JP2002513770A - How to breed animals for edible meat production - Google Patents

Abstract

  (57) [Summary] A method of breeding a non-castrated male animal for edible meat production is disclosed. In these methods, a composition comprising a GnRH immunogen is used. These methods are useful for producing portions of edible meat with enhanced organoleptic quality.

Description

DETAILED DESCRIPTION OF THE INVENTIONTechnical field The present invention generally relates to a method of breeding animals for edible meat production. further
In particular, the present invention relates to GnRH immunogens that reduce circulating gonadal steroid levels.
Primary vaccination followed by one or more androgen steroids
Slaughter that substantially reduces the levels of steroids and / or non-androgenic steroids
A method for immunizing an animal by revaccination with the immediately preceding GnRH immunogen.
Background of the Invention Male cattle, pigs, and sheep are typically more highly muscular,
Has a larger mature body than females. According to conventional descriptions, male animals are sexually active.
Testosterone, an anabolic steroid produced by the testes when it reaches ripeness
Secretion increases muscle protein deposition and reduces fat. Also, many laboratories
That males use feed more efficiently than females during the growth period
Was established (Field, RA, Journal Animal Soc)
. (1971)32: 849-858). These differences occur around the spring
Most obvious, and testosterone plays an important role in these changes
Have. In males, androgenic steroids, testosterone and androster
Are involved in the regulation of two different biological processes. One action is physiological
Increases muscle deposition, reduces fat synthesis, and increases feed utilization
Let it. Testosterone inhibits the growth of gonads such as the seminal vesicles, prostate and testes.
And has a similar direct effect. These effects are due to the combination of androgen and target pairs.
Includes direct interaction with the receptor in the tissue. Androgen's second one
The general effect is to cause male sexual and other behavioral changes. Them
The effects are transmitted through the central nervous system. These various actions are performed by different molecular mechanisms.
Some of them androgen
It may be maintained at low plasma concentrations, but other effects may require higher levels.
There is. Androgens maintain optimal growth, muscle deposition and feed efficiency in life
It is generally assumed that there is a measurable amount of androgen
Even so, that assumption may not be true. Or the androgen sensitive group
Weaves, e.g., muscles, are earlier in life than in puberty or later.
May be much more responsive to lower androgens
You. Allrich et al., Journal Animal Soc. (1982)
55: 1391-1146, as a function of age and testosterone concentration.
The rate of increase in body weight and the weight of testosterone-sensitive tissue in rats were compared. In particular, a comparison of the growth rates of different tissues at different ages indicates that testosterone
Sensitive tissues and testosterone levels may change with age
showed that. For example, on days 40-100, serum testosterone was 3.1 times higher.
But the body weight increased by a factor of 3.2, the testes weight increased by a factor of 3.6, and accumulated.
The authors showed that seminal vesicles increased 4.0-fold. Also, these data show
Thus, early adolescent testosterone levels are low but easily detectable, and
Body weight increases at about the same rate as other tissues in the presence of low levels of testosterone
Was. Pigs are sexually mature at approximately 130-150 days of age (65-85 kg body weight)
, And testosterone levels increase significantly at that time. No.
From 100 to 190 days, serum testosterone increases by a factor of 4.1,
Weight, testes weight, and seminal vesicle weights were 2.6 times, 13.5 times, and
And 9.2-fold increase. Further, Knudson et al., Journal Animal Soc. (19
85)61: 789-796 shows that castrated male pigs become fully male until about 90 days of age
In comparison, they gained weight at a similar rate, but beyond that age, full males were castrated.
Grown more efficiently than males. This feature is a flock of animals
It is especially important for infertility and is especially important for the
"Boar rot" produced by sex steroids in functional testes
aint) "is particularly important where prevention is desired. For example,
See the following reference: Meloen et al., Vaccine (1994).12(
8): 741-746. As a method of improving growth rate and feed efficiency in animals, GnRH immunogens
Numerous studies have been conducted in pigs and cattle to investigate the use of
I have. See, for example, the following documents: Adams and Adams, Journa.
l Animal Soc. (1992)701691-1698; Cara
ty and Bonneau, C.I. R. Acad. Sc. Paris (1986
)303: 673-676; Chaffaux et al., Recueil de Me.
decision Veterinaire (1985)161133-145;
J. Finnerty et al. Repro. Fertil. (1994)101: 3
33-343. Many of the goals of these studies have been moving toward the end of the fattening period.
Was to grow things as full males and then immunologically castrate them
. Towards the end of the fattening period and just prior to slaughter,
To prime the immune system with one or more vaccinations early in life
Thus, towards the end of the fattening period, the animals were
Be responsive. The first vaccination is based on the immune system against the GnRH immunogen.
Anti-GnRH antibody potency to prime but reduce serum testosterone levels
Avoid induction of titers or serum test procedures when animals approach puberty.
Designed to prevent Ron levels from increasing. This is because serum testosterone
The belief that reducing will reduce growth rate or feed efficiency in young animals
Based on For example, Meloen et al., Vaccine (1994).12: 741-746
Is administered in two doses to reduce boar rot in pigs, G
Describes the use of the nRH associated vaccine. Testicle size reduced until after second immunization
Little did not happen. In addition, International Publication No. WO 90/11298, 1990
Published on October 4th, see. Falvo et al., Journal Animal Soc
. (1986)63: 986-994 describes the presence of boar spoilage and carcass characteristics
Use of GnRH vaccines to study various effects in pigs, including
Reporting. The authors reported that plasma testosterone 2 weeks after the first booster injection
The level was reported to be significantly reduced. Similarly, US Pat. No. 5,573,76
No. 7 describes a method for improving sensory irritation quality of edible meat using GnRH immunization
About. This method involves two immunizations, one immunization to gonad steroids
And a second immunization is performed prior to sacrifice.
