Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/08—Peptides having 5 to 11 amino acids
  • A61K38/09—Luteinising hormone-releasing hormone [LHRH], i.e. Gonadotropin-releasing hormone [GnRH]; Related peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0005—Vertebrate antigens
  • A61K39/0006—Contraceptive vaccins; Vaccines against sex hormones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
  • A61P15/16—Masculine contraceptives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
  • A61P15/18—Feminine contraceptives
• • 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
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• the present invention relates generally to compositions and methods for hormone modulation. More particularly, the invention is directed to active and passive GnRH immunization to achieve prolonged suppression of reproductive behavior and/or fertility. The invention also relates to the use of GnRH agonists and antagonists to reduce circulating GnRH levels.
• gonadotropin-releasing hormone In the adult animal, gonadotropin-releasing hormone (GnRH) is produced in the hypothalamus (Schally et al . , Science (1973) 179:341-350) and causes the release of the gonadotropin hormones, luteinizing hormone (LH) and follicle stimulating hormone (FSH) , from the pituitary gland. These, in turn, control the gonadal production of sex steroids; testosterone in males and estrogen and progesterone in females. Therefore, in adults, the hypothalamic-pituitary-gonadal (H-P-G) axis is responsible for sexual function.
• GnRH gonadotropin-releasing hormone
• LH luteinizing hormone
• FSH follicle stimulating hormone
• the onset of puberty appears to be driven by the hypothalamus, however the detailed mechanisms of this process are not well understood.
• the H-P-G axis is functional and appears to play a key role in the onset of puberty.
• the first clinical signs of puberty are preceded by increased pulsatile secretion of GnRH, followed by increased pituitary responsiveness to GnRH (Apter D., Ann. NY Acad. Sci . (1997) 816:9-21) .
• Gonadotropin secretion from the pituitary gland increases progressively during the peripubertal period, ultimately resulting in gonadal stimulation, secretion of sex hormones and progressive occurrence of sexual activity and physical maturation, which are recognized as the events referred to collectively as puberty.
• GnRH analogues before puberty has been shown to delay the onset of puberty in some species. However, the mechanism by which this occurs has not been well described. In humans, long-term administration of GnRH agonists has been shown to be efficacious in the treatment of precocious puberty by inhibiting progression of puberty ( ⁇ eely et al . , J. Ped. (1992) 121 :634-640) .
• GnRH analogue conjugate vaccine human serum albumin- Cys-Gly-GnRH
• GnRH analogue conjugate vaccine induced and maintained sufficient anti-GnRH titers to delay puberty for 175 days (Prendiville et al . , J. Animal Sci . (1995) 73:3030-3037).
• continued suppression of reproductive function was not demonstrated in the face of declining antibody titers.
• this study did not evaluate whether the heifers were able to become pregnant and reproduce.
• Prepubertal immunization with an ovalbumin- GnRH analogue conjugate vaccine impaired testes function and affected the development of social and sexual behavior of young bulls until 17 months of age (Jago et al . , J. Animal Sci . (1997) 75:2609-2619). After 17 months of age, testicular function and reproductive behavior returned to normal and immunized bulls could not be differentiated from normal untreated bulls of the same age.
• a study in sheep showed significant delay in testicular growth and function for up to 115 weeks of age when rams were actively immunized against an ovalbumin- GnRH analogue conjugate vaccine soon after birth (prepubertal) or around puberty (peripubertal) (Brown et al .
• the present invention is directed to methods for reducing the levels of GnRH in a prepubertal vertebrate.
• the methods result in prolonged reduction in reproductive behavior and/or fertility, for example, a prolonged suppression of testicular function and/or development in males, or ovarian function and/or development in females.
• the invention is particularly suitable for use with domestic animals such as cats, dogs and horses, and wild-life, such as deer, and provides a viable and desirable alternative to surgical forms of sterilization that are currently used.
