg of body weight (day 115 to 125). The pigs in group 1 received the second immunization approximately one week earlier when the pigs in group 2 were surgically castrated. This was done in order to allow the GnRH antibody titers generated by the second immunization to reach biologically effective levels at approximately the same time that the animals in group 2 were surgically castrated.

In order to simplify the analysis of the data and presentation, day 120 is referred to as the time of the "events", that is, when the animals received either the second injection (all groups) or were castrated surgically (group 2). ). All data collected subsequent to the "events" are described in relation to the "events", that is, 7 days after the "events" refers to day 127, 14 days after the "events" refers to day 134 , Blood samples were obtained from all pigs by jugular vein perforation at approximately 28-day intervals between days 28 and 120. Subsequently, blood was obtained at weekly intervals of all pigs until the animals were sacrificed on day 162 of the experiment (42 days after the "events"). The blood was allowed to clot at room temperature, it was centrifuged and the serum was frozen in the space of 24 hours after sampling. The individual weight gains were determined on a monthly basis by weighing all the animals from day 0 to the "event" time after which they were weighed weekly until slaughter. Samples of subcutaneous fat (approximately 5 g) were obtained for measurements of androstenone under local anesthesia of alternating sides of the neck of all pigs at the time of the "events" and at weekly intervals until killing (day 162). The fat samples were immediately cooled and frozen in the space of 4 hours after the biopsy. Measurements in time of slaughter They included dead body weight, depth of back fat at the level of the tenth rib, testicular weight and length of the vulvo-urethral gland. The GnRH antibody titers were determined by a modified radioimmunoassay procedure. Synthetic GnRH (Bachem, Inc.) was cleaved with I 125 (Amersham, Oakville, Ontario). Serum dilutions were added to test tubes followed by a normal amount of 125 I-labeled GnRH to give a final incubation volume of 0.7 ml. A suspension of carbon in assay buffer was added at the end of a 24 hour incubation at 2-6 ° C to adsorb the non-antibody bound GnRH-I 125. After centrifugation, the radioactivity in the carbon fraction was measured. The data was presented as a numerical value that is the% of a normal dose (approximately 12,000 cpm) of the GnRH-I 125 bound to the antibody at a specified serum dilution. Serum testosterone was measured using a Total Coat-A-Count testosterone kit (DPS, Los Angeles, CA). This trial was based on I125-testosterone and antibodies that have a high specificity of testosterone.

Fat androstenone concentrations were determined using a color-neutral method. The final primary measurements included GnRH antibody titers measured as the% binding at a serum dilution of 1: 5000 in group 1 and a serum dilution of 1: 100 in groups 2, 3, and 4. Serum testosterone concentrations , fat androstenone, body weight, back fat, testicular weight and length of retral bulboure t were also measured. All pigs in group 1 developed GnRH antibody titers that were readily detectable at a serum dilution of 1: 100 after primary immunization (see Table 3). Additionally, immunization of pigs at 21 and at approximately 140 days of age generated GnRH antibody titers that resulted in a decline in serum testosterone and androstenone concentrations of fat equivalent to those seen in early and late castrated pigs in the life. A significant reduction in the size of the testes and bulbourethral glands was also seen in pigs immunized, compared to intact males.

EXAMPLE 6 Immunization of Bull GnRH This experiment was carried out with 58 pre-pubertal male calves. Twenty-eight male calves in group 1 were vaccinated subcutaneously jaa- twice with a vaccine composition comprising 200 μg of the GnRH immunogen derived from the plasmid pCB122 in the adjuvant VSA-3 (day 0 and day 56) and 30 control bulls in group 2 were vaccinated with a placebo. Group 1 vaccinations resulted in significant titers against GnRH by day 42, significant reductions in scrotal circumferences by day 84 and significantly reduced levels of testosterone by day 98 (Table 4). Despite these significant anti-GnRH and reduced testosterone titers, no differences in daily gain or feeding efficiency were observed in the period from day 0 to day 84 (Table 5).

* Measured as% binding of GnRH-I at a serum dilution of 1: 1,000. Statistical comparisons were made between control groups and immunized with GnRH. Values with different superscripts (a against b) differ (p < 0.5).

These new findings indicate that there is an important utility for a GnRH vaccine that gives a sufficiently strong immune response after primary immunization to result in antibody titers that give a detectable reduction in serum testosterone but do not significantly reduce growth or power efficiency. These findings are particularly new because they show that the temporary suppression of androgen secretion during the early growth period does not suppress body growth or feed efficiency. This has a particular priority when Vaccination particles are used that require subsequent immunization later in life in order to achieve a strong secondary response.

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, Manassas, VA. The indicated access number was assigned after the successful viability test and the necessary cups were paid. The deposits were made under the conditions of the Budapest Treaty in the international recognition of deposit of microorganisms for the purpose of patent procedure and regulations in accordance with it (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 later; of the most recent request for the submission of a deposit sample by the depositary. The agencies will be available through the ATCC under the terms of the Budapest treaty, which ensures the permanent and unrestricted availability of crops not yet determined by the North American Patent and Trademark Commissioner to be hereby titled in accordance with 35 U.S.C. § 122 and the rules of the Commissioner according to the same (including 37 C.F.R. § 1.12). In the granting of a patent, all restrictions on the availability to the public of the crops deposited will be irrevocably removed. These deposits are specifically provided as convenience to those skilled in the art, and it is not an admission that a deposit is required under 35 U.C. §112. 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 controlled in the case of any conflict with the description herein. A license may be required to make, use or sell the deposited materials and this license is not granted in this way.

- ~ - Strain No. ATCC deposition date pAA352 in E. ccli W1485 March 30, 1990 68283 pCE113 in E. coli JM105 February 1, 1995 69749 pCBll in E. coli JM105 February 1, 1995 69748 In this way, methods of immunization against GnRH are described. Although the 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. 116 < 223 > Xaa is acid p3.roglutami.co < 400 > 2 Xaa Trp Ser Tyr Xaa Leu Arg Pro Gly Xaa Xaa < 210 > 3 < 211 > 17 * 212 > PRT < 213 > GnRH < 400 > 3 Cyß Pro Pro Pro Pro Ser Glu Trp Ser Tyr Gly Leu Arg Pro 1 5 10 15 Gly < 210 > 4 < 211 > 17 < 212 > PRT < 213 > GnRH < 220 > < 221 > KOD_RES < 222 > (1) < 223 > Xaa is pyroglutamic acid < 400 > 4 Xaa His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Ser Pro Pro Pro Pro 1 5 10 15 Cye < 210 > 5 < 211 > 16 < 212 > PRT < 213 > GnRH < 220 > < 221 > M0D_RES < 222 > (1) < 223 > Xaa is pyroglutamic acid < 400 > 5 Xaa His Trp Ser Tyr Gly Leu Arg Pro Gly Arg Pro Pro Pro CyB < 210 > 6 < 211 > 24 < 212 > DNA '213 - synthetic construction < 400 > 6 atggctactg tcatagatcg atct < 210 > 7 211 > 8 < 212 > PRT < 213 > GnRH < 400 > 7 Het Ala Thr Val lie Asp Arg Ser 1 5 < 210 > 8 < 211 > 30 < 212 > DNA < 213 > GnRH < 220 > < 221 > CDS < 222 > (1) .. (30) < 400 > 8 cag cat tgg age tac ggc ctg cgc ect ggc Gln His Trp Ser Tyr Gly Leu Arg Pro Gly 1 5 10 < 210 > 9 < 211 > 10 < 212 > PRT < 213 > GnRH < 400 > 9 Gln His Trp Ser Tyr Gly Leu Arg Pro Gly < 210 > 10 < 211 > 147 < 212 > DNA < 213 > GnRH < 220 > < 221 > CDS < 222 > (1) .. (147) < 400 > 10 cag cat tgg age tac ggc ctg cgc ect ggc age ggt tet caa gat tgg 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 tet age cag cat tgg age tac ggc Ser Tyr Gly Leu Arg Pro Gly sly Being Ser Gln His Trp Ser Tyr Gly 20 25 30 ctc cgc ect ggc age ggt age ca gt gat age tac ggc ctg cgt ceg Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr Gly Leu Arg Pro Gly ggt gcg ggt ttt gaa ttg gca aac ca gtt gtt ggt aat att acc aaa Gly Wing Gly Phe Glu Leu Wing Gln Val Val Gly Aan He Thr Lyß 340 345 350 gcc gtt tet tet tac att tta gcc ca gt gca cgt gca ggt tta tet Wing Val Ser Ser Tyr He Leu Wing Gln Arg Val Wing Wing Gly Leu Ser 355 360 365 tea act ggg ect gtg gct gct tta att gct tet act gtt tet ett gcg Ser Thr Gly Pro Val Wing Ala W Leu Wing Ser Thr Val Ser Leu Wing 370 375 380 att age cea tta gca ttt gcc ggt att gcc gat aaa ttt aat cat gca He Ser Pro Leu Wing Phe Wing Gly He Wing Asp Lyß Phe Asn His Wing 3T5 390 395 400 aaa agt tta gag agt tat gcc gaa cgc ttt aaa aaa tta ggc tat gac Lys Ser Leu Glu Ser Tyr Wing 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 Gly Asp Asn Leu Leu Wing Glu Tyr Gln Arg Gly Thr Gly Thr He Asp 420 425 430 gca teg gtt act gca att aat acc gca gt tcg gct gct att gct ggt Wing Ser Val Thr Wing He Asn Thr Ala Leu Wing Wing He Wing Gly Gly 435 440 445 gtg tet gct gct gca gca gcc gat tta here ttt gaa aaa gtt aaa cat aat Val Ser Wing Wing Wing Wing Asp Leu Thr Phe Glu Lys Val Lys His Asn 450 455 460 ett gtc ate acg aat age aaa aaa gag aaa gtg acc att caa aac tgg Leu Val He Thr Asn Ser Lya Lys Glu Lys Val Thr He Gln Asn Trp 465 470 475 480 ttc cga gag gct gat ttt gct aaa gaa gtg ect aat tat aaa gca act Phe Arg Glu Wing Asp Phe Wing Lys Glu Val Pro Asn. Tyr Lys Wing Thr 485 490 495 aaa gat gag aaa ate gaa gaa ate ate ggt caa aat ggc gag cgg ate Lyß Asp Glu Lys He Glu Glu He He Gly Gln Asn Gly Glu Arg He 500 505 510 tea aag caa gtt gat gat ett ate gca aaa ggt aac ggc aaa att Thr Ser Lys Gln Val Asp Asp Leu He Wing Lys Gly Asn Gly Lya He 515 520 525 acc cat gat gag cta tea aaa gtt gtt gat aac tat gaa ttg etc aaa 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 ate tea tet gta Ser Lys Asn Val Thr Asn Ser Leu Asp Lys Leu He Ser Ser Val 545 550 555 560 agt gca ttt acc teg tet aat gat teg aga aat gta tta gtg gct cea Be Ala Phe Thr Ser Ser Asn Asp Ser Arg Asn Val Leu Val Ala Pro 565 570 575 act tea atg ttg gat caá agt tta tet tet ett caà ttt gct agg gga 1776 Thr Ser Met Leu Asp Gln Ser Leu Ser Ser Leu Gln Phe Wing Arg Gly 580 585 590 tet cag cat tgg age tac ggc ctg cgc ect ggc age ggt tet 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 tet 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 ect ggc age ggt age cag gat tgg age tac ggc ctg cgt 1920 Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr Gly Leu Arg 625 630 635 640 ceg ggt gga tet cag cat tgg age tac ggc ctg cgc ect ggc age ggt 1968 Pro Gly Gly Ser Gln His Trp Be Tyr Gly Leu Arg Pro Gly Be Gly 645 650 655 tet ca gat tgg age tac ggc ctg cgt ceg ggt ggc tet age cag cat 2016 Ser Gln Asp Trp Ser Tyr Gly Leu Arg Pro Gly Gly Ser Gln His 660 665 670 tgg age tac ggc cgc ect ggc age ggt age ca gat tgg age tac 2064 Tip Ser Tyr Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr 675 680 6B5 ggc ctg cgt ceg ggt gga tec tag 20B8 Gly Leu Arg Pro Gly Gly Ser 690 695 < 210 > 13 < 211 > 695 < 212 > PRT < 213 > GnRH < 400 > 13 Met Ala Thr Val He Asp Arg Ser Gln His Trp Ser Tyr Gly Leu Arg 1 5 10 15 Pro Gly Ser Gly Ser Gln Aßp 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 Trp Ser Tyr Gly Leu Arg Pro Gly Gly Ser Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr Gly Leu Arg 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 T r Gly Wing Lye Lys He He Leu Tyr He Pro Gln Asn Tyr 115 120 125 Gln Tyr Asp Thr Glu Gln Gly Asn Gly 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 1B5 190 Gln Lys Thr Lys Wing Gly Gln Wing Leu Gly Wing Glu Wing Being He Val 195 200 205 Gln Asn Wing Asn Lys Wing 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 Ala Leu Gln Asn 225 230 235 240 Asn Ser Asn Gln His Wing Leu Wing Lys Wing Gly Leu Glu Leu Thr Aan 245 250 255 Ser Leu He Glu Asn He Wing Asn Ser Val Lys Thr Leu Asp Glu Ptie 260 265 270 Gly Glu Gln Gln Be Ser Gln Phe Gly Ser Lys Leu Gln Asn He Lys Gly 275 280 285 Leu Gly Thr Leu Gly Asp Lys Leu Lys Asn He Gly Gly Leu Asp Lys 290 295 300 Wing Gly Leu Gly Leu Asp Val He Ser Gly Leu Leu Ser Gly Ala Ttir 305 310 315 320 Ala Ala Leu Val Leu Ala Asp Lys Asn Ala Ser Thr Ala Lys Lye Vial 325 330 335 Gly Ala Gly Phe Glu Leu Ala Asn Gln Val Val Gly Asn He Thr Lys 340 345 350 Wing Val Ser Ser Tyr He Leu Wing Gln Arg Val Wing Wing Gly Leu Ser 355 360 365 Ser Thr Gly Pro Wing Wing Wing Leu Wing Wing Ser Thr Val Ser Leu Wing 370 375 380 Wing Being Pro Leu Wing Phe Wing Gly Wing 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 Gly Asp Asn Leu Leu Wing Glu Tyr Gln Arg Gly Thr Gly Thr He A_sp 420 425 430 Ala Ser Val Thr Ala He Asn Thr Ala Leu Ala Ala Ala Ala Gly Gly 435 440 445 Val Ser Ala Ala Ala Ala Asp Leu Thr Phe Glu Lyß Val Lys Hiß Aßn 450 455 460 Leu Val He Thr Asn Ser Lys Lys Glu Lyß Val Thr He Gln Asn Trp 465 470 475 480 Phe Arg Glu Wing Asp Phe Wing Lys Glu Val Pro Asn Tyr LyB Wing Thr 485 490 495 Lys Asp Glu Lys He Glu Glu He He Gly Gln Gly Glu Arg 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 5S0 Being Wing Phe Thr Ser Being Asn Asp Being Arg Asn Val Leu Val Wing Pro 565 570 575 Thr Ser Met Leu Aßp Gln Ser Leu Ser Ser Leu Gln Phe Wing Arg Gly 580 585 590 Be Gln His Trp Be Tyr Gly Leu Arg Pro Gly Be 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 Pro Gly Ser Gly Ser Gln Aap Trp Ser Tyr Gly Leu Arg 62S 630 635 640 Pro Gly Gly Ser Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly 645 650 655 Ser Gln Asp Trp Ser Tyr Gly Leu Arg Pro Gly Gly Ser Ser Gln Hiß 660 665 670 Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr 675 680 6B5 Gly Leu Arg Pro Gly Gly Ser 690 695 < 210 > 14 < 211 > 6 < 212 > PRT < 213 > RTX consensus sequence < 220 > < 221 > MOD RES < 222 > (3) < 223 > Xaa IE Lys, Asp, Val or Asn < 220 > < 221 > MOD_RES < 222 > (5) < 223 > Xaa? S Lys, Asp, Val or Asn < 400 > 1 Gly Gly Xaa Gly Xaa Asp 1 5

Claims (3)

12E CLAIMS 1. The use of a first and second GnRH immunogen for the preparation of a first and second vaccine compositions for the use of a method for raising a food producing animal, male, non-castrated for the production of meat, the method which comprises vaccinating the animal with the first vaccine composition before or during the fattening period of the animal to cause a reduction in circulating levels of testosterone and to vaccinate the animal with the second vaccine composition at about 2 to about 8 weeks before killing the animal to substantially reduce the level of one or more androgenic and / or non-androgenic steroids.

2. The use according to claim 1, wherein the first vaccine composition is administered to the animal at a time between the birth of the animal and about 10 weeks of age.

3. The use according to claim 1, wherein the first and second vaccine compositions additionally comprise an immunological adjuvant. TO . The use according to claim 3, wherein the immunological adjuvant in the first and / or second vaccine composition comprises an oil and dimethyldioctadecylammonium bromide. The use according to claim 3, wherein the GnRH immunogen in the first and second vaccine compositions is the same. 6. The use according to claim 3, wherein the GnRH immunogen in the first and second vaccine compositions is different. The use according to claim 1, wherein the administration of the first vaccine composition results in the production of antibodies that cross-react with endogenous GnRH of the animal and the second vaccine composition is administered after the antibodies have declined. antibody levels. The use according to claim 3, wherein the GnRH immunogen in the first and / or second vaccine composition is a multirower of GnRH comprising the General Formula (GnRH-X-GnRH) and wherein: GnRH is an immunogen of GnRH; X is one or more molecules selected from the group consisting of a bond peptide, a group of amino acid separators, 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. 9. The use according to claim 8, wherein the carrier molecule is a leukotoxin polypeptide. The use according to claim 1, wherein the GnRH immunogen is a nucleic acid molecule 11. The use according to claim 1, wherein the second vaccine composition is administered at about 4 to about 6 weeks prior to killing. of the animal 12. The use according to claim 11, wherein the immunological adjuvant in the second vaccine composition is an aqueous adjuvant. 13. The use of the first and second GnRH mmunogens for the preparation of a first and a second vaccine compositions for use in a method for breeding a bovine, ovine or porcine, male, non-castrated animal for the production of meat comprising vaccinate the animal with the first vaccine composition or during the fattening period of the animal to cause a reduction in the circulating levels of testosterone and to vaccinate the animal with the second vaccine composition at about 2 to about 8 weeks before slaughter of the animal, to substantially reduce the level of one or more androgenic and / or non-androgenic steroids. The use according to claim 13, wherein the first and second vaccine compositions additionally comprise an immunological adjuvant. 15. The use according to claim 14, wherein the GnRH immunogen in the first and second vaccine compositions is the same. 16. The use according to claim 14, wherein the GnRH immunogen in the first and second vaccine compositions is different. The use according to claim 13, wherein the GnRH immunogen in the first and / or second vaccine composition is a multimer of GnRH comprising the General Formula (GnRH-X-GnRH) and, wherein: GnRH is a GnRH immunogen; X is one or more molecules selected to from the group consisting of a peptide bond, an amino acid spacer group, a carrier molecule and [GnRH] ", where n is an integer greater than or equal to 1; and y is an integer greater than or equal to 1. 18. The use according to claim 17, wherein the carrier molecule is a leukotoxin polypeptide. 19. The use according to claim 13, wherein the second vaccine composition is administered at about 4 to about 6 weeks prior to slaughter of the animal. 20. The use of the first and second GnRH multimer in the preparation of the first and second vaccine compositions for use in a method for breeding a bovine, ovine, porcine, male, non-castrated animal for the production of meat, the first and second vaccine compositions comprising an adjuvant, the first and second GnRH multimer comprising the General Formula (GnRH-X-GnR H) and, wherein: GnRH is a GnRH immunogen; X is one or more molecules selected from the group consisting of a bond peptide, an amino acid spacer group, a leukotoxin polypeptide and [GnRH] ", where n is an integer greater than or equal to 1; and y is an integer greater than or equal to 1, wherein the first vaccine composition is administered before or during the fattening period of the animal to cause a reduction in testosterone circulation levels and the second vaccine composition is administered approximately 2 up to about 8 weeks before slaughter of the animal, to substantially reduce the level of one or more androgenic and / or non-androgenic steroids. The use according to claim 20, wherein the first and second vaccine compositions comprise the same GnRH multimer. The use according to claim 20, wherein the GnRH multimer in the first and / or second vaccine composition comprises the amino acid sequence represented in Figures 3A-3F (SEQ ID Nos. 12 and 13), or a sequence of amino acids with at least about 75% sequence identity to the same. 23. The use according to claim 22, in wherein the GnRH multimer comprises the amino acid sequence depicted in Figures 3A-3F (SEQ ID Nos. 12 and 13). 24. The use according to claim 20, wherein the second vaccine composition is administered at about 4 to about 6 weeks prior to slaughter of the animal. 25. The use of the first and second GnRH multimer in the preparation of the first and second vaccine compositions for use in a method for breeding a bovine, ovine or porcine, male, non-castrated animal for the production of meat, the first and second vaccine compositions comprising an adjuvant, the first and second GnRH multimer comprising the amino acid sequence depicted in Figures 3A-3F (SEQ ID Nos. 12 and 13), or an amino acid sequence with at least about 75 % sequence identity therein, wherein the first vaccine composition is administered before or during the fattening period of the animal to cause a reduction in circulating levels of testosterone and the second vaccine composition is administered approximately 2 to approximately 8 weeks before slaughter of the animal, to substantially reduce the level of one or more androgenic and / or non-androgenic steroids. 26. The use according to claim 25, wherein the GnRH multimer in the first and second vaccine composition comprises the amino acid sequence depicted in Figures 3A-3F SEQ ID Nos. 12 and 13). The use according to claim 25, wherein the second vaccine composition is administered about 4 to about 6 weeks prior to slaughter of the animal. The use according to claim 25, wherein the immunological adjuvant in the first and / or second vaccine composition comprises a light mineral oil and dimethyldioctadecylammonium bromide.