Designed to counteract the effects of steroids and nonandrogenic steroids
ing. In addition, previous attempts at immune infertility have shown that GnRH peptides and / or
The associated carrier systems are poorly immunogenic and the resulting various previous GnRHs
-Based vaccines can elicit a sufficient immune response against endogenous GnRH
Was not possible, and did not produce uniform results. For example, the following document
See Robertson, Vet. Record (1981)108 : 381-382. Therefore, a reliable method of immuno-sterility in food-producing animals is
Would be desirable.Disclosure of the invention The present invention provides a reliable, reproductive method for breeding food-producing male animals for edible meat production.
Based on available methods. In particular, as opposed to the dominant belief, the tests described herein
Early in the life cycle to produce commercially acceptable quantities of edible meat based on
It is unnecessary to avoid reducing testosterone substantially in
And an anti-cancer drug with a measurable effect on gonadal steroid secretion during the fattening period
Primary GnRH immunization to induce the body significantly reduces growth rate or feed efficiency
No, it seems achievable. After primary immunization, later in life,
Eliminates the effects of androgenic and / or non-androgenic steroids
A second immunization can be performed. Thus, in one aspect, the present invention provides a castration for the production of edible meat.
Prior to or during the fattening period of the male food producing animal, the animal may be provided with a first vaccine composition
To reduce circulating testosterone levels and kill the animals
About 2 to about 8 weeks prior to sacrifice, the animal is vaccinated with the second vaccine composition for one time.
Or higher androgenic steroids and / or non-androgenic steroids
Castration for edible meat production, comprising substantially reducing the level of
A method of breeding male food producing animals. In a particularly preferred embodiment,
The first and / or second vaccine composition may be an immunological adjuvant, for example,
Contains adjuvant comprising oil and dimethyldioctadecyl ammonium bromide
. Further, the GnRH immunogen in the first and / or second vaccine composition is generally
Can be a GnRH multimer having the formula (GnRH-X-GnRH) y
Wherein GnRH is a GnRH immunogen, X is a peptide bond, an amino acid spacer group, a carrier molecule and [GnRH] n
One or more molecules selected from the group consisting of:
Y is an integer equal to or equal to, and y is an integer greater than or equal to 1. In some embodiments, administration of the first vaccine composition cross-links endogenous GnRH in the animal.
After producing reactive antibodies and antibody levels are reduced, the second vaccine composition is
Administer. In another embodiment, the present invention relates to a non-castrated male cow, hen for production of edible meat.
Before or during the fattening period of the azalea or pig animal, the animal may be given a first vaccine composition
Vaccinated to reduce circulating testosterone levels, and
About 2 to about 8 weeks before sacrifice, the animals are vaccinated with the second vaccine composition and
Or higher androgenic steroids and / or non-androgenic steroids
A method for producing edible meat comprising substantially reducing the level of id.
The present invention relates to a method of breeding male bovine, ovine or porcine animals. First and
And / or the second vaccine composition may further comprise an immunological adjuvant.
it can. In still other embodiments, the present invention provides: (a) non-castrated male bovine, ovine or porcine animal for edible meat production;
Before or during the fattening period, the animals are vaccinated with the first vaccine composition and circulated.
Reducing the level of cyclic testosterone, wherein the first vaccine composition comprises an immunological adjuvant.
A bunt and a GnRH multimer, wherein the GnRH multimer has the general formula (
GnRH-X-GnRH) y, where GnRH is a GnRH immunogen, X is a peptide bond, an amino acid spacer group, a leukotoxin polypeptide and
And [GnRH] n, one or more molecules selected from the group consisting of
Wherein n is an integer greater than or equal to 1 and y is an integer greater than or equal to 1 and (b) at about 2 to about 8 weeks prior to sacrifice of the animal, A second vaccine composition for animals
Vaccinated with one or more androgenic steroids and / or non-
Substantially reducing the level of an androgenic steroid, said second vaccine composition
A product comprising an adjuvant and a GnRH multimer;
Has the general formula (GnRH-X-GnRH) y, where GnRH is a GnRH immunogen, X is a peptide bond, an amino acid spacer group, a leukotoxin polypeptide and
And [GnRH] n, one or more molecules selected from the group consisting of
Where n is an integer greater than or equal to 1 and y is an integer greater than or equal to 1 for breeding non-castrated male cattle, sheep or pig animals for edible meat production
About the method. In still other embodiments, the present invention provides: (a) non-castrated male bovine, ovine or porcine animal for edible meat production;
Before or during the fattening period, the animals are vaccinated with the first vaccine composition and circulated.
Reducing cyclic testosterone levels, wherein the first vaccine composition is immunologically
An adjuvant and a GnRH multimer, wherein the GnRH multimer is a second
The amino acid sequence depicted in FIGS. 3A-3F (SEQ ID NO: _), or
An amino acid sequence having at least about 75% sequence identity thereto.
And (b) about 2 to about 8 weeks before sacrifice of the animal, the animal is provided with a second vaccine composition.
Vaccinated with one or more androgenic steroids and / or non-
Substantially reducing the level of an androgenic steroid, wherein said second vaccine
The composition comprises an immunological adjuvant and a GnRH multimer.
The H multimer is the amino acid sequence depicted in FIGS. 3A-3F (SEQ ID NO: _).
Sequence or amino acid sequence having at least about 75% sequence identity thereto
Non-castrated male cattle, sheep or pigs for edible meat production, comprising a row
And a method for breeding animals. In the first and / or second vaccine composition
Immunological adjuvant uses light mineral oil and dimethyldioctadecyl ammonium bromide
Can comprise. These and other aspects of the invention will be apparent to those skilled in the art in light of the disclosure herein.
Will be readily apparent toDetailed description In the practice of the present invention, unless otherwise specified, molecular biology, microbiology, virology
Using conventional techniques of recombinant DNA technology, and immunology, which are within the skill of the artisan.
Within the range. Such techniques are explained fully in the literature. For example,
See Sambrook, Fritsch and Maniati.
s, Molecular Cloning: A Laboratory Man
ua; DNA Cloning, Vol. I and II (DN Clove)
r); Oligonucleotide Synthesis (MJ Ga)
it edition); Nucleic Acid Hybridization (BD).
Hames & S.M. J. Higins); Perbal, A Prac
physical Guide to Molecular Cloning; the
 Series, Methods in Enzymology (S. Colo)
wick and N.W. Kaplan, Ed., Academic Press, Inc.