• Long-term immunosterilization may be achieved by active or passive immunization against GnRH during the prepubertal or peripubertal period. Without being bound by a particular theory, there appears to be a critical period in early life during which normal and sustained gonadal development requires gonadotropic stimulation, particularly by LH from the pituitary gland.
• GnRH therapy GnRH agonists or antagonists
• GnRH therapy prepubertally or peripubertally, results in a reduction in circulating GnRH concentrations which in turn may cause down-regulation of pituitary GnRH receptors and a reduction in gonadatropins during this critical time period. This may lead to long-term impairment of gonadal development and function by the permanent inability of the pituitary gland to respond to GnRH.
• the present invention is directed to a method for prolonged suppression of reproductive behavior and/or fertility.
• the method comprises administering to the vertebrate subject, prepubertally, a composition comprising an effective amount of a GnRH immunogen, GnRH analogue, or antibodies that cross-react with endogenous GnRH of said vertebrate subject.
• the composition optionally includes an immunological adjuvant, preferably comprising an oil and dimethyldioctadecylammonium bromide .
• the method results in a prolonged, long-term suppression of gonadal development and/or function, such as a suppression of testicular development and/or function in males or prolonged long-term suppression of ovarian development and/or function in females.
• the method may further comprise administering to the vertebrate subject a second composition comprising an effective amount of a GnRH immunogen.
• the GnRH immunogen may be the same or different in the compositions.
• the invention is directed to a method for prolonged suppression of reproductive behavior and/or fertility in a feline, canine, equine or cervine subject.
• the method comprises administering to the subject, prepubertally, a vaccine composition comprising an effective amount of 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.
• the invention is directed to a method for prolonged suppression of reproductive behavior and/or fertility in a feline subject. The method comprises:
• 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 ;
• 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.
• the first and second vaccine compositions comprise the same GnRH multimer and the GnRH multimer comprises the amino acid sequence depicted in Figures 6A-6F (SEQ ID NO: ) , or an amino acid sequence with at least about 75% sequence identity thereto.
• the invention is directed to a method for prolonged suppression of reproductive behavior and/or fertility in a feline subject.
• the method comprises:
• a first vaccine composition comprising an immunological adjuvant which comprises a light mineral oil and dimethyldioctadecylammonium bromide, and an effective amount of a GnRH multimer comprising the amino acid sequence depicted in Figures 6A-6F (SEQ ID NO: ) , or an amino acid sequence with at least about 75% sequence identity thereto; and
• a second vaccine composition comprising an immunological adjuvant which comprises a light mineral oil and dimethyldioctadecylammonium bromide, and an effective amount of a GnRH multimer comprising the amino acid sequence depicted in Figures
• the invention is directed to a method for suppressing reproductive behavior and/or fertility in a feline, canine, equine or cervine subject for at least 10 months.
• the method comprises administering to the subject, a first composition comprising an effective amount of a GnRH immunogen.
• a second vaccine composition is administered which comprises an effective amount of a GnRH immunogen.
• the GnRH immunogen in the first and second compositions may be the same or different .
• both the first and second compositions are administered prepubertally, or postpubertally, or the first composition is administered prepubertally and the second composition is administered postpubertally.
• the method further comprises administering additional compositions comprising an effective amount of a GnRH immunogen at least about 6 to about 12 months subsequent to the prior administration.
• the additional compositions are administered at about yearly intervals.
• Figure 1 depicts the relationship between GnRH antibody titers (mean + SEM) and serum testosterone in cats immunized with GnRH vaccines.
• Figure 2 shows GnRH antibody titers (mean +
• Figure 3 shows the effect of treatment with leuprolide acetate on serum LH concentrations in 10 month old kittens immunized at 8 , 12 and 16 weeks of age with a GnRH vaccine.
• Figure 4 shows GnRH antibody titers (mean +
• Figures 5A and 5B show the nucleotide sequences and amino acid sequences of the GnRH constructs used in the chimeric leukotoxin-GnRH polypeptide gene fusions herein.
• Figure 5A depicts a single copy of a GnRH decapeptide.
• Figures 6A through 6F show the nucleotide sequence and predicted amino acid sequence of the LKT- GnRH chimeric protein from pCB130.
• Figure 7 depicts serum testosterone concentrations in kittens immunized at 8 , 12 and 16 weeks of age with a GnRH fusion protein described in the examples. Arrows represent time of immunization.
• Figure 8 depicts serum estradiol concentrations in kittens immunized at 8 , 12 and 16 weeks of age with a GnRH fusion protein described in the examples. Arrows represent time of immunization.
• GnRH Gonadotropin releasing hormone
• LH luteinizing hormone
• FSH follicle stimulating hormone
• GnRH derived from most mammals including human, bovine, porcine and ovine GnRH 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) 311:666-668) .
• 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, or which have the ability to act as GnRH agonists or antagonists that bind to, and ultimately down- regulate, GnRH receptors, or otherwise block the action of GnRH such as by competing for GnRH receptors.
• 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 Figure 5B) ; a GnRH analogue with an N-terminal addition such as
• Cys-Gly-GnRH (see, e.g., Prendiville et al . , J. Animal Sci . (1995) 21:3030-3037); a carboxyl -containing GnRH analogue (see, e.g., Jago et al . , J " . Animal Sci . (1997) 75 . :2609-2619; Brown et al . , J. Reproduc . Fertil . (1994) 101:15-21); the GnRH analogue (D-Trp6- Pro9-ethyl amide)GnRH (see, e.g., Tilbrook et al .
• 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-
• Arg-Pro-Pro-Pro-Pro-Cys SEQ ID NO: ) ; the GnRH analogue known as Deslorelin, commercially available from Apeptech (Australia) , and OvuplantTM; and molecules with other amino acid additions, substitutions and/or deletions which retain the ability to either elicit formation of antibodies that cross-react with naturally occurring GnRH, or molecules that act as GnRH agonists or antagonists.
• GnRH Representative agonists of GnRH include the compounds LupronTM and Lupron DepotTM, both available from TAP Pharmaceuticals, Inc. (Deerfield, IL) , with the chemical formula 6-Oxo-L-propyl-L-histidyl-L- tryptophyl-L-seryl-L-tyrosyl-D-leucyl-L-leucyl-L- arginyl-N-ethyl-L-prolinamide acetate, ZoladexTM, a goserelin acetate implant, available from Zeneca Pharmaceuticals (Wilmington, DE) .
• Representative antagonists include N- terminus-modified analogues, such as those described in U.S. Patent No.
• analogues described in U.S. Patent No. 4,740,500 including an analog with the formula Ac-3-D-NAL-R2-D-3PAL-Ser-Arg-R6-Leu-Arg-Pro- R10, wherein R2 is Cl-D-Phe, F-D-Phe, N0 2 -D-Phe, Br-D- Phe, 3,4C1 2 -D-Phe or C Me-Cl-D-Phe; R6 is D-3PAL, D- Trp, For-D-Trp, N0 2 -D-Trp, (imBzl) D-His, D-Tyr or ⁇ -O- NAL, and R10 is Gly-NH 2 , NHCH 2 CH 3 , NHNHC0NH 2 or D-Ala- NH 2 , j ⁇ -D-NAL is the D-isomer of alanine which is substituted by naphthyl on the 3-carbon atom and D- 3PAL is
• 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 Proteins having substantially the same amino acid sequence as the reference molecule, but possessing 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, so long as the molecule retains the desired activity. Epitopes of GnRH are also captured by the definition.
• 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 5B herein. Such multimers are described more fully below.
• a 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.
• Particular 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) y 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 . Ma th . 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, Wl) 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.
• identity can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
• DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al . , supra; DNA Cloning, supra ; Nucleic Acid Hybri dization, s upra .
• 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 Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey.
• 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.
• Hopp and Woods Proc . Natl . Acad . Sci . USA (1981) 78:3824-3828, can also be used to determine antigenic portions of a given molecule.
• 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.
• 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.
• the term includes both full-length and partial sequences, as well as analogues.
• native full-length leukotoxins display cytotoxic activity
• 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
• 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 ⁇ os . 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 lktA 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 adjuvant refers to 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.
• derived f om denotes an actual or theoretical source or origin of the subject molecule or immunogen.
• 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 .
• vertebrate subject any member of the subphylum cordata, including, without limitation, mammals such as ovine, bovine, porcine, equine, cervine subjects, including cattle, sheep, pigs, goats, horses, deer and humans; domestic animals such as domestic canines and felines (dogs and cats) ; and birds, including domestic, wild and game birds such as cocks and hens including chickens, turkeys and other gallinaceous birds; fish, rodents such as hamsters, guinea pigs, gerbils, ground hogs, gophers, lagomorphs, rabbits, ferrets, squirrels and reptilian subjects.
• mammals such as ovine, bovine, porcine, equine, cervine subjects, including cattle, sheep, pigs, goats, horses, deer and humans
• domestic animals such as domestic canines and felines (dogs and cats)
• birds including domestic, wild and game birds such as cocks and hens including chickens, turkey
• suppression of reproductive behavior and/or fertility in a vertebrate subject is meant a significant reduction in mating behavior and/or fertility in an animal treated under the invention, as compared to the mating behavior and/or fertility normally expected from a postpubertal animal of the same species.
• a suppression in reproductive behavior and/or fertility can result in a reduction in the number of attempted matings, or a reduction in successful matings, such as a reduction in the number of conceptions, in the case of a female, or fertilizations, in the case of a male, as compared to a typical, untreated animal of the same species.
• the suppression can result in complete sterility of the animal in question.
• the effect may be the result of either suppression at the hypothalamic pituitary or suppression at the gonadal level, such as a suppression of testicular development or function in the male and ovarian development or function in the female.
• prolonged or “long-term suppression” of reproductive behavior and/or fertility is meant a suppression of reproductive behavior and/or fertility, as described above, that persists after GnRH antibody titers have waned, or in the case of a GnRH analogue, such as a GnRH agonist or antagonist, that persists after the circulating blood level of the substance has declined below a pharmacologically active level.
• Such an effect may be permanent, or persist for six months to one year or more after GnRH antibody titers or the circulating blood level of the GnRH analogue have declined, more preferably eight or ten months to one year or more, even up to the lifespan of the animal.
• Such a prolonged effect can be readily determined by measuring GnRH antibody titers or blood levels of the analogue using methods well known in the art, such as those described in the examples below.
• prepubertal is meant anytime prior to the onset of puberty.
• prepubertal also includes the peripubertal period.
• the onset of puberty is indicated by the ability of an animal in question to conceive, in the case of a female, e.g., the onset of ovulation, or to fertilize, e.g., the ability to produce sperm, in the case of a male.
• the typical timing for the onset of puberty for a given species is well known in the art.
• the onset of puberty is accompanied by the presence of various circulating hormones in the subject of interest.
• clinical signs of puberty may be preceded by increased pulsatile secretion of GnRH produced in the hypothalamus, followed by increased pituitary responsiveness to GnRH which causes the release of LH and FSH from the pituitary gland.
• GnRH produced in the hypothalamus
• pituitary responsiveness to GnRH which causes the release of LH and FSH from the pituitary gland.
• Gonadotropin secretion, particularly LH increases progressively during the peripubertal period resulting in gonadal stimulation, secretion of sex hormones and progressive physical maturation.
• puberty The levels of these hormones associated with puberty are known in the art and will vary from species to species. For a detailed description of puberty and the hormonal changes associated therewith, see, e.g., Apter D., Ann. NT Acad . Sci . (1997) 816:9-21.
• Hormone levels can be measured using standard techniques such as radioimmunoassays (see, e.g., Lee et al . , J. Reproduc . Fertil . (1976) 46 . : 1-6; Bremner et al . , Endocrin . (1980) 106:329-336) , as well as highly sensitive immunoflurometric assays (see, e.g., Apter et al . , J. Clin . Endocrinol . Metab . (1989) 68 . : 53-57; Bis et al., Pediatr. Res . (1990) 27:215-219; Gon et al . ,
• estradiol secretion can be measured using various well-known assays such as recombinant cell bioassays (see, e.g., Klein, et al . , J. Clin . Invest . (1994) 94:2475-2480) .
• the compositions described herein surprisingly provide a means for producing a long-term immunological response in a subject immunized therewith. If immunization is used, the method entails one or more primary immunizations before the onset of puberty, optionally followed by one or more boosts, with the same or different GnRH composition, to cause a long-term suppression as described above.
• the boosts may be given prior to, or after the onset of puberty.
• a primary vaccination will generally be administered at about 0 to about 35 weeks of age, more preferably about 3 to about 15 weeks, preferably about 5 to about 12 weeks of age, and even more preferably at about 6 to about 10 weeks of age.
• One or more booster treatments may be given, also prior to puberty, at an appropriate time after the primary immunization, generally at about 2 to about 10 weeks following the primary immunization, preferably at about 3 to about 4 weeks following the primary immunization.
• additional immunizations at regular intervals can be given in order to ensure a prolonged effect on reproductive behavior and/or fertility. For example, in one embodiment, additional immunizations are given at least 6 months to 1 year following the second immunization and can be administered annually thereafter.
• the animal may be immunized postpubertally in order to achieve a prolonged effect.
• the vaccine compositions of the present invention are administered directly to the animal in question (active immunization) or to laboratory animals in order to produce antibodies which in turn can be used to immunize the animal in question (passive immunization) .
• passive immunization can be achieved using monoclonal antibodies, 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, described in greater detail below.
• 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 . Natl . 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 . Natl . Acad . Sci . U. S . A . 79:569- 573), ovalbumin, leukotoxin polypeptides, and
• 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. 4,722,840. Still other suitable carriers include cells, such as lymphocytes, since presentation in this form mimics the natural mode of presentation in the subject, which gives rise to the immunized state. Alternatively, the GnRH immunogens may be coupled to erythrocytes, preferably the subject's own erythrocytes .
• 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
• 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 5B.
• 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 adjacent GnRH sequences (without intervening spacer sequences) .
• 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.
• sequences for the GnRH immunogens 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.
• GnRH immunogen portions of the chimera can be fused 5' and/or 3' to a desired ancillary sequence or carrier molecule.
• 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; ⁇ ambair et al . (1984) Science 223:1299; and Jay et al . (1984) J. Biol . Chem. 259:6311.
• control elements for expression in suitable host tissue in vivo .
• 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 metallothionem 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. 4:761), the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus (Gorman et al . (1982) Proc . Natl . 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 258:1745; 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 5A and 5B 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 expression vector which already contains the control sequences and an appropriate restriction site.
• polypeptides may be desirable to add sequences which cause the secretion of the polypeptide from the host organism, with subsequent cleavage of the secretory signal. It may also be desirable to produce mutants or analogues of the polypeptide.
• Mutants or analogues 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 . , supra; DNA Cloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra ; Kunkel, T.A. Proc . Natl . Acad. Sci .
• 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, 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 (e.g., Hep G2), Madin-Darby bovine kidney (“MDBK”) cells, as well as others.
• ATCC American Type Culture Collection
• CHO Chinese hamster ovary
• HeLa cells HeLa cells
• BHK baby hamster kidney
• COS monkey kidney cells
• MDBK Madin-Darby bovine kidney
• bacterial hosts such as E. coli , Bacillus subtilis, and Streptococcus spp . , will find use with the present expression constructs.
• 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, or block the action of GnRH in a subject vertebrate .
• 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. This can be accomplished in any one of several ways known to those of skill in the art.
• 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 intramuscu- larly) .
• 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 vi tro 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. Typically, 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").
• 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) .
• 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 Fschreib.
• 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. For example, if the antibodies are to be used in human subjects, 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. (1991) Nature 349:293-299; Jones, P.T. et al . (1986) Nature 321:522-525; Riechmann, L. et al . (1988) 33_2:323-327; and Carter, P. et al . (1992) Proc . Natl . Acad. Sci . USA 89:4285-4289.
• 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.
• 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, chitosan-based adjuvants, and any of the various saponins, oils, and other substances known in the art.
• compounds which may serve as emulsifiers herein 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 (e.g., glyceryl monostearate) , polyoxyethylene glycol esters and ethers, and the sorbitan fatty acid esters (e.g., sorbitan monopalmitate) and their polyoxyethylene derivatives (e.g., polyoxyethylene sorbitan monopalmitate) .
• 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 ) propanediamine ("avridine”).
• DDA dimethyldioctadecylammonium bromide
• avridine N,N-dioctadecyl-N,N- bis (2 -hydroxyethyl ) propanediamine
• 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) .
• DDA distyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-a modified form of the EMULSIGEN PLUSTM adjuvant which includes DDA (see, allowed U.S. Patent Application Serial No. 08/463,837) .
• DDA U.S. Patent Application Serial No. 08/463,837
• the 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 liposome vehicles or other particulate carriers used.
• the 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.
• 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, before puberty, 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 either prepubertally, postpubertally, or both, in order to result in a prolonged suppression of testicular development or function in males or ovarian development or function in females.
• any suitable pharmaceutical delivery means may be employed to deliver the compositions to the vertebrate subject.
• suitable pharmaceutical delivery means for example, 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 . ) , and particle injectors (U.S. Patent Nos.
• 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
• Plasmid pCB130 was used to produce a GnRH fusion protein for use in the examples described below.
• This GnRH construct 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. See, Figures 5A and 5B .
• 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
• 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.
• the GnRH- leukotoxin fusion protein produced by plasmid pCB130 is shown in Figures 6A through 6F.
• Plasmid pCB130 was prepared as follows. The leukotoxin gene was isolated as described in U.S.
• Patent Nos. 5,476,657 and 5,837,268 are 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 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.
• pAA114 This construct contained the entire leukotoxin gene sequence.
• 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.
• LKT 352 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 5B) inserted twice. Both these plasmids are described in U.S. Patent No. 5,837,268 and produce shortened leukotoxin polypeptides termed 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 pCB130 which produces the LKT 114 polypeptide fused to 16 copies of GnRH polypeptide.
• the nucleotide sequence of the recombinant LKT-GnRH fusion of pCB130 is shown in Figures 6A through 6F.
• Example 2 Purification of LKT-antigen Fusions
• the recombinant LKT-GnRH fusion from 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 ⁇ g/mL of ampicillin and incubated at 37°C for 6 hours on a G10 shaker, 220 rpm. Four mL of this culture was diluted into each of two baffled Fernbach flasks containing 400 mL of TB broth + ampicillin and incubated overnight as described above.
• Cells were harvested by centrifugation for 10 minutes at 4,000 rpm in polypropylene bottles, 500 mL volume, using a Sorvall GS3 rotor. 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.
• IPTG isopropyl-B,D-thiogalactopyranoside
• 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
• 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.
• fusion protein was expressed at high levels as inclusion bodies.
• Example 3 Immunization of Mature Cats by GnRH Vaccination
• the following experiment utilizes an immunological approach to demonstrate the use of GnRH vaccines in order to achieve prolonged immunological castration.
• Five mature male cats were immunized twice, 67 days apart, with a GnRH vaccine.
• vaccines derived from cultures containing the pCB130 plasmid described above were formulated to contain 50 ⁇ g of the GnRH multimer fusion molecules (36 ⁇ g total of GnRH) in a .5 mL final volume of VSA-3 adjuvant, (a modified EMULSIGEN PLUSTM adjuvant which contains a light mineral oil and dimethyldioctadecylammonium bromide, see, allowed U.S. Patent Application Serial No. 08/463,837).
• VSA-3 adjuvant a modified EMULSIGEN PLUSTM adjuvant which contains a light mineral oil and dimethyldioctadecylammonium bromide, see, allowed U.S. Patent Application Serial No. 08/463,837.
• GnRH titers were measured as % binding at 1:5000 dilution and testicular size determined.
• Immunological activity of the GnRH fusion protein vaccine was assayed by measuring anti-GnRH antibody titers using a standard radioimmunoassay procedure at a 1:5000 serum dilution.
• anti-GnRH antibody titers increased dramatically in the immunized animals and remained at levels significantly in excess of the minimal amount required to produce a biological effect (approximately 30% binding in Figure 2) .
• Biological activity of the GnRH fusion protein vaccine was also assayed by measuring serum testosterone concentration in the males and serum estradiol concentration in the females using standard radioimmunoassay procedures.
• testosterone was measured using a Coat-A-Count Total Testosterone KitTM (Diagnostic Products Corporation, Los Angeles, CA) .
• the 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.
• 125 I-labeled testosterone was competed for 3 hours at 37°C with testosterone in the test sample for antibody sites.
• the tube was decanted to separate bound from free, and counted in a gamma counter.
• the amount of testosterone present in the test sample was determined from a calibration curve.
• Serum estradiol was measured using a Double Antibody Estradiol KitTM (Diagnostic Products Corporation, Los Angeles, CA) .
• the test is a sequential radioimmunoassay in which the test sample was preincubated with anti-estradiol antibodies.
• Mating and androgen dependent behavior was assessed by exposing the male kittens to a queen in estrus at approximately 9 months of age, by which age males would normally be sexually active.
• Reproductive function in the females was assessed by exposing the female kittens to an intact male cat at approximately 10 months of age by which time the females would normally be sexually active. No sexual behavior was evident in 9 month old male kittens when exposed to a queen in estrus.
• the control adult female showed signs of estrus within 5 days after the test . None of the kittens showed signs of estrus and the immunized male kitten showed no signs of sexual behavior.
• the lack of sexual behavior seen in these kittens at an age when the onset of sexual function was expected could be attributed to the continued high circulating levels of GnRH antibodies binding to endogenous GnRH and preventing activation of the pituitary GnRH receptors.
• Low levels of endogenous GnRH are also required for the maintenance of normal concentrations of pituitary GnRH receptors. The removal of GnRH by circulating antibodies may result in down-regulation of the pituitary receptors, with subsequent inability of the pituitary to respond to exogenous GnRH.
• a prolonged reduction in the number of pituitary GnRH receptors may have a permanent effect on the maturation of hypothalamic-pituitary-gonadal axis and lead to long-term sterilization.
• this test can be repeated again when antibody titers have declined.
• the GnRH vaccine described above was used to immunize two kittens (one female and one male) from one litter group at 6 and 10 weeks of age.
• the litter group also included one age- and sex-matched untreated control kitten.
• Serum antibody titers to GnRH, serum testosterone concentrations, serum estradiol concentrations and LH concentrations in response to an exogenous GnRH challenge test were measured as described above.
• pituitary responsiveness to exogenous GnRH is assessed by a GnRH challenge test, also as described above.
• Mating and androgen dependent behavior is assessed by exposing the male kittens to a queen in estrus at approximately 9 months of age, by which age males would normally be sexually active.
• Reproductive function in the females is assessed by exposing the female kittens to an intact male cat at approximately 10 months of age by which time the females would normally be sexually active. Vaginal cytology to assess the reproductive cycle is also obtained.
• 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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