EP1094836A2 - Procedes d'elevage d'animaux pour la production de viande - Google Patents

Procedes d'elevage d'animaux pour la production de viande

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Publication number
EP1094836A2
EP1094836A2 EP99917704A EP99917704A EP1094836A2 EP 1094836 A2 EP1094836 A2 EP 1094836A2 EP 99917704 A EP99917704 A EP 99917704A EP 99917704 A EP99917704 A EP 99917704A EP 1094836 A2 EP1094836 A2 EP 1094836A2
Authority
EP
European Patent Office
Prior art keywords
gnrh
animal
vaccine composition
vaccine
immunogen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99917704A
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German (de)
English (en)
Inventor
Jack G. Manns
Stephen D. Acres
Richard Harland
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MetaMorphix International Inc
Original Assignee
BIOSTAR Inc
Biostar Inc Canada
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BIOSTAR Inc, Biostar Inc Canada filed Critical BIOSTAR Inc
Publication of EP1094836A2 publication Critical patent/EP1094836A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0006—Contraceptive vaccins; Vaccines against sex hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]

Definitions

  • the present invention relates generally to methods for raising animals for meat production. More particularly, the invention is directed to methods of immunizing animals with a primary vaccination of a GnRH immunogen which causes a reduction in circulating gonadal steroid levels, followed by revaccination with a GnRH immunogen shortly before slaughter to substantially reduce the level of one or more androgenic and/or non-androgenic steroids.
  • Testosterone has similar direct effects on growth of sex glands such as seminal vesicles, the prostate gland and testes . These actions involve direct interaction of the androgen with receptors in target tissues.
  • the second general effect of androgens is to cause sexual and other behavioral changes typical of males . Those effects are mediated through the central nervous system. It is possible that because these various effects occur in different tissues by different molecular mechanisms, some of them may be maintained at low plasma concentrations of androgen whereas other effects may require higher levels.
  • the animals are vaccinated one or more times earlier in life to prime the immune system so 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 to the GnRH antigen but to avoid inducing high anti-GnRH antibody titers which would reduce serum testosterone levels or prevent it from increasing as animals approach puberty. This was based on the belief that reducing serum testosterone would also reduce growth rate or feed efficiency in young animals.
  • the present invention is based on a reliable, reproducible method for raising a food- producing male animal for meat production.
  • avoiding a substantial reduction in testosterone early in life is not necessary in order to produce commercially acceptable quantities of meat, and that primary GnRH immunization that induces antibodies that have a measurable effect on gonadal steroid secretion during the fattening period can be achieved without significant loss of growth rate or feed efficiency.
  • the primary immunization can be followed later in life with a secondary immunization that abolishes the action of androgenic and/or non-androgenic steroids.
  • the invention is directed to a method of raising an uncastrated male food-producing animal for meat production comprising vaccinating the animal with a first vaccine composition comprising a GnRH immunogen prior to or during the fattening period of the animal to cause a reduction in circulating testosterone levels, and vaccinating the animal with a second vaccine composition comprising a GnRH immunogen 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 first and/or second vaccine compositions comprise an immunological adjuvant such as an adjuvant comprising an oil and dimethyldioctadecylammonium bromide.
  • the GnRH immunogen in the first and/or second vaccine composition may be a GnRH multimer comprising the general formula (GnRH-X-GnRH) y wherein: GnRH is a GnRH immunogen;
  • X is one or more molecules selected from the group consisting of a peptide linkage, 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.
  • administration of the first vaccine composition results in the production of antibodies that cross-react with endogenous GnRH of the animal and the second composition is administered after the antibody levels have declined.
  • the invention is directed to a method of raising an uncastrated male bovine, ovine or porcine animal for meat production comprising vaccinating the animal with a first vaccine composition comprising a GnRH immunogen prior to or during the fattening period of said animal to cause a reduction in circulating testosterone levels, and vaccinating the animal with a second vaccine composition comprising a GnRH immunogen 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 first and/or second vaccine compositions may further comprise an immunological adjuvant.
  • the invention is directed to a method of raising an uncastrated male bovine, ovine or porcine animal for meat production comprising: (a) vaccinating the animal with a first vaccine composition comprising an immunological adjuvant and a GnRH multimer comprising the general formula (GnRH-X-GnRH) y wherein:
  • GnRH is a GnRH immunogen
  • X is one or more molecules selected from the group consisting of a peptide linkage, an amino acid spacer group, a leukotoxin 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 said first vaccine composition is administered prior to or during the fattening period of the animal to cause a reduction in circulating testosterone levels; and (b) vaccinating the animal with a second vaccine composition comprising an immunological adjuvant and a GnRH multimer comprising the general formula (GnRH-X-GnRH) y wherein:
  • GnRH is a GnRH immunogen
  • X is one or more molecules selected from the group consisting of a peptide linkage, an amino acid spacer group, a leukotoxin 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 said second vaccine composition is administered 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 invention is directed to a method of raising an uncastrated male bovine, ovine or porcine animal for meat production comprising:
  • vaccinating 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 NO: ) , or an amino acid sequence with at least about 75% sequence identity thereto, wherein the second vaccine composition is administered 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 adjuvant in the first and/or second vaccine composition may comprise a light mineral oil and dimethyldioctadecylammonium bromide.
  • Figure 1 depicts the relationship between antibody titers before booster vaccination on Day 35 of the trial when pigs were 63 days of age, and 14 days after booster injection at Day 49 of the trial, when animals were 77 days of age, as described in the examples .
  • Figures 2A and 2B show the nucleotide sequences and amino acid sequences of the GnRH constructs used in the chimeric leukotoxin-GnRH polypeptide gene fusions herein.
  • Figure 2A depicts a single copy of a GnRH decapeptide.
  • Figures 3A through 3F show the nucleotide sequence and predicted amino acid sequence of the LKT- GnRH chimeric protein from plasmids pCB122 and pCB130.
  • Figure 4 shows body weight as a function of age in pigs treated with GnRH vaccines according to the invention (immunocastrates) , as compared to castrated male pigs (barrows) and uncastrated male pigs (boars) .
  • GnRH Gonadotropin releasing hormone
  • LH luteinizing hormone
  • FSH follicle stimulating hormone
  • GnRH The amino acid sequence of GnRH is highly conserved among vertebrates, and especially in mammals.
  • GnRH derived from most mammals including human, bovine, porcine and ovine GnRH (formerly designated LHRH) has the amino acid sequence pyroGlu- His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 (SEQ ID NO:l) (Murad et al . , Hormones and Hormone Antagonists, in The Pharmacological Basis of Therapeutics, Sixth Edition (1980) and Seeburg et al . , Nature (1984) 3J ,: 666-668) .
  • GnRH polypeptide includes a molecule derived from a native GnRH sequence, as well as recombinantly produced or chemically synthesized GnRH polypeptides having amino acid sequences which are substantially homologous to native GnRH and which remain immunogenic, as described below.
  • the term encompasses derivatives and analogues of GnRH including any single or multiple amino acid additions, substitutions and/or deletions occurring internally or at the amino- or carboxy-termini of the peptide.
  • a “GnRH polypeptide” includes molecules having the native sequence as well as analogues of GnRH.
  • GnRH analogues include an analogue with an N-terminal Gin or Glu residue rather than a pyroGlu residue, an analogue having Asp at amino acid position 2 instead of His (see Figures 2A and 2B) ; a GnRH analogue with an N-terminal addition such as Cys-Gly-GnRH (see, e.g., Prendiville et al . , J “ . Animal Sci . (1995) 73 . : 3030-3037) ; a carboxyl- containing GnRH analogue (see, e.g., Jago et al . , J " . Animal Sci .
  • GnRH analogues with the first, sixth and/or tenth normally occurring amino acids replaced by Cys and/or wherein the N-terminus is acetylated and/or the C-terminus is amidated (see, e.g., U.S. Patent Nos . 4,608,251 and 4,975,420); the GnRH analogue pyroGlu-His-Trp-Ser-Tyr-
  • X-Leu-Arg-Pro-Gly-Y-Z (SEQ ID NO: ) wherein X is Gly or a D-amino acid, Y is one or more amino acid residues which may be the same or different, preferably 1-3 Gly residues, and Z is Cys or Tyr (see, UK Patent Publication No. GB 2196969) ; GnRH analogues described in U.S. Patent No. 5,688,506, including the GnRH analogue Cys-Pro-Pro-Pro-Pro-Ser-Ser-Glu-His-Trp- Ser-Tyr-Gly-Leu-Arg-Pro-Gly (SEQ ID NO: ) , pyroGlu-
  • GnRH polypeptide includes a
  • GnRH molecule differing from the reference sequence by having one or more amino acid substitutions, deletions and/or additions and which has at least about 50% amino acid identity to the reference molecule, more preferably about 75-85% identity and most preferably about 90-95% identity or more, to the relevant portion of the native polypeptide sequence in question.
  • the amino acid sequence will have not more than about 1-5 amino acid substitutions, or not more than about 1-3 amino acid substitutions. Particularly preferred substitutions will generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids.
  • amino acids are generally divided into four families: (1) acidic -- aspartate and glutamate; (2) basic -- lysine, arginine, histidine; (3) non-polar -- alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar -- glycine, asparagine, glutamine, cystine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.
  • GnRH polypeptide also includes peptide fragments of the reference GnRH molecule, so long as the molecule retains the desired activity. Epitopes of GnRH are also captured by the definition.
  • GnRH including repeating 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 herein.
  • Such multimers are described more fully below.
  • GnRH polypeptide may be derived from any of the various known GnRH sequences, described above, including without limitation, GnRH polypeptides derived from human, bovine, porcine, ovine, canine, feline, cervine subjects, rodents such as hamsters, guinea pigs, gerbils, ground hogs, gophers, lagomorphs, rabbits, ferrets, squirrels, reptilian and avian subjects.
  • GnRH peptide is a GnRH polypeptide, as described herein, which includes less than the full- length of the reference GnRH molecule in question and which includes at least one epitope as defined below.
  • a vaccine composition comprising a GnRH peptide would include a portion of the full-length molecule but not the entire GnRH molecule in question.
  • GnRH peptides for use herein include, for example, GnRH peptides with 5, 6 or 7 amino acids, particularly those peptides which 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, e.g., International Publication No. WO 88/05308).
  • 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 selected GnRH polypeptide, GnRH immunogen, GnRH peptide or epitope.
  • the GnRH multimer may correspond to a molecule with repeating units of the general formula (GnRH-X-GnRH) wherein GnRH is a GnRH polypeptide, X is one or more molecules selected from the group consisting of a peptide linkage, an amino acid spacer group, a carrier molecule and [GnRH] n , where n is an integer greater than or equal to 1, y is an integer greater than or equal to 1, and further wherein "GnRH” may comprise any GnRH polypeptide. Y may therefore define 1-40 or more repeating units, more preferably, 1-30 repeating units and most preferably, 1-20 repeating units.
  • the selected GnRH sequences may all be the same, or may correspond to different derivatives, analogues, variants or epitopes of GnRH, so long as they retain the ability to elicit an immune response.
  • GnRH units are linked either chemically or recombinantly to a carrier
  • GnRH molecules may be linked to either the 5' -end, the 3'- end, or may flank the carrier in question.
  • the GnRH multimer may be located at sites internal to the carrier. GnRH multimers are discussed in further detail below.
  • GnRH immunogen refers to GnRH polypeptides, as described above, that elicit an immunological response without an associated immunological carrier, adjuvant or immunostimulant, as well as GnRH polypeptides capable of being rendered immunogenic, or more immunogenic, by way of association with a carrier molecule, adjuvant or immunostimulant, or by mutation of a native sequence, and/or by incorporation into a molecule containing multiple repeating units of at least one epitope of a GnRH molecule.
  • the term may be used to refer to an individual macromolecule or to a homogeneous or heterogeneous population of antigenic macromolecules derived from GnRH.
  • GnRH immunogen will elicit formation of antibodies that cross-react with the naturally occurring, endogenous GnRH of the vertebrate species to which such an immunogen is delivered.
  • GnRH immunogen also refers to nucleic acid molecules, such as DNA and RNA molecules encoding GnRH polypeptides which are capable of expression in vivo, when administered using nucleic acid delivery techniques described further below.
  • Homology refers to the percent identity between two polynucleotide or two polypeptide moieties.
  • Two DNA, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 75%-85%, preferably at least about 90%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules.
  • substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence.
  • Percent "identity" between two amino acid or polynucleotide sequences can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100.
  • Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl . 3_: 353-358, National biomedical Research Foundation, Washington, DC, which adapts the local homology algorithm of Smith and Waterman (1981) Advances in Appl . Math . 2:482-489 for peptide analysis.
  • nucleotide sequence identity Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, WI) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.
  • an “epitope” refers to any portion or region of a molecule with the ability or potential to elicit, and combine with, a GnRH-specific antibody.
  • 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 could comprise nearly the full-length of a protein sequence, or even a fusion protein comprising two or more epitopes of a protein in question.
  • epitopes in polypeptide molecules can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epi tope Mapping
  • linear epitopes may be determined by e.g., 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 still attached to the supports.
  • Such techniques are known in the art and described in, e.g., U.S. Patent No. 4,708,871; Geysen et al . (1984) Proc . Natl . Acad . Sci .
  • conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epi tope Mapping Protocols , supra .
  • hydropathy scales from the amino acid sequence of the protein, utilizing the hydrophobic and hydrophilic properties of each of the 20 amino acids, as described in, e.g., Kyte et al . , J. Mol . Biol . (1982) 157:105-132; and Hopp and Woods, Proc . Natl . Acad . Sci . USA (1981) 78 . : 3824-3828 , can also be used to determine antigenic portions of a given molecule. For example, the technique of Hopp and Woods assigns each amino acid a numerical hydrophilicity value and then repetitively averages these values along the peptide chain. The points of highest local average hydrophilicities are indicative of antigenic portions of the molecule.
  • immunological carrier any molecule which, when associated with a GnRH immunogen of interest, imparts immunogenicity to that molecule, or enhances the immunogenicity of the molecule.
  • suitable carriers include large, slowly metabolized macromolecules such as: proteins ; polysaccharides, such as sepharose, agarose, cellulose, cellulose beads and the like; polymeric amino acids such as polyglutamic acid, polylysine, and the like; amino acid copolymers ; inactive virus particles; bacterial toxins such as toxoid from diphtheria, tetanus, cholera, leukotoxin molecules, and the like. Carriers are described in further detail below.
  • a GnRH immunogen is "linked" to a specified carrier molecule when the immunogen is chemically coupled to, or associated with the carrier, or when the immunogen is expressed from a chimeric DNA molecule which encodes the immunogen and the carrier of interest .
  • an “immunoconjugate” is a GnRH immunogen such as a GnRH peptide or multimer which is linked to a carrier molecule, as defined above.
  • leukotoxin polypeptide or "LKT polypeptide” intends a polypeptide which is derived from a protein belonging to the family of molecules characterized by the carboxy-terminus consensus amino acid sequence Gly-Gly-X-Gly-X-Asp (Highlander et al . (1989) DNA 8 . :15-28), wherein X is Lys , Asp, Val or Asn.
  • Such proteins include, among others, leukotoxins derived from P . haemolytica and Actinobacillus pleuropneumoniae, as well as E. coli alpha hemolysin (Strathdee et al . (1987) Infect . Immun .
  • leukotoxin polypeptide refers to a leukotoxin polypeptide which is chemically synthesized, isolated from an organism expressing the same, or recombinantly produced. Furthermore, the term intends an immunogenic protein having an amino acid sequence substantially homologous to a contiguous amino acid sequence found in the particular native leukotoxin molecule. Thus, the term includes both full-length and partial sequences, as well as analogues. Although native full-length leukotoxins display cytotoxic activity, the term “leukotoxin” also intends molecules which remain immunogenic yet lack the cytotoxic character of native leukotoxins.
  • nucleotide sequences and corresponding amino acid sequences for several leukotoxins are known. See, e.g., U.S. Patent Nos . 4,957,739 and 5,055,400; Lo et al . (1985) Infect . Immun . 50:667-67; Lo et al. (1987) Infect . Immun . 55. : 1987-1996 ; Strathdee et al . (1987) Infect . Immun . 55:3233-3236; Highlander et al . (1989) DNA 8:15-28; and Welch (1991) Mol . Microbiol . 5:521-528.
  • leukotoxin chimeras having a selected leukotoxin polypeptide sequence that imparts enhanced immunogenicity to one or more GnRH multimers fused thereto.
  • immunogenic leukotoxin polypeptides for use in the present invention are truncated leukotoxin molecules described in U.S. Patent Nos . 5,476,657 and 5,837,268. These truncated molecules include LKT 352, LKT 111 and LKT
  • LKT 352 is derived from the lktA gene present in plasmid pAA352 (ATCC Accession No. 68283) .
  • the nucleotide sequence and corresponding amino acid sequence of this gene are described in U.S. Patent 5,476,657.
  • the gene encodes a truncated leukotoxin, having 914 amino acids and an estimated molecular weight of around 99 kDa .
  • LKT 111 is a leukotoxin polypeptide derived from the IJtA gene present in plasmid pCBlll (ATCC Accession No. 69748) .
  • the nucleotide sequence of this gene and the corresponding amino acid sequence are disclosed in U.S. Patent No.
  • the gene encodes a shortened version of leukotoxin which was developed from the recombinant leukotoxin gene present in 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 99 kDa LKT 352 polypeptide) , but retains portions of the LKT 352 N- terminus containing T-cell epitopes which are necessary for sufficient T-cell immunogenicity, and portions of the LKT 352 C-terminus containing convenient restriction sites for use in producing fusion proteins for use in the present invention.
  • LKT 114 is derived from the gene present in plasmid pAA114 (described in U.S. Patent No. 5,837,268). LKT 114 differs from LKT 111 by virtue of an additional amino acid deletion from the internal portion of the molecule .
  • immunological adjuvants an agent which acts in a nonspecific manner to increase an immune response to a particular antigen, thus reducing the quantity of antigen necessary 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, e.g., A.C. Allison J. Reticuloendothel . Soc . (1979) 26 : 619-630.
  • “Native” proteins, polypeptides or peptides are proteins, polypeptides or peptides isolated from the source in which the proteins naturally occur.
  • “Recombinant” polypeptides refer to polypeptides produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide.
  • “Synthetic” polypeptides are those prepared by chemical synthesis.
  • polynucleotide is meant a sequence of nucleotides including, but is not limited to, RNA such as mRNA, cDNA, genomic DNA sequences and even synthetic DNA sequences. The term also captures sequences that include any of the known base analogues of DNA and RNA.
  • an immunogen that is "derived from” a particular GnRH molecule will bear close sequence similarity with a relevant portion of the reference molecule.
  • an immunogen that is "derived from” a particular GnRH molecule may include all of the wild- type GnRH sequence, or may be altered by insertion, deletion or substitution of amino acid residues, so long as the derived sequence provides for an immunogen that corresponds to the targeted GnRH molecule.
  • Immunogens derived from a denoted molecule will contain at least one epitope specific to the denoted molecule .
  • food-producing animal is meant an animal intended for consumption by humans or domestic pets such as cats and dogs.
  • animals include, without limitation, mammals such as ovine, bovine, porcine, and cervine subjects, including sheep, cattle, pigs and deer.
  • enhancing the organoleptic qualities of meat is meant improving the smell, taste and/or tenderness of meat from an animal treated under the invention as compared to meat from a typical uncastrated member of the same species that has not been so treated.
  • Meat from uncastrated males generally suffers from several drawbacks. In this regard, meat derived from uncastrated male pigs and sheep often has an unpleasant taste and smell.
  • Boar taint refers to a urine-like odor found in cooked meat of uncastrated pigs. Boar taint is produced by steroids stored in tissues in male piglets with normally functioning testicles. See e.g. Brooks et al . , J. Anim . Sci . (1986) 62 . : 1279. The presence of androstenone in boar carcasses is one measure of boar taint and is considered a measure of gonadal steroid production. Additionally, skatole may contribute to boar taint. See, e.g., Mortensen and Sorensen, Proc .
  • a "reduction in circulating testosterone” is meant a statistically significant reduction in serum testosterone levels as measured using a standard assay, such as an RIA as described herein, as compared with the serum testosterone levels expected in a typical uncastrated, untreated male, of the same age and species.
  • “Androgenic” steroids include androstenone, androstenedione, androstenediol and/or testosterone. Androgenic steroids can be measured using well known techniques. For example, testosterone and the other androgenic steroids can be measured using ELISAs and RIAs well known in the art. Particularly convenient measures may be made using commercially available test kits, e.g., the Coat-A-Count Total Testosterone KitTM (Diagnostic Products Corporation, Los Angeles, CA) . This kit is a solid-phase RIA designed for the quantitative measurement of testosterone in serum, based on testosterone-specific antibody 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.
  • Test kits e.g., the Coat-A-Count Total Testosterone KitTM (Diagnostic Products Corporation, Los Angeles, CA) . This kit is a solid
  • Non-androgenic steroids include the 16- androstene derivatives, including 5 ⁇ androstenone (5 ⁇ androst-16-en-3-one) .
  • Non-androgenic steroids can be measured using techniques well known in the art, such as by ELISAs and RIAs. See, e.g., Claus et al . , Archiv fuer Lebens i ttelhygiene (1988) 39:87-90.
  • 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-adrogenic steroid is at least about 50% less than expected in a typical uncastrated, untreated male, of the same age and species, preferably at least about 75% less, and more preferably at least about 80% to 90% or less.
  • the term "fattening period” intends the period from weaning up 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 raised. Thus, the fattening period is largely a matter of choice and one of skill in the art can readily determine the appropriate fattening period for a given animal .
  • 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 of circulating testosterone levels, but generally does not result in complete immunocastration.
  • the primary vaccination is followed with a boost with the same or different GnRH composition shortly before slaughter, to substantially reduce the level of one or more androgenic and/or non-androgenic steroids.
  • compositions described herein surprisingly provide a means for producing an adequate immunological response in a subject immunized therewith.
  • the timing of the 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 prior or during the fattening period of the animal. For example, in pigs and sheep, the primary immunization will generally be given 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, the primary immunization will generally be given at a time between birth and about 48 weeks of age.
  • booster treatments are given before slaughter.
  • the timing of the booster will also depend on the animal in question. For example, in pigs and sheep, the booster will generally be at about 1 to about 12 weeks prior to slaughter, preferably about 2 to about 8 weeks prior to slaughter and most preferably about 4 to about 6 weeks prior to slaughter, and even 2 to about 3 weeks prior to slaughter. In cows, it may be preferable to administer the second vaccine composition several months prior to slaughter.
  • the subsequent immunization (s) is given after GnRH antibodies, raised against the primary immunization, have declined, i.e., to a level at least about 50% below the maximum antibody levels detected, preferably decreased at least about 75% below the maximum levels detected.
  • the vaccine compositions of the present invention employ GnRH polypeptides, as defined above, optionally linked to carrier molecules in order to enhance immunogenicity thereof.
  • GnRH Immunoconjugates As explained above, GnRH is an endogenous molecule and, as such, it may be desirable to further increase the immunogenicity of the GnRH polypeptides (or multimers described below) by linking them to carriers to form GnRH immunoconjugates. This is especially necessary if the GnRH immunogen will be administered to the same species from which it is derived.
  • Suitable carriers are generally polypeptides which include antigenic regions of a protein derived from an infectious material such as a viral surface protein, or a carrier peptide sequence. These carriers serve to non-specifically stimulate T-helper cell activity and to help direct an immunogen of interest to antigen presenting cells (APCs) for processing and presentation at the cell surface in association with molecules of the major histocompatibility complex (MHC) .
  • APCs antigen presenting cells
  • 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 . Na tl . Acad . Sci . U. S . A . 79:569- 573), ovalbumin, leukotoxin polypeptides, and sperm whale myoglobin, to produce an immune response.
  • 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 . Na tl . Acad . Sci . U. S . A . 79:569- 573), ovalbumin, leukotoxin poly
  • Suitable carriers for use with the present invention include VP6 polypeptides of rotaviruses, or functional fragments thereof, as disclosed in U.S. Patent Number 5,071,651. Also useful is a fusion product of a viral protein and one or more epitopes from GnRH, which fusion products are made by the methods disclosed in U.S. Patent No.
  • GnRH immunogens may be coupled to erythrocytes, preferably the subject's own erythrocytes .
  • Methods of coupling peptides to proteins or cells are known to those of skill in the art .
  • Delivery systems useful in the practice of the present invention may also utilize particulate carriers. For example, pre- formed particles have been used as platforms onto which immunogens can be coupled and incorporated. Systems based on proteosomes (Lowell et al . (1988) Science 240:800-802) and immune stimulatory complexes (Morein et al . (1984) ature 308 :457-460) are also known in the art.
  • Carrier systems using recombinantly produced chimeric proteins that self-assemble into particles may also be used with the present invention.
  • the yeast retrotransposon, Ty encodes a series of proteins that assemble into virus like particles (Ty-VLPs; Kingsman et al . (1988) Vaccines 6 . :304-306).
  • Ty-VLPs proteins that assemble into virus like particles
  • a gene, or fragment thereof, encoding the GnRH immunogen of interest may be inserted into the TyA gene and expressed in yeast as a fusion protein.
  • the fusion protein retains the capacity to self assemble into particles of uniform size.
  • Other useful virus-like carrier systems are based on HBsAg, (Valenzuela et al . (1985) Bio/Technol .
  • Especially preferred carriers include serum albumins, keyhole limpet hemocyanin, ovalbumin, sperm whale myoglobin, leukotoxin molecules as described above, and other proteins well known to those skilled in the art.
  • chimeric systems using a leukotoxin polypeptide, as defined above, such as a Pasteurella haemolytica leukotoxin (LKT) polypeptide fused to the antigen of interest can also be used herein.
  • LLT Pasteurella haemolytica leukotoxin
  • the nucleotide sequences and corresponding amino acid sequences for several leukotoxin carriers are known. See, e.g., U.S. Patent Nos. 5,422,110, 5,708,155, 5,723,129 and International Publication Nos.
  • 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 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 99 kDa LKT 352 polypeptide) , but retains portions of the LKT 352 N- terminus containing T-cell epitopes which are necessary for sufficient T-cell immunogenicity, and portions of the LKT 352 C- terminus containing convenient restriction sites for use in producing the fusion proteins of the present invention.
  • LKT 114 differs from LKT 111 by virtue of an additional amino acid deletion from the internal portion of the molecule. See, e.g., U.S. Patent No. 5,837,268 and International Publication Nos. WO 98/06848 and WO 96/24675 for descriptions of these molecules.
  • Protein carriers may be used in their native form or their functional group content may be modified by, for example, succinylation of lysine residues or reaction with Cys-thiolactone .
  • a sulfhydryl group may also be incorporated into the carrier (or antigen) by, for example, reaction of amino functions with 2-iminothiolane or the N-hydroxysuccinimide ester of 3- (4-dithiopyridyl propionate .
  • Suitable carriers may also be modified to incorporate spacer arms (such as hexamethylene diamine or other bifunctional molecules of similar size) for attachment of peptide immunogens. Carriers can be physically conjugated to the GnRH immunogen of interest, using standard coupling reactions.
  • 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, encoding for a selected GnRH immunogen.
  • the GnRH portion can be fused either 5' or 3 ' to the carrier portion of the molecule, or the GnRH portion may be located at sites internal to the carrier molecule.
  • the GnRH immunogens can also be administered via a carrier virus which expresses the same.
  • Carrier viruses which will find use herein include, but are not limited to, the vaccinia and other pox viruses, adenovirus, and herpes virus.
  • vaccinia virus recombinants expressing the proteins can be constructed as follows. The DNA encoding a particular protein is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK) . This vector is then used to transfect cells which are simultaneously infected with vaccinia.
  • TK thymidine kinase
  • Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the desired immunogen into the viral genome.
  • the resulting TK—recombinant can be selected by culturing the cells in the presence of 5- bromodeoxyuridine and picking viral plaques resistant thereto.
  • Immunogenicity of the GnRH immunogens may also be significantly increased by producing immunogenic forms of the molecules that comprise multiple copies of selected epitopes. In this way, endogenous GnRH may be rendered an effective autoantigen.
  • vaccine compositions containing GnRH immunogen multimers are provided in either nucleic acid or peptide form.
  • the GnRH multimer will have more than one copy of selected GnRH immunogens, peptides or epitopes, as described above, or multiple tandem repeats of a selected GnRH immunogen, peptide or epitope.
  • the GnRH multimers may comprise either multiple or tandem repeats of selected GnRH sequences, multiple or tandem repeats of selected GnRH epitopes, or any conceivable combination thereof.
  • GnRH epitopes may be identified using techniques as described in detail above.
  • the GnRH multimer may correspond to a molecule with repeating units of the general formula (GnRH-X-GnRH) y wherein GnRH is a GnRH immunogen, X is selected from the group consisting of a peptide linkage, an amino acid spacer group, a carrier molecule and [GnRH] n , where n is an integer greater than or equal to 1, y is an integer greater than or equal to 1, and further wherein "GnRH” may comprise any GnRH immunogen.
  • the GnRH multimer may contain from 2-64 or more GnRH immunogens, more preferably 2-32 or 2-16 GnRH immunogens.
  • the selected GnRH immunogen sequences may all be the same, or may correspond to different derivatives, analogues, variants or epitopes of GnRH so long as they retain the ability to elicit an immune response. Additionally, if the GnRH immunogens are linked either chemically or recombinantly to a carrier, GnRH immunogens may be linked to either the 5' -end, the 3 '-end, or may flank the carrier in question. Further, the GnRH multimer may be located at sites internal to the carrier.
  • One particular carrier for use with the present GnRH multimers is a leukotoxin polypeptide as described above . As explained above, spacer sequences may be present between the GnRH moieties.
  • Ser- Gly-Ser trimers and Gly-Ser dimers are present in the GnRH multimers exemplified herein which provide spacers between repeating sequences of the GnRH immunogens. See, e.g., Figure 2B.
  • the strategic placement of various spacer sequences between selected GnRH immunogens can be used to confer increased immunogenicity on the subject constructs.
  • a selected spacer sequence may encode a wide variety of moieties such as a single amino acid linker or a sequence of two to several amino acids .
  • Selected spacer groups may preferably provide enzyme cleavage sites so that the expressed multimer can be processed by proteolytic enzymes in vivo (by APCs , or the like) to yield a number of peptides, each of which contain at least one T-cell epitope derived from the carrier portion, and which are preferably fused to a substantially complete GnRH polypeptide sequence.
  • the spacer groups may be constructed so that the junction region between selected GnRH moieties comprises a clearly foreign sequence to the immunized subject, thereby conferring enhanced immunogenicity upon the associated GnRH immunogens.
  • spacer sequences may be constructed so as to provide T-cell antigenicity, such as those sequences which encode amphipathic and/or ⁇ -helical peptide sequences which are generally recognized in the art as providing immunogenic helper T-cell epitopes.
  • T-cell antigenicity such as those sequences which encode amphipathic and/or ⁇ -helical peptide sequences which are generally recognized in the art as providing immunogenic helper T-cell epitopes.
  • the choice of particular T-cell epitopes to be provided by such spacer sequences may vary depending on the particular vertebrate species to be vaccinated.
  • particular GnRH portions are exemplified which include spacer sequences, it is also an object of the invention to provide one or more GnRH multimers comprising directly
  • the GnRH multimeric sequence thus produced renders a highly immunogenic GnRH antigen for use in the compositions of the invention.
  • the GnRH polypeptides, immunoconjugates and multimers can be produced using the methods described below, and used for nucleic acid immunization, gene therapy, protein-based immunization methods, and the like.
  • nucleic acid-based vaccines for use with the present invention will include relevant regions encoding a GnRH immunogen, with suitable control sequences and, optionally, ancillary therapeutic nucleotide sequences.
  • the nucleic acid molecules are prepared in the form of vectors which include the necessary elements to direct transcription and translation in a recipient cell.
  • the nucleic acid molecules can be administered in conjunction with ancillary substances, such as pharmacological agents, adjuvants, or in conjunction with delivery of vectors encoding biological response modifiers such as cytokines and the like.
  • ancillary substances include, but are not limited to, substances to increase weight gain, muscle mass or muscle strength, such as growth hormones, growth promoting agents, beta antagonists, partitioning agents and antibiotics.
  • Nucleotide sequences selected for use in the present invention can be derived from known sources, for example, by isolating the same from cells or tissue containing a desired gene or nucleotide sequence using standard techniques, or by using recombinant or synthetic techniques.
  • GnRH immunogens Once coding sequences for the GnRH immunogens have been prepared or isolated, such sequences can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Ligations to other sequences, e.g., ancillary molecules or carrier molecules, are performed using standard procedures, known in the art. One or more GnRH immunogen portions of the chimera can be fused 5' and/or 3' to a desired ancillary sequence or carrier molecule. Alternatively, one or more GnRH immunogen portions may be located at sites internal to the carrier molecule, or such portions can be positioned at both terminal and internal locations in the chimera .
  • DNA sequences encoding the GnRH immunogens of interest can be prepared synthetically rather than cloned.
  • the DNA sequences can be designed with appropriate codons for the particular sequence.
  • the complete sequence of the immunogen is then assembled from overlapping oligonucleotides prepared by standard 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 the control of suitable control elements for expression in suitable host tissue in vivo .
  • control elements will depend on the subject being treated and the type of preparation used. Thus, if the subject's endogenous transcription and translation machinery will be used to express the immunogens, control elements compatible with the particular subject will be utilized.
  • promoters for use in mammalian systems are known in the art.
  • typical promoters for mammalian cell expression include the SV40 early promoter, a CMV promoter such as the CMV immediate early promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP) , and the herpes simplex virus promoter, among others.
  • Other nonviral promoters such as a promoter derived from the murine metallothionein gene, will also find use for mammalian expression.
  • transcription termination and polyadenylation sequences will also be present, located 3' to the translation stop codon.
  • a sequence for optimization of initiation of translation located 5' to the coding sequence, is also present.
  • transcription terminator/polyadenylation signals include those derived from SV40, as described in Sambrook et al . , supra , as well as a bovine growth hormone terminator sequence.
  • Introns, containing splice donor and acceptor sites, may also be designed into the constructs for use with the present invention.
  • Enhancer elements may also be used herein to increase expression levels of the constructs. Examples include the SV40 early gene enhancer (Dijkema et al. (1985) EMBO J.
  • the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus (Gorman et al . (1982) Proc . Na tl . Acad . Sci . USA 79:6777) and elements derived from human CMV (Boshart et al . (1985) Cell 41:521), such as elements included in the CMV intron A sequence.
  • LTR long terminal repeat
  • elements derived from human CMV Boshart et al . (1985) Cell 41:521
  • the nucleic acid vaccine compositions can be delivered to the subject using known methods.
  • various techniques for immunization with antigen-encoding DNAs have been described. See, e.g., U.S. Patent No. 5,589,466 to Feigner et al . ; Tang et al . (1992) Nature 358 :152 ; Davis et al . (1993) Hum. Molec . Genet . 2:1847; Ulmer et al. (1993) Science 2 . 58: 1745; Wang et al . (1993)
  • nucleic acid vaccine compositions can be delivered in either liquid or particulate form using a variety of known techniques. Typical vaccine compositions are described more fully below.
  • Protein-based compositions can also be produced using a variety of methods known to those skilled in the art.
  • GnRH polypeptides can be isolated directly from native sources, using standard purification techniques.
  • the polypeptides can be recombinantly produced using nucleic acid expression systems, well known in the art and described in, e.g., Sambrook et al . , supra .
  • GnRH polypeptides can also be synthesized using chemical polymer syntheses such as solid phase peptide synthesis. Such methods are known to those skilled in the art. See, e.g., J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis , 2nd Ed.
  • GnRH polypeptides for use in the compositions described herein may also be produced by cloning the coding sequences therefor into any suitable expression vector or replicon.
  • Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice.
  • Examples of recombinant DNA vectors for cloning, and host cells which they can transform, include the bacteriophage lambda (E. coli ) , pBR322 (E. coli ) , pACYC177 (E.
  • the coding sequences for porcine, bovine and ovine GnRH have been determined (Murad et al . (1980) Hormones and Hormone Antagonists, in The Pharmacological Basis of Therapeutics, Sixth Edition) , and the cDNA for human GnRH has been cloned so that its sequence has been well established (Seeburg et al . (1984) Nature 311:666-668) . Additional GnRH polypeptides of known sequences have been disclosed, such as the GnRH molecule occurring in salmon and chickens (International Publication No. WO 86/07383, published 18 December 1986).
  • GnRH coding sequences for use with the present invention are shown in Figures 2A and 2B herein.
  • the GnRH coding sequence is highly conserved in vertebrates, particularly in mammals, and porcine, bovine, ovine and human GnRH sequences are identical to one another.
  • 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, e.g., 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 sequence may or may not contain a signal peptide or leader sequence.
  • the polypeptides can be expressed using, for example, the E . coli tac promoter or the protein A gene (spa) promoter and signal sequence.
  • Leader sequences can be removed by the bacterial host in post-translational processing. See, e.g., U.S. Patent Nos. 4,431,739; 4,425,437; 4,338,397. Ancillary sequences, such as those described above, may also be present.
  • regulatory sequences which allow for regulation of the expression of the polypeptide sequences relative to the growth of the host cell.
  • Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene 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 sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the "control" of the control sequences (i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence) .
  • control sequences i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence
  • Modification of the sequences encoding the particular GnRH polypeptide may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it can be attached to the control sequences in the appropriate orientation; i.e., to maintain the reading frame.
  • control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above.
  • a vector such as the cloning vectors described above.
  • the coding sequence can be cloned directly into an expres- sion vector which already contains the control sequences and an appropriate restriction site.
  • mutants or analogues of the polypeptide may be prepared by the deletion of a portion of the sequence encoding the reference polypeptide, or if present, a portion of the sequence encoding the desired carrier molecule, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence.
  • Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, and the like, are well known to those skilled in the art. See, e.g., Sambrook et al .
  • the 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.
  • insect cell expression systems such as baculovirus systems
  • baculovirus systems are known to those of skill in the art and described in, e.g., Summers and Smith, Texas Agricul tural Experiment Station Bulletin No . 1555 (1987) .
  • Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia , Invitrogen, San Diego CA ("MaxBac" kit) .
  • bacterial and mammalian cell expression systems are well known in the art and described in, e.g., Sambrook et al . , supra .
  • Yeast expression systems are also known in the art and described in, e.g., Yeast Genetic Engineering (Barr et al . , eds., 1989) Butterworths, London.
  • mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC) , such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS) , human hepatocellular carcinoma cells
  • ATCC American Type Culture Collection
  • CHO Chinese hamster ovary
  • HeLa HeLa
  • BHK baby hamster kidney
  • COS monkey kidney cells
  • human hepatocellular carcinoma cells such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS) , human hepatocellular carcinoma cells
  • Yeast hosts useful in the present invention include inter alia , Saccharomyces cerevisiae, Candida albicans, Candida mal tosa , Hansenula polymorpha, Kluyveromyces fragilis , Kluyveromyces lactis, Pichia guillerimondii , Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica .
  • Insect cells for use with baculovirus expression vectors include, inter alia , Aedes aegypti , Autographa calif ornica, Bombyx mori , Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni .
  • the GnRH polypeptides are produced by growing host cells transformed by an expression vector described above under conditions whereby the polypeptide is expressed. The expressed polypeptide is then isolated from the host cells and purified. If the expression system secretes the polypeptide into growth media, the product can be purified directly from the media. If it is not secreted, it can be isolated from cell lysates . The selection of the appropriate growth conditions and recovery methods are within the skill of the art.
  • the GnRH polypeptides may be formulated into compositions, such as vaccine compositions as described further below, in order to elicit antibody production.
  • the subject GnRH immunogens can be used to generate antibodies for use in passive immunization methods.
  • peptides useful for producing antibodies will usually be at least about 3-5 amino acids in length, preferably 7-10 amino acids in length.
  • Antibodies against the subject immunogens include polyclonal and monoclonal antibody preparations, monospecific antisera, as well as preparations including hybrid antibodies, altered antibodies, F(ab') 2 fragments, F(ab) fragments, F v fragments, single domain antibodies, chimeric antibodies, humanized antibodies, and functional fragments thereof, which retain specificity for the target molecule in question.
  • an antibody can include variable regions, or fragments of variable regions, which retain specificity for the molecule in question.
  • the remainder of the antibody can be derived from the species in which the antibody will be used.
  • the antibody can be "humanized” in order to reduce immunogenicity yet retain activity.
  • chimeric antibodies see, e.g., Winter, G. and
  • Such chimeric antibodies may contain not only combining sites for the target molecule, but also binding sites for other proteins. In this way, bifunctional reagents can be generated with targeted specificity to both external and internal antigens.
  • a selected mammal e.g., mouse, rabbit, goat, horse, etc.
  • the desired antigen or its fragment, or a mutated antigen, as described above.
  • immunization it may be desirable to further increase the immunogenicity of a particular immunogen.
  • immunization for the production of antibodies is generally performed 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, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly) .
  • a suitable excipient such as saline
  • an adjuvant such as Freund' s complete adjuvant, or any of the adjuvants described below
  • the animal is generally boosted 2-6 weeks later with one or more injections of the protein in saline, preferably using Freund' s incomplete adjuvant, or the like.
  • Antibodies may also be generated by in vitro immunization, using methods known in the art. Polyclonal antisera is then obtained from the immunized animal and treated according to known procedures.
  • 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.
  • a mouse or rat is immunized as described above.
  • the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells.
  • the spleen cells may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen.
  • B-cells, expressing membrane-bound immunoglobulin specific for the antigen will bind to the plate, and are not rinsed away with the rest of the suspension.
  • Resulting B-cells, or all dissociated spleen cells are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium (e.g., hypoxanthine , aminopterin, thymidine medium, "HAT") .
  • the resulting hybridomas are plated by limiting dilution, and are assayed for the production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens) .
  • the selected monoclonal antibody-secreting hybridomas are then cultured either in vi tro (e.g., in tissue culture bottles or hollow fiber reactors) , or in vivo (as ascites in mice) .
  • Panels of monoclonal antibodies produced against the GnRH immunogen of interest, or fragment thereof, can be screened for various properties; i.e., for isotype, epitope, affinity, etc.
  • Functional fragments of the antibodies can also be made against the GnRH immunogen of interest and can be produced by cleaving a constant region, not responsible for antigen binding, from the antibody molecule, using e.g., pepsin, to produce F(ab') 2 fragments. These fragments will contain two antigen binding sites, but lack a portion of the constant region from each of the heavy chains.
  • Fab fragments comprising a single antigen binding site, can be produced, e.g., by digestion of polyclonal or monoclonal antibodies with papain. Functional fragments, including only the variable regions of the heavy and light chains, can also be produced, using standard techniques. These fragments are known as F v . Chimeric or humanized antibodies can also be produced using the subject immunogens. These antibodies can be designed to minimize unwanted immunological reactions attributable to heterologous constant and species-specific framework variable regions typically present in monoclonal and polyclonal antibodies.
  • chimeric antibodies can be created by replacing non-human constant regions, in either the heavy and light chains, or both, with human constant regions, using techniques generally known in the art. See, e.g., Winter, G. and Milstein, C.
  • GnRH polypeptides or antibodies are formulated into compositions for delivery to a vertebrate subject.
  • the relevant GnRH molecule is administered alone, or mixed with a pharmaceutically acceptable vehicle or excipient.
  • Suitable vehicles are, for example, water, saline, dextrose, glycerol , ethanol , or the like, and combinations thereof.
  • the vehicle may contain minor amounts of auxiliary substances 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 can also include ancillary substances, such as pharmacological agents, cytokines, or other biological response modifiers.
  • vaccine compositions of the present invention may include adjuvants to further increase the immunogenicity of the GnRH immunogen.
  • Adjuvants may include for example, emulsifiers, muramyl dipeptides, avridine, aqueous adjuvants such as aluminum hydroxide and any of the various saponins, chitosan-based adjuvants, oils, and other substances known in the art.
  • emulsifiers include natural and synthetic emulsifying agents, as well as anionic, cationic and nonionic compounds.
  • anionic emulsifying agents include, for example, the potassium, sodium and ammonium salts of lauric and oleic acid, the calcium, magnesium and aluminum salts of fatty acids (i.e., metallic soaps), and organic sulfonates such as sodium lauryl sulfate.
  • Synthetic cationic agents include, for example, cetyltrimethylammonium bromide, while synthetic nonionic agents are exemplified by glyceryl esters
  • Natural emulsifying agents include acacia, gelatin, lecithin and cholesterol.
  • Suitable adjuvants can be formed with an oil component, such as a single oil, a mixture of oils, a water-in-oil emulsion, or an oil-in-water emulsion.
  • the oil may be a mineral oil, a vegetable oil, or an animal oil.
  • Mineral oil, or oil-in-water emulsions in which the oil component is mineral oil are preferred.
  • a “mineral oil” is defined herein as a mixture of liquid hydrocarbons obtained from petrolatum via a distillation technique; the term is synonymous with “liquid paraffin,” “liquid petrolatum” and “white mineral oil.”
  • the term is also intended to include "light mineral oil,” i.e., an oil which is similarly obtained by distillation of petrolatum, but which has a slightly lower specific gravity than white mineral oil. See, e.g., Remington ' s Pharmaceutical Sciences , supra .
  • a particularly preferred oil component is the oil-in- water emulsion sold under the trade name of EMULSIGEN PLUSTM (comprising a light mineral oil as well as 0.05% formalin, and 30 mcg/mL gentamicin as preservatives) , available from MVP Laboratories, Ralston, California.
  • Suitable animal oils include, for example, cod liver oil, halibut oil, menhaden oil, orange roughy 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, peanut oil, safflower oil, sesame oil, soybean oil, and the like.
  • aliphatic nitrogenous bases can be used as adjuvants with the vaccine formulations.
  • known immunologic adjuvants include amines, quaternary ammonium compounds, guanidines, benzamidines and thiouroniums (Gall, D. (1966) Immunology 11:369-386) .
  • Specific compounds include dimethyldioctadecylammonium bromide (DDA) (available from Kodak) and N, N-dioctadecyl-N, N- bis (2 -hydroxyethyl ) ropanediamine ("avridine").
  • DDA dimethyldioctadecylammonium bromide
  • Avridine is also a well-known adjuvant. See, e.g., U.S. Patent No. 4,310,550 to Wolff, III et al .
  • VSA-3 is a modified form of the EMULSIGEN PLUSTM adjuvant which includes DDA (see, allowed U.S. Patent Application Serial No. 08/463, 837) .
  • composition or formulation to be administered will contain a quantity of the GnRH polypeptide adequate to achieve the desired state in the subject being treated.
  • compositions of the present invention are normally prepared as injectables, either as liquid solutions or suspensions, or as solid forms which are suitable for solution or suspension in liquid vehicles prior to injection.
  • the preparation may also be emulsified or the active ingredient encapsulated in liposo e vehicles or other particulate carriers used.
  • compositions may also be prepared in solid form.
  • solid particulate formulations can be prepared for delivery from commercially available needleless injector devices.
  • solid dose implants can be provided for implantation into a subject.
  • Controlled or sustained release formulations may also be used and are made by incorporating the GnRH polypeptides into carriers or vehicles such as liposomes, nonresorbable impermeable polymers such as ethylenevinyl acetate copolymers and Hytrel ® copolymers, swellable polymers such as hydrogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures .
  • the polypeptides may 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 may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol , histidine, procaine, and the like.
  • composition is formulated to contain an effective amount of the GnRH polypeptide, the exact amount being readily determined by one skilled in the art, wherein the amount depends on the animal to be treated, in the case of a vaccine composition, the capacity of the animal's immune system to synthesize antibodies, and the degree of immunoneutralization of GnRH desired.
  • formulations including approximately 1 ⁇ g to about 2 mg, more generally about 5 ⁇ g to about 800 ⁇ g, and even more particularly, 10 ⁇ g to about 400 ⁇ g of GnRH polypeptide per mL of injected solution should be adequate to raise an immunological 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 such molecules.
  • the ratio of GnRH to leukotoxin in the vaccine formulation will vary based on the particular leukotoxin and GnRH polypeptide moieties selected to construct those molecules .
  • One preferred vaccine composition contains a leukotoxin-GnRH chimera having about 1 to 90% GnRH, preferably about 3 to 80% and most preferably about 10 to 70% GnRH polypeptide per fusion molecule. Increases in the percentage of GnRH present in the LKT-GnRH fusions reduce the amount of total antigen which must be administered to a subject in order to elicit a sufficient immunological response to GnRH.
  • the subject is administered one of the above-described compositions e.g., 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 with one or more boosts with the same or different GnRH composition shortly 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 may be employed to deliver the compositions to the vertebrate subject.
  • conventional needle syringes, spring or compressed gas (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 .
  • particle injectors U.S. Patent Nos. 5,149,655 to McCabe et al . and 5,204,253 to Sanford et al .
  • the composition is administered intramuscularly, subcutaneously, intravenously, subdermally, intradermally, transdermally or transmucosally to the subject.
  • a jet injector is used, a single jet of the liquid vaccine composition is ejected under high pressure and velocity, e.g., 1200-1400 PSI, thereby creating an opening in the skin and penetrating to depths suitable for immunization.
  • high pressure and velocity e.g. 1200-1400 PSI
  • Example 1 Construction of pCB122 and pCB130 Plasmids pCB122 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 GnRH construct in both plasmids contains 8 tandem repeats of the GnRH sequence fused to both the 5' and 3' ends of a DNA sequence coding for a carrier leukotoxin polypeptide. Each alternating GnRH sequence has a change in the fourth base in the sequence from cytosine to guanosine. This results in a single amino acid change in the second amino acid of the GnRH molecule from His to Asp.
  • the leukotoxin portion of the construct encodes a shortened version of leukotoxin which was developed from the recombinant leukotoxin gene present in plasmid pAA352 (ATCC Accession No. 68283 and described in U.S. Patent 5,476,657) by removal of an internal DNA fragment of approximately 1300 bp in length.
  • the leukotoxin polypeptide has an estimated molecular weight of 52 kDa and contains convenient restriction sites for use in producing the fusion proteins of the present invention.
  • the chimeric construct is under the control of the Tac promoter and induction is controlled through the use of Lac I .
  • Plasmid pCB122 was prepared as follows.
  • the leukotoxin gene was isolated as described in U.S. Patent Nos. 5,476,657 and 5,837,268.
  • gene libraries of P. haemolytica Al were constructed using standard techniques. See, Lo et al . , Tnfect. 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 constructed in the bacteriophage lambda gtll. The resulting clones were used to transform E. coli and individual colonies were pooled and screened for reaction with serum from a calf which had survived a P. haemolytica infection and that had been boosted with a concentrated culture supernatant of P. haemolytica to increase anti-leukotoxin antibody levels. Positive colonies were screened for their ability to produce leukotoxin by incubating cell lysates with bovine neutrophils and subsequently measuring release of lactate dehydrogenase from the latter.
  • lktA a Mael restriction endonuclease fragment from pAA114 which contained the entire leukotoxin gene, was treated with the Klenow fragment of DNA polymerase I plus nucleotide triphosphates and ligated into the Smal site of the cloning vector pUC13.
  • This plasmid was named pAA179. From this, two expression constructs were made in the ptac-based vector pGH432:lacI digested with Smal . One, pAA342, consisted of the 5'-A aIII fragment of the lktA gene while the other, pAA345, contained the entire Mael fragment described above.
  • the clone pAA342 expressed a truncated leukotoxin peptide at high levels while pAA345 expressed full length leukotoxin at very low levels. Therefore, the 3' end of the lktA gene (StyI BamUI fragment from pAA345) was ligated to Styl BamHI- digested pAA342, yielding the plasmid pAA352.
  • the P. haemolytica leukotoxin produced from the pAA352 construct 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 internal DNA fragment of approximately 1300 bp in length from the recombinant LKT gene as follows.
  • the resultant linearized plasmid was then digested with mung-bean nuclease (Pharmacia) to remove the single stranded protruding termini produced by the BstBl digestion.
  • the blunted DNA was then digested with the restriction enzyme Nael (New England Biolabs) , and the digested DNA was loaded onto a 1% agarose gel where the DNA fragments were separated by electrophoresis .
  • a large DNA fragment of approximately 6190 bp was isolated and purified from the agarose gel using a Gene Clean kit (Bio 101) , and the purified fragment was allowed to ligate to itself using bacteriophage T4 DNA ligase (Pharmacia) .
  • the resulting ligation mix was used to transform competent E. coli JM105 cells, and positive clones were identified by their ability to produce an aggregate protein having an appropriate molecular weight .
  • the recombinant plasmid thus formed was designated pCBlll, (ATCC Accession No. 69748), and produces a shortened leukotoxin polypeptide (hereinafter referred to as LKT 111) fused to four copies of GnRH polypeptide.
  • Plasmid pCB114 has the multiple copy GnRH sequence (corresponding to the oligomer of Figure 2B) inserted twice. Both these plasmids are described in U.S. Patent No.
  • LKT 111 and LKT 114 respectively.
  • the synthetic nucleic acid molecule encodes an eight amino acid sequence
  • the resulting recombinant molecule thus contains in the order given in the 5' to 3 ' direction: the synthetic nucleic acid molecule; a nucleotide sequence encoding a first 8 copy GnRH multimer; a nucleotide sequence encoding the shortened LKT peptide (LKT 114) ; and a nucleotide sequence encoding a second 8 copy GnRH multimer.
  • the recombinant molecule was circularized, and the resulting molecule was used to transform competent E. coli JM105 cells. Positive clones were identified by their ability to produce an aggregate protein having a molecular weight of approximately 74 KDa.
  • the recombinant plasmid thus formed was designated pCB122 which produces the LKT 114 polypeptide fused to 16 copies of GnRH polypeptide.
  • plasmid pCB130 For plasmid pCB130, the amp r gene or pCB122 was replaced with the tet r gene. Thus, the plasmid is under tetracycline selection.
  • the nucleotide sequence of the recombinant LKT-GnRH fusion of plasmids pCB122 and pCB130 is shown in Figures 3A through 3F.
  • Example 2 Purification of LKT-antigen Fusions The recombinant LKT-GnRH fusion from Example
  • the pellet was resuspended in an equal volume of TB broth containing ampicillin which had been prewarmed 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. Cultures were incubated for two hours.
  • Cells were harvested by centrifugation as described above, resuspended in 30 mL of 50 mM Tris-hydrochloride , 25% (w/v) sucrose, pH 8.0, and frozen at -70°C.
  • the frozen cells were thawed at room temperature after 60 minutes at -70°C, and 5 mL of lysozyme (Sigma, 20 mg/mL in 250 mM Tris-HCl, pH 8.0) was added.
  • the mixture was 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 stirring with a 2 mL pipette.
  • the beaker containing the lysed cell suspension was placed on ice and sonicated for a total of 2.5 minutes (5-30 second bursts with 1 minute cooling between each) with a Braun sonicator, large probe, set at 100 watts power. Equal volumes of the solution were placed in Teflon SS34 centrifuge tubes and centrifuged for 20 minutes at 10,000 rpm in a Sorvall SS34 rotor. The pellets were resuspended in a total of 100 mL of sterile double distilled water by vortexing at high speed, and the centrifugation step repeated. Supernatants were discarded and the pellets combined in 20 mL of 10 mM Tris-HCl, 150 mM NaCl , pH 8.0 (Tris-buffered saline) and the suspension 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 was added quickly. This mixture was stirred at room temperature for an additional 2 hours.
  • the buffer was replaced with Tris-buffered saline (no guanidine) , and dialysis continued for 12 hours. This was repeated three more times.
  • the final solution was poured into a 2000 mL plastic roller bottle (Corning) and 13 mL of 100 mM PMSF (in ethanol) was added to inhibit protease activity. The solution was stored at -20°C in 100 mL aliquots.
  • Example 3 Antibody Titers Following GnRH Immunization on Pigs This trial was designed to evaluate variables including volume, site of the second injection relative to the first and the number (one vs two) injections for the primary vaccination. For the study, 160 pigs, 28 days of age and weighing 3 to 4 kg, were assigned to one of eight treatment groups
  • GnRH vaccines were made using the GnRH immunogen from plasmid pCB122, described above.
  • 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 EMULSIGEN PLUSTM adjuvant which includes DDA (see, allowed U.S. Patent Application Serial No. 08/463,837).
  • the GnRH vaccines were prepared by combining the stock solution of 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 dose of GnRH immunogen for Groups 1, 2, 3, 4, and 6 was 40 ⁇ g, 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 ⁇ g of the GnRH immunogen/0.25 mL while Group 8 received 40 ⁇ g of the GnRH immunogen from plasmid pCB130 in 0.4 mL of adjuvant. In all instances the ratio of VSA-3 adjuvant to the aqueous phase (phosphate buffered saline) remained at 1:1 (v/v) . Adjustments were made by altering the volume of stock immunogen solution.
  • the vaccines were all administered with a Biojector 2000 needleless injection device manufactured by Bioject Inc., Portland, Oregon, USA. This device utilizes a gas cylinder to inject the vaccine under high pressure through a small opening. The vaccine penetrates through the skin and is deposited subcutaneously .
  • the first injection was given when the pigs were 28 days old and the second was given 35 days later. Injections were given on the outer surface of the pinna of the ear except for the second injection in Group 7 which was given on the dorsal midline 10-15 cm behind the head. Blood was collected by jugular veinpuncture at Days 35, 49 and 63 of the trial (relative to the beginning of the study (Day 0) ) .
  • GnRH antibody titers were determined by a modified radioimmunoassay procedure. Synthetic GnRH (Bachem, Inc.) was iodinated with I 125 (Amersham, Oakville, Ontario) . Dilutions of serum were added to tet tubes followed by a standard amount of I 125 labeled GnRH to give a final incubation volume of 0.7 mL.
  • Figure 1 shows the relationship between antibody titer before the booster vaccination on Day 35 of the trial, when animals were 63 days of age, and 14 days after booster injection, Day 49 of the trial when animals were 77 days of age. Animals that had titers greater than 10% binding at 1:5000 on Day 35 gave a better response to the booster vaccination than animals that had a weaker response to the primary injection. Based on other experiments, we know that binding of approximately 20% at a 1:5000 dilution will give partial suppression of testosterone secretion. These results indicate the utility of a strong response to the primary immunization providing there is no effect on growth or efficiency of feed utilization.
  • Example 5 Immunocastration of Sexually Mature Pigs by GnRH Vaccination
  • the objects of this study were to determine if GnRH vaccination decreased serum testosterone and fat androstenone concentrations in sexually mature male pigs to values equivalent to those seen in surgically castrated pigs and to determine the kinetics of GnRH antibody response, serum testosterone concentrations and fat androstenone levels after a primary and secondary immunization.
  • 24 intact male pigs were assigned randomly prior to Day 0 to one of three treatment groups (Groups 1, 2 and 3) as shown in Table 2.
  • Six age- and litter- matched pigs which had been surgically castrated at less than 1 week of age were assigned to a fourth treatment group (Group 4 - early castrates) .
  • Pigs were housed 10 animals per pen until they were approximately 60 kg in weight at which time they were housed 2 animals per pen. Pigs were provided free access to feed and water and were cared for using standard operating procedures documented by the Prairie Swine Centre, an animal facility affiliated with the University of Saskatchewan and inspected by the Canadian Council on Animal Care.
  • GnRH vaccines were made using the GnRH immunogen from plasmid pCB122, dissolved at a concentration of 28 mg/ml in 4M 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 ⁇ g GnRH immunogen per 0.5 ml dose and was administered IM.
  • the placebo contained phosphate buffered saline and VSA-3.
  • Pigs were given two IM injections of vaccine or placebo in the neck. The first injection was given at Day 0 of the experiment at which time the pigs were 21 days of age. The second injection was given when the pigs were approaching sexual maturity at which time they were approximately 100 kg of body weight (Day 110 - Day 120) (Table 2) . Pigs in Group 2 (late castrates) were castrated surgically when they reached sexual maturity which is influenced strongly by body weight and occurs at approximately 110 kg in body weight (Day 115 to 125) . Pigs in Group 1 received the second immunization approximately 1 week prior to when the pigs in Group 2 were castrated surgically. 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.
  • Day 120 was referred to as the time of the "events", i.e., when the animals received either the second injection (all groups) or were surgically castrated (Group 2). All data collected subsequent to the “events” are described relative to the “events” , i.e. 7 days after the "events” is referred to as Day 127, 14 days after the "events” is referred to as Day 134, etc.
  • Blood samples were obtained from all pigs by jugular veinpuncture at approximately 28 day intervals between Days 28 and 120. Thereafter, blood was obtained at weekly intervals from all pigs until animals were killed on Day 162 of the experiment (42 days after the "events") . Blood was allowed to clot at room temperature, centrifuged and the serum was frozen within 24 hours after sampling.
  • Subcutaneous fat samples (approximately 5 g) for androstenone measurements were obtained under local anesthesia from alternate sides of the neck of all pigs at the time of the "events" and at weekly intervals until slaughter (Day 162) . Fat samples were chilled immediately and frozen within 4 hours after biopsy.
  • Measurements at the time of slaughter included carcass weight, backfat depth at the level of the 10th rib, testicular weight and bulbo-urethral gland length.
  • GnRH antibody titers were determined by a modified radioimmunoassay procedure. Synthetic GnRH (Bachem, Inc.) was iodinated with I 125 (Amersham,
  • Serum testosterone was measured using a Coat-A-Count total testosterone kit (DPS, Los Angeles, CA) . This assay is based on I 12s -testosterone and antibodies that have a high specificity of testosterone .
  • Fat androstenone concentrations were determined using a colorimetric method.
  • GnRH antibody titers measured as % binding at a serum dilution of 1:5000 in Group 1, and at a serum dilution of 1:100 in Groups 2, 3 and 4. Serum testosterone concentrations, fat androstenone, body weight, backfat, testicular weight and bulbourethrethral length were also measured. All pigs in Group 1 developed GnRH antibody titers that were readily detectable at 1:100 serum dilution after primary immunization (see Table 3) .
  • Example 6 GnRH Immunization of Bulls This experiment was conducted with 58 prepubertal bull calves. Twenty-eight bull calves in Group I were vaccinated subcutaneously twice with a vaccine composition comprising 200 ⁇ g of the GnRH immunogen derived from plasmid pCB122 in VSA-3 adjuvant (Day 0 and Day 56) and 30 control bulls in Group 2 were vaccinated with a placebo. Vaccinations of Group I resulted in significant titers against GnRH by Day 42, significant reductions in scrotal circumferences by Day 84 and significantly reduced testosterone levels by Day 98 (Table 4) . Despite these significant anti-GnRH titers and reduced testosterone, no differences in daily gain or feed efficiency were observed in the period from Day 0 to 84 (Table 5) .
  • 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 accession number indicated was assigned after successful viability testing, and the requisite fees were paid. The deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty) . This assures maintenance of viable cultures for a period of thirty (30) years from the date of deposit and at least five (5) years after the most recent request for the furnishing of a sample of the deposit by the depository.

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Abstract

L'invention concerne des procédés d'élevage d'animaux mâles non castrés pour la production de viande. Ces procédés mettent en application des compositions contenant des immunogènes de GnRH. Ils sont utiles pour produire de la viande possédant des qualités organoleptiques améliorées.
EP99917704A 1998-05-05 1999-05-05 Procedes d'elevage d'animaux pour la production de viande Withdrawn EP1094836A2 (fr)

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