); And Handbook of Experimental Immuno
logic, Vol. I-IV (DM Weir and CC Blackwee)
ll, Blackwell Scientific Publication
s). Before describing the present invention in detail, the present invention contemplates certain formulations or process parameters.
It is to be understood that these are not limited to
is there. Also, the terms used in the specification are intended to describe specific embodiments of the present invention.
It should be understood that they are intended to be limiting and not limiting.1. Definition In describing the present invention, the following terms will be used, as defined below:
Intend. The term "conadotropin releasing hormone" or "GnRH"
Luteinizing hormone (LH) and follicle stimulating hormone (FSH)
Mean decapeptide secreted by the hypothalamus that controls release (F
Ink, G .; , British Medical Bulletin (1979)
)35: 155-160). The amino acid sequence of GnRH is vertebrate, especially mammalian
Is highly conserved between In this regard, humans, cattle, pigs and pigs
GnRH from most mammals, including the azalea GnRH (formally LH
RH) is the amino acid sequence pyroGlu-His-Trp-Ser
-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂(SEQ ID NO: 1)
(Murad et al., Hornes and Hornes Ant
agonists, inThe Pharmacological Basi
s of Therapeutics, 6th edition (1980) and Seebur
g et al., Nature (1984)311: 666-668). As used herein, a “GnRH polypeptide” is a native GnRH
Molecules derived from the sequence, as described below, substantially against native GnRH
Recombinantly produced, having amino acid sequences that are homologous and maintain immunogenicity
Or chemically synthesized GnRH polypeptides. Like this
This term is used internally or at the amino or carboxy terminus of the peptide.
Including the addition, substitution and / or deletion of any single or multiple amino acids present.
GnRH derivatives or analogs. Therefore, in the present invention
, “GnRH polypeptides” are molecules having a native sequence as well as GnRH analogs.
Rogue. Representative GnRH analogs include the following analogs: pyroGlu
Rather, an analog having an N-terminal Gln or Glu residue at amino acid position 2
An analog with Asp instead of His (see FIGS. 2A and 2B);
GnRH analogs with an N-terminal addition, such as Cys-Gly-GnRH (
See, for example, the following document: Prendiville et al., Journal.
 Animal Soc. (1995)73: 3030-3037);
GnRH analog containing sill (see, eg, Jag
o et al., Journal Animal Soc. (1997)75: 2609-
2619; Brown et al. Reproduc. Fertil. (1994)
101GnRH analog (D-Trp6-Pro9-ethyl)
Amides) GnRH (see, eg, Tilbrook et al., H.
ormoes and Behavior (1993)27: 5-28) or
(D-Trp6) GnRH (for example, see the following document: Chaffau)
x, et al., Recueil de Medicine Veterinaire (1
985)161: 133-145); the first, sixth and / or tenth normal
Having an amino acid substituted with an existing Cys and / or N-terminally acylated
GnRH analogs that are amidated and / or amidated at the C-terminus (eg,
See U.S. Pat. No. 4,608,251 and U.S. Pat.
No. 4,975,420); GnRH analog pyroGlu-His-Trp
-Ser-Tyr-X-Leu-Arg-Pro-Gly-YZ (SEQ ID NO: _
Where X is a Gly or D-amino acid and Y is the same or different
One or more amino acid residues, preferably 1 to 3 Gly residues
And Z is Cys or Tyr (UK Patent Publication No. GB21
No. 96969); the GnRH solution described in US Pat. No. 5,688,506.
Narrow, GnRH analog Cys-Pro-Pro-Pro-Pro-Se
r-Ser-Glu-His-Trp-Ser-Tyr-Gly-Leu-Ar
g-Pro-Gly (SEQ ID NO: _), pyroGlu-His-Trp-Ser
-Tyr-Gly-Leu-Arg-Pro-Gly-Ser-Ser-Pro
-Pro-Pro-Pro-Cys (SEQ ID NO: _), pyroGlu-His-
Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Arg-Pro-
Deslorel, including Pro-Pro-Pro-Cys (SEQ ID NO_).
in, commercially available from Apeptech, Australia
And Ovplant^TMGnRH analog known as;
And additions, substitutions and / or deletions of other amino acids and naturally occurring G
A molecule that retains the ability to induce the formation of antibodies that cross-react with nRH. Thus, the term "GnRH polypeptide" refers to one or more amino acids.
Differs from the reference sequence by having substitutions, deletions and / or additions and is referenced
Identity to the molecule of at least about 50 amino acids, more preferably the natural
About 75-85% identity to the relevant part of the oligonucleotide sequence, most preferably
Or GnRH molecules having about 90-95% identity. Amino acid sequence
Has about 1 to 5 amino acid substitutions, or about 1 to 3 amino acid substitutions.
There will be. Particularly preferred substitutions will generally be conserved, ie,
It may be a substitution within the amino acid range of Millie. In this regard, one amino acid
It is generally divided into four families: (1) acid--aspartate and
Lutemate; (2) basic--lysine, arginine, histidine; (3) non-polar
Sex-alanine, valine, leucine, isoleucine, proline, phenylalanine
And methionine, tryptophan; and (4) uncharged polar--glycine,
Paragine, glutamine, cysteine, serine, threonine, tyrosine. Phenyl
Alanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids
Is done. For example, an isolated substitution of leucine by isoleucine or valine,
Or vice versa; replacement of aspartate by glutamate or vice versa;
Substitution of leonine by serine or vice versa; or structurally related amino acids
Similar conservative substitutions with amino acids have no major effect on activity.
Reasonably predictable. Has substantially the same amino acid sequence as the reference molecule,
Proteins with small amino acid substitutions that retain the desired activity are therefore
Within the definition of a GnRH polypeptide. A “GnRH polypeptide” can also be used as long as the molecule retains the desired activity.
Includes peptide fragments of the reference GnRH molecule. In addition, episodes of GnRH
Loops are captured by definition. A multimer of GnRH containing a repeat sequence of a GnRH polypeptide, eg, one or more.
Or more, 2, 4, 8, 16, 35 copies of a GnRH polypeptide and more.
Multimers, optionally including a spacer sequence, e.g., international publications
No. It is described in WO 98/06848 and WO 96/24675 and
The one shown in FIG. 2B is particularly contemplated in the present invention. like this
Various multimers are described more fully below. For the purposes of the present invention, a GnRH polypeptide may be any of the various known Gs described above.
It can be derived from nRH sequences and includes, but is not limited to: human
, Cow, pig, sheep, dog, cat, deer subject, rodent, eg, hamster
-, Guinea pig, gerbil, aardvark, gopher, rabbit (lagomo)
rphs), rabbits, ferrets, squirrels, reptiles and birds
A GnRH polypeptide derived from a subject. A “GnRH peptide” has a length that is less than the full length of the reference GnRH molecule in question.
And at least one epitope as defined below,
The GnRH polypeptide described. Thus, including the GnRH peptide
The resulting vaccine composition comprises a portion of the full-length molecule, but the entire GnRH molecule in question.
Will not be included. Particular GnRH peptides for use in the present invention are
GnRH peptides having 5, 6 or 7 amino acids, especially the amino terminus
Or a peptide containing a carboxy terminus, such as amino acids 1-5, 1
Gn including ６6, 1-7, 2-8, 3-8, 3-10, 4-10 and 5-10
RH peptide (see, for example, International Publication No. WO88 / 05308).
). “GnRH multimer” refers to a selected GnRH polypeptide, GnRH immunity,
Molecule having two or more copies of the original, GnRH peptide or epitope, or
Is a selected GnRH polypeptide, GnRH immunogen, GnRH peptide or
It means a multimeric tandem repeat of the pitope. The GnRH multimer has the general formula (Gn
RH-X-GnRH) y, which can correspond to a molecule having a repeating unit
nRH is a GnRH polypeptide, X is a peptide bond, an amino acid spacer
One or more selected from the group consisting of groups, carrier molecules 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
"H" can comprise any GnRH polypeptide. Therefore, y
Is 1 to 40 or more repeating units, more preferably 1 to 30 repeating units, most
Also preferably, 1 to 20 repeating units can be defined. In addition, the selected GnRH
All of the sequences can be identical or different derivatives of GnRH,
Can correspond to analogs, variants or epitopes, provided that
Retains the ability to elicit an immune response. Furthermore, conversion of GnRH units to carriers
When chemically or recombinantly linked, the GnRH molecule has a 5'-end, 3'-end
Or the carrier in question can be flanked. Furthermore, G
The nRH multimer can be located at a site internal to the support. GnRH
Multimers are discussed in further detail below. The term "GnRH immunogen" refers to a related immunological carrier, adjuvant or immunological agent.
A GnRH polypeptide, as described above, that elicits an immune response in the absence of a stimulator
Means tide and associates with a carrier molecule, adjuvant or immunostimulator
And / or GnRH content
Incorporation of at least one epitope of a child into a molecule containing multiple repeating units
GnRH polypeptides that can be made immunogenic, or more immunogenic
Means tide. This term refers to an individual macromolecule or a highly antigenic substance derived from GnRH.
It can be used to mean a homogeneous or heterogeneous population of molecules. Generally, GnRH immunogens are derived from the native vertebrate species to which such immunogens are delivered.
Elicit antibodies that cross-react with endogenous GnRH present in The term "GnRH license
"Primogen" is also administered using the nucleic acid delivery technique described further below.
Sometimes encoding a GnRH polypeptide that can be expressed in vivo
Nucleic acid molecules, such as DNA and RNA molecules. "Homology" is the identity between two nucleotides or two polypeptide moieties
Means percentage. Two DNA sequences or two polypeptide sequences have the sequence
At least about 75% to 85%, preferably less, over a defined length of the molecule.
At least about 90%, most preferably at least about 95% to 98% sequence identity;
When indicated, they are "substantially homologous" to each other. As used herein,
Substantially homologous also refers to a specified DNA or polynucleotide sequence.
Mean sequences that show complete identity. The "identity" percentage between two amino acid or polynucleotide sequences is
Align the columns, count the exact number of matches between the two aligned sequences, and
By dividing by the length of the sequence and multiplying the obtained value by 100,
It can be determined by direct comparison of the sequence information. Easily available
Facilitate the analysis using a simple computer program, for example:
ALIGN, Dayhoff, M.E. O. , Atlas of Pr
protein Sequence and Structure, M.E. O. Day
Hoff, 5 Suppl.3: 353-358, National Res
ear Foundation, Washington, D.C. C. This is Smith
And Waterman (1981) Advance in Appl. Math
.2: 482-489 local homology algorithm adopted for peptide analysis
I do. Programs for determining nucleotide sequence identity, eg, BESTFI
T, FASTA and GAP programs (which also include Smith and Wa
terman's algorithm) is a Wisconsin sequence analysis package (
Wisconsin Sequence Analysis Package)
, Version 8 (Genetics Computer Group, Wisco
(Madison, N.C.). these
The program is recommended by the manufacturer and is described in the Wisconsin Sequence Analysis Package above.
Cage (Wisconsin Sequence Analysis Pack)
It is easily used with the default parameters described in (age).
For example, the percentage identity of a particular nucleotide sequence to a reference sequence can be expressed as Smit
h and Waterman homology algorithms with default score tables and
Decision to use with gap penalty at 6 nucleotide positions
it can. "Epitope" refers to the ability to elicit and bind to a GnRH-specific antibody or
It refers to any part or region of a molecule having potential. For the purpose of the present invention
Thus, the epitope of the polypeptide is at least about 3 amino acids of the reference molecule, preferably
Or at least about 5 amino acids. For fragment length
There is no critical upper limit, and fragments can be protein sequences or the protein in question.
The fusion protein comprising two or more epitopes of the same quality.
It can be. GnRH is a very small molecule and can elicit an antibody response
Identification of that epitope is accomplished using techniques well known in the art.
Can be easily achieved. For example, an epitope in a polypeptide molecule may be
Using multiple epitope mapping techniques well known in the art
Can be identified. For example, see the following document: Epitope M
applying Protocols in Methods in Molec
ullar Biology, Vol. 66 (Glenn E. Morris ed.)
, 1996) Humana Press, Totowa, NJ. For example,
Linear epitopes can be used, for example, to simultaneously synthesize multiple peptides on a solid support,
Where the peptide corresponds to a part of the protein molecule and the peptide is still bound to the support
By reacting the peptide with the antibody while
. Such techniques are known in the art and are described, for example, in
No. 4,708,871; Geysen et al. (198).
4) Proc. Natl. Acad. Sci. USA81: 3998-400
2: Geysen et al. (1986) Molec. Immunol.23: 709-
715. Similarly, a conformational epitope is a spatial conformation of amino acids.
By determining the conformation, for example, X-ray crystallography and two-dimensional nuclear magnetic
It is easily identified by resonance. For example, Epitope Mapping Pr
See autocols, supra. In addition, protein amino acids utilizing the hydrophobicity and hydrophilicity of each of the 20 amino acids are used.
Computer program for formulating hydropathic scales from amino acid sequences (
See, for example, Kyte et al. Mol. Biol. (1982)157: 105-1
32; and Hopp and Woods, Proc. Natl. Acad. Sc
i. USA (1981)78: 3824-3828) to obtain the desired molecule.
Can determine the antigenic portion. For example, the techniques of Hopp and Woods
The technique assigns each amino acid to a number of hydrophilicity values, and then
The values are averaged repeatedly. Highest local average hydrophilicity point indicates antigenic portion of molecule
You. An "immunological carrier" is one that, when associated with the GnRH immunogen in question,
Any molecule that imparts immunogenicity to the offspring or enhances the immunogenicity of the molecule.
means. Examples of suitable carriers are: large, slowly metabolizing macromolecules
Eg, proteins; polysaccharides, eg, Sepharose, agarose, cellulose
, Cellulose beads and others; polymeric amino acids, such as polyglutamids
Acid, polystyrene, and others; amino acid copolymers; inactivated virus particles
Offspring; bacterial toxins such as diphtheria, tetanus, cholera, leukotoxin
Toxoids from molecules, and others. Carriers are described further below. The GnRH immunogen is chemically coupled to a carrier or associated therewith.
Chemical DNA molecules that encode the immunogen and the carrier in question
GnRH immunogen is "bound" to a specific carrier molecule when the immunogen is expressed from
You. An “immune complex” is a Gn bound to a carrier molecule, as defined above.
An RH immunogen, for example, a GnRH peptide or multimer. The terms "leukotoxin polypeptide" or "LKT polypeptide"
Consensus amino acid sequence Gly-Gly-X-Gly-X-As of boxy end
A family of molecules characterized by p (Highlander et al. (1989)
) DNA8: Intended for polypeptides derived from proteins belonging to 15-28)
Where X is Lys, Asp, Val or Asn. Such a tampa
The substances include, inter alia, the following: Pasteurella haemolytica (P.ha)
emolitica) and Actinobacillus priperneumoniae (Act)
Inobacillus pleuropneumoniae
Cotoxin, as well as E. coli alpha hemolysin (Strat)
hdee et al. (1987) Infect. Immun.55: 3233-3236
Lo (1990) Can. J. Vet. Res.54: S33-S35; We
Ich (1991) Mol. Microbiol.5: 521-528). Ibis
This family of syn is known as the "RTX" family of toxins (
Lo (1990) Can. J. Vet. Res.54: S33-S35). Further
In addition, the term “leukotoxin polypeptide” is chemically synthesized and expressed
Leukotoxin polypeptide isolated or recombinantly produced from a living organism
Means tide. In addition, the term is found within certain natural leukotoxin molecules.
Having an amino acid sequence substantially homologous to the adjacent amino acid sequence
Means a sex protein. Thus, the term covers both full-length and partial sequences
, As well as analogs. Natural full-length leukotoxin is cytotoxic
However, the term "leukotoxin" also limits its immunogenicity and
A molecule that lacks the cytotoxic properties of leukotoxin. The nucleotide sequences and corresponding amino acid sequences of some leukotoxins are
Is known. See, for example, US Pat. No. 4,957,739.
No. 5,055,400; Lo et al. (1985) Infect.
Immun.50: 667-67; Lo et al. (1987) Infect. Immu
n.55: 1987-1996; Strathdee et al. (1987) Infec.
t. Immun.55: 3233-3236; Highlander et al. (198
9) DNA8: 15-28; and Welch (1991) Mol. Micr
obiol.5: 521-528. In a preferred embodiment of the present invention,
Syn chimeras are provided, wherein these chimeras have one or more fused thereto.
Selected, which confer enhanced immunogenicity to the above GnRH multimers
It has a leukotoxin polypeptide sequence. Specific examples of immunogenic leukotoxin polypeptides for use in the present invention include:
Described in U.S. Patent No. 5,476,657 and U.S. Patent No. 5,837,268
Is a truncated leukotoxin molecule. These truncated molecules
Include LKT 352, LKT 111 and LKT 114. LKT 352 is plasmid pAA352 (ATCC Accession No. 68283).
From the 1 ktA gene present in The nucleotide sequence and
And corresponding amino acid sequences are described in U.S. Patent No. 5,476,657.
This gene has 914 amino acids and an estimated molecular weight of about 99 kDa
Encodes a truncated leukotoxin. LKT 111 is the plasmid pC
1 ktA remains in B111 (ATCC Accession No. 69748)
It is a leukotoxin polypeptide derived from a gene. The nucleotide of this gene
The sequence and the corresponding amino acid sequence are described in US Pat. No. 5,837,268.
ing. This gene encodes an internal DNA fragment approximately 1300 bp in length.
By removal, plasmid pAA352 (ATCC Accession No. 68)
283), which is generated from the recombinant leukotoxin gene present in
It is a shortened version of Shin. LKT 111 polypeptide is a putative 52 kDa protein
Has a determined molecular weight (compared to a 99 kDa LKT 352 polypeptide)
Is a LKT3 containing T cell epitopes required for sufficient T cell immunogenicity
52 N-terminal portion and for the production of the fusion protein used in the present invention
Retain the C-terminal portion of LKT 352 containing the usual restriction sites to use.
LKT114 is derived from a gene present in plasmid pAA114 (US
No. 5,837,268). LKT 114 has the additional amino acids deleted from an internal portion of the LKT 111 molecule by:
Different from LKT111. "Immunological adjuvant" is a non-specific method of measuring the immune response to a specific antigen.
The amount of antigen required in any given vaccine
And / or generate an appropriate immune response against the antigen in question
Is a factor that reduces the frequency of injections required for For example, see the following document.
And: A. C. Allison, J.M. Reticuloendothel. Soc
. (1979)26: 619-630. A “native” protein, polypeptide or peptide is a protein in which the protein is found in nature.
A protein, polypeptide or peptide isolated from an existing source. "set
A "recombinant" polypeptide has been produced by recombinant DNA technology;
Produced from cells transformed with an exogenous DNA construct encoding a polypeptide
Means a polypeptide produced. "Synthetic" polypeptides are synthesized by chemical synthesis.
It is manufactured. "Polynucleotide" refers to RNA, for example, mRNA, cDNA, genomic D
Nucleic acids, including but not limited to NA sequences and further synthetic DNA sequences
Means the sequence of leotide. The term also refers to known salts of DNA and RNA.
A sequence encompassing the base analog is meant. The term "derived from", as used herein, refers to the subject molecule or
Represents the actual or theoretical source or origin of the immunogen. For example, a specific GnRH molecule
An immunogen "derived from" supports contiguous sequences similar to the relevant portion of the reference molecule
Will be. Thus, an immunogen "derived from" a particular GnRH molecule is a wild-type G
Can include all of the nRH sequences or can be derived from the target
Insertion of amino acid residues, so long as it provides an immunogen corresponding to the Ted GnRH molecule;
It can be changed by deletion or substitution. Immunogen derived from the indicated molecule
Contains at least one epitope specific for the indicated molecule.
There will be. "Food producing animal" refers to a human or domestic pet, such as a cat or dog.
Means an animal intended for consumption by. Such animals include, without limitation,
Includes mammalian, e.g., sheep, cow, pig, and deer subjects,
, Cattle, pigs and deer. "Enhancing the quality of sensory irritation in edible meat
'' Means food from a typical non-castrated member of the same species that is not so treated
Odor, taste and taste of edible meat from animals treated according to the invention compared to meat
And / or to improve fragility. In general, food from males that do not castrate
Animal meat suffers from several disadvantages. In this regard, non-castrated boars and
Edible meat from sheep often has an unpleasant taste and smell. For example,
"Bork rot" is a urine-like odor found in cooked edible meat of non-castrated pigs
Means Boar spoilage is found in tissues in boars with normally functioning testes.
Produced by stored steroids. For example, Brooks et al., Jou
rnal Animal Soc. (1986)62: 1279. Do not castrate
The presence of androstenone in boars is a measure of boar rot.
And is a measure of gonadal steroid production. In addition, skatole is a male
May contribute to pig rot. For example, see the following document: Morte
nsen and Sorensen, Proc. 30th European M
eating of Meat Research Workers, Chen
t Belgium (1986) pp. 394-396, skatole in fat
How to assay for Similarly, bulls that do not castrate have higher muscle mass.
Because of the increase, they often produce lean but tough edible meat. Thus,
Edible meat with improved organoleptic properties has a more desirable odor, fragility and
It is edible meat with a taste and / or taste. "Reduction of circulating testosterone" refers to standard assays, for example, as described herein.
A typical castration of the same age and species, as measured using the RIA described
No, when compared to expected serum testosterone levels in untreated males,
Means a statistically significant decrease in serum testosterone levels. "Androgenic" steroids include androstenone, androstenedione,
Androstenediol and / or testosterone. Androgue
Steroids can be measured by well-known techniques. For example,
Stosterone and other androgenic steroids are well known in the art.
Can be measured using standard ELISA and RIA. Especially convenient
Rigorous measurements are performed using commercially available test kits, such as Coat-A-Count Total
Stosterone kit (Coat-A-Count Total Testest)
eron Kit^TM) (Diagnostic Products Corp)
oration, Los Angeles, CA)
You. This kit contains a testosterone-specific antibody immobilized on a polypropylene tube wall.
Designed for body-based, quantitative measurement of testosterone in serum,
It is a solid phase RIA. Also, the description of direct RIA to measure testosterone level
For the reference see the following documents: Schanbacher and D'Oc
chio, J. et al. Andrology (1982)3: 45-51. An ELISA for measuring androstenone levels is described, for example, in Abouzied
Et al. Agric. Food Chem. (1990)38: 331-335
It is described in. See also the following documents: Meloen et al., Vacc.
ine (1994)12(8): 741-746; and a
Booth et al., Anni, describing ELISA performed on nandrostenone.
m. Prod. (1986)42145-152. Androstenone level
The RIA to measure is also known. For example, Andersen, 0. , Acta
. Vet. Scand. (1979)20: 343-350. “Non-androgenic” steroids are 5α-androstenone (5α-androst)
-16-en-3-one) and 16-androstene derivatives
. Non-androgenic steroids are well known in the art, such as
For example, it can be measured using ELISA and RIA. For example, Cla
us et al., Archiv fuel Lebensmittelhyginene
(1988)38: 87-90. One or more androgenic steroids and / or non-androgens
A “substantially reduced” level of teroid is defined as at least one androgen
Levels of teroids or non-androgenic steroids may be of the same age and species
At least about 50% of the expected level in untreated males who have not been castrated
Low, preferably less than at least about 75%, more preferably at least about 8
Mean 0% to 90% or less. The term “feeding period” refers to the period from weaning to slaughter and, thus,
Before, around and after the period. Typical fattening periods vary from species to species, within one species
Even depending on the preferences of food producing animals and the country in which they are raised.
There will be. Thus, the fattening period is primarily a matter of choice, and those skilled in the art
An appropriate fattening period of the animal can be easily determined.2. General method Before describing the present invention in detail, the present invention has been described in connection with specific recipes or process parameters.
It is to be understood that the invention is not limited to such examples, but can, of course, vary.
Further, the terms used in the present specification are only for describing specific embodiments of the present invention.
It is to be understood that for purposes of limitation, this is not meant to be limiting. A number of compositions similar or equal to those described herein, and
Although methods can be used in the practice of the present invention, preferred materials and methods
Is described herein. The present invention provides edible meat by modulating the secretion of gonadal steroids.
Based on the discovery of ways to improve the quality of This method can be used before the animal fattening period or
One or more primary immunizations with a GnRH formulation during the
Reduces melanin levels measurably but generally does not produce complete castration
It is included. After primary vaccination, use the same or different composition just before sacrifice.
Booster to administer one or more androgenic steroids and / or
Substantially reduces the levels of non-androgenic steroids. GnRH is generally recognized as "self" and is therefore non-immunogenic,
The compositions described herein are surprisingly found in subjects immunized therewith.
To generate an appropriate immunological response. The timing of vaccination depends on the animal in question, and such animals are generally
Sheep, cows or pigs, as well as favorite food producing animals. However,
The first vaccination will be performed before or during the fattening period of the animal. example
For example, in pigs and sheep, primary immunization generally occurs at animal birth and about 15 weeks of age.
And preferably between the birth of the animal and about 10 weeks of age. female
In cattle, primary immunization is generally performed at a time between animal birth and about 48 weeks of age.
It is. One or more booster treatments are given prior to sacrifice. Booster administration
The timing also depends on the animal in question. For example, pigs and sheep
And the booster dose is generally about 1 to about 12 weeks before slaughter, preferably about 2 weeks before slaughter.
About 8 weeks before, most preferably about 4 to about 6 weeks before slaughter, and even about 3 weeks before slaughter
is there. Preferred administration of the second vaccine several months before slaughter in cows
There is. In some embodiments, one or more subsequent immunizations is different from the primary immunization.
After the decrease in the amount of GnRH antibody generated, ie, the maximum antibody level detected
After decreasing to a level below at least about 50% of the
It is performed after reducing to at least about 75% of the antibody level. The vaccine composition of the present invention increases its immunogenicity as defined above.
Use a GnRH polypeptide, optionally linked to a carrier molecule to enhance
You.GnRH immune complex As explained above, GnRH is an endogenous molecule and as such bears GnRH.
By binding to the body to form a GnRH immune complex,
Desirable to increase the immunogenicity of the tide (or multimer described below)
There is. When administering a GnRH immunogen to the same species from which the GnRH immunogen is derived,
This is particularly necessary. Suitable carriers are generally proteins derived from infectious agents, such as viruses.
Polypeptides comprising the antigenic region of a surface protein or the sequence of a carrier peptide
Is. These carriers act to stimulate T helper cell activity non-specifically.
And associates with major histocompatibility complex (MHC) molecules
For processing and presentation at the surface, the immunogen in question is transformed into antigen presenting cells (A
PC). Several carrier systems have been developed for this purpose. For example, a small pen
The peptide hapten is a protein carrier, such as keyhole limpet hemocyanin
(Bittle et al. (1982) Nature298: 30-33), bacterial ibis
Syn, for example, tetanus toxoid (Muller et al. (1982) Proc.
tl. Acad. Sci. U. S. A.79: 569-573), ovalbumin
, Leukotoxin polypeptides, and sperm whale myoglobin
Coupling produces an immune response. Typically, these couplings
The reaction results in the incorporation of several moles of peptide hapten / mole of carrier protein. Other suitable carriers for use with the present invention are described in US Pat. No. 5,071,
No. 651, the VP6 polypeptide of rotavirus, or
Include their functional fragments. In addition, viral proteins and GnRH
Fusion products with one or more epitopes are useful and these fusion products
The article is made by the method disclosed in U.S. Pat. No. 4,722,840. What
Other suitable carriers include cells, for example, lymphocytes. Because of this form
Presentation mimics the natural presentation mode in the subject, resulting in an immunized state
This is because that. Alternatively, the GnRH immunogen is converted to red blood cells, preferably the subject's own red blood cells.
It can be coupled to blood cells. Transfer peptide to protein or cell
Methods for pulling are known in the art. Delivery systems useful in the practice of the present invention also employ particulate carriers. For example,
Preformed particles allow the immunogen to be coupled and incorporated thereon
It has been used as a platform. Proteosome-based systems
(Lowell et al. (1988) Science240: 800-802) and
And immunostimulatory complexes (Morein et al. (1984) Nature308: 457
-460) are also known in the art. We also use recombinantly produced chimeric proteins that self-assemble into particles.
A carrier system can be used in the present invention. For example, yeast retrotrans
Poson Ty encodes a series of proteins that assemble like viruses (Ty
-VLP; Kingsman et al. (1988) Vaccine.6: 304-30
6). Thus, the gene encoding the GnRH immunogen in question, or its flag
Insert into the TyA gene and express it as a fusion protein in yeast
be able to. This fusion protein has the ability to self-assemble into uniformly sized particles
Hold. Other useful virus-like carrier systems are based on: HBsA
g (Valenzuela et al. (1985) Bio / Technol.3: 323
-326; U.S. Pat. No. 4,722,840; Delpeyroux et al.
6) Science233: 472-475), hepatitis B core antigen (Clar
ke et al. (1988) Vaccine 88 (editor: H. Ginsberg et al.)
pp. 127-131), poliovirus (Burke et al. (1988) Natu).
re332: 81-82), and tobacco mosaic virus (Haynes et al.)
(1986) Bio / Technol.4: 637-641). Particularly preferred carriers are serum albumin, keyhole limpet as described above.
Hemocyanin, ovalbumin, sperm whale myoglobin, leukotoxin
And other proteins well known to those skilled in the art. For example, above
Chimeric systems using leukotoxin polypeptides, as defined in
For example, Pasteurella haemolytica (Pasteurella) fused with the antigen of interest.
 haemlytica) leukotoxin (LKT) polypeptide
Can also be. In this regard, the nucleotide sequences of some leukotoxin carriers
And the corresponding amino acid sequence are known. For example, US Pat. No. 5,422,1
10, US Patent No. 5,708,155, US Patent No. 5,723,129 and
And International Publication No. See WO 98/24675. For use in the present invention
Specific examples of immunogenic leukotoxin polypeptides of the invention include LKT 342, LKT 352, LKT 111, LKT 326 and LKT 101.
These are described in the patents and publications cited above. LKT 111 and
LKT 114 is particularly preferred. The gene encoding LKT111 is pAA
352 (ATCC Accession No. 68283)
Removal of internal DNA fragments of approximately 1300 bp in length from toxins
Caused by The LKT 111 polypeptide is a putative molecule of 52 kDa.
Amount (compared to a 99 kDa LKT 352 polypeptide) but sufficient
N-terminal of LKT 352 containing T cell epitopes required for T cell immunogenicity
And convenient restriction sites used to produce the fusion proteins of the invention.
Retain the C-terminal part of LKT 352 containing. LKT 114 is a molecule
Differs from LKT111 due to additional amino acid sequence deletions from parts of the region. this
For a description of these molecules, see, for example, US Pat. No. 5,837,268 and
Published No. See WO98 / 06848 and WO96 / 24675.
When. Protein carriers can be used in their natural form or
The group content can be determined, for example, by succinylation of lysine residues or with Cys-thiolactone.
It can be modified by the reaction. Sulfhydryl groups are, for example,
Functional group is 2-iminothiolane or 3- (4-dithiopyridylpropionate)
By reacting with N-hydroxysuccinimide ester of
Or antigen). Suitable carriers also include a peptide immunogen.
A spacer arm (e.g., hexamethylenediamine or
Other bifunctional molecules of similar size) can be modified to incorporate them. The carrier is physically attached to the GnRH immunogen in question using standard coupling reactions.
Can be compounded. Alternatively, for example, encoding a suitable polypeptide carrier
Is selected from the group consisting of one or more genes encoding the selected GnRH immunogen.
By fusing to a copy of the fragment or a fragment thereof.
Chimeric molecules can be produced recombinantly for use in GnRH
Is fused 5 'or 3' to the carrier portion of the molecule, or G
A portion of the nRH can be located at a site internal to the carrier molecule. In addition, administration of a GnRH immunogen via a carrier virus expressing the GnRH immunogen
can do. The carrier virus used in the present invention is vaccinia and
And other poxviruses, adenoviruses, and herpes viruses
However, it is not limited to these. One example is vaccinia wis, which expresses proteins.
Rus recombinants can be constructed as follows. First, a specific protein
DNA adjacent to the vaccinia promoter and the vaccinia DNA sequence
Sequence, eg, flanking the sequence encoding thymidine kinase (TK).
, And insert it into the appropriate vector. The cells are then used to
Transfect and simultaneously infect these cells with vaccinia
. Homologous recombination involves the vaccinia promoter and the gene encoding the desired immunogen.
And insert it into the viral genome. Cells are treated with 5-bromodeoxyuri
Grow in the presence of gin and pick up viral plaques resistant to it
Can select the resulting TK-recombinant.GnRH multimer It also produces immunogenic forms of molecules comprising multiple copies of the selected epitope.
Can significantly increase the immunogenicity of GnRH immunogens
. In this way, endogenous radiation hybrids can be used as effective autoantigens.
can do. Thus, in one aspect of the invention, a multimer of the GnRH immunogen is contained.
The resulting vaccine composition is provided in the form of a nucleic acid or peptide. GnRH Multi
The mer can be selected from a selected GnRH immunogen, peptide or epitope as described above.
More than one copy of the selected GnRH immunogen, peptide or epitome
Will have multiple tandem repeats of loops. Thus, the GnRH multimer was selected
Multiple or tandem repeats of GnRH sequence, multiple or multiplex of selected GnRH epitopes
Can comprise tandem repeats, or any possible combination thereof
. GnRH epitopes can be identified using the techniques detailed above.
Can be. For example, the GnRH multimer is a repeating unit of the general formula (GnRH-X-GnRH) y
Wherein GnRH is a GnRH immunogen;
X represents a peptide bond, an amino acid spacer group, a carrier molecule and [GnRH] n
One or more molecules selected from the group consisting of: where n is greater than 1
Or an integer equal to it, and y is greater than 1 or equal to it
And "GnRH" should not include any GnRH immunogens.
Can be Thus, the GnRH multimer has 2 to 64 or more G
nRH immunogen, more preferably containing 2 to 32 or 2 to 16 GnRH immunogens
can do. Furthermore, can all of the selected GnRH immunogenic sequences be identical,
Alternatively, different derivatives of GnRH, as long as they retain the ability to elicit an immune response,
It can correspond to an analog, variant or epitope. Furthermore, GnR
When the H immunogen is chemically or recombinantly linked to a carrier,
The origin may be attached to the 5'-end, 3'-end or flank the carrier in question
Can be In addition, the GnRH multimer is located at an internal site for loading.
can do. One for use with the GGnRH multimers of the invention
One particular carrier is the leukotoxin polypeptide described above. As explained above, a spacer sequence can exist between GnRH molecules.
You. For example, Ser-Gly-Ser trimer and Gly-Ser dimer are described in the present invention.
Among the GnRH multimers exemplified in the textbook, these are GnRH immunogens.
Provides a spacer between the native repeats. See, for example, FIG. 2B. Selected Gn
Strategic placement of various spacers between the RH immunogens provides immunity to the subject constructs.
Epidemiology can be increased. Therefore, in the present invention, the selected
Protein sequences can include a wide variety of components, such as a single amino acid linker or two
~ Can encode a sequence of multiple amino acids. The chosen spacer group is good
Preferably, the multimer expressed is i
Proteolytic enzymes (APC or other) in vivo
Is processed into a number of polypeptides, each of which is derived from a carrier moiety
Contains at least one T cell epitope, and preferably is substantially complete
Fused to GnRH polypeptide sequence. The binding region between the selected GnRH moieties is distinctly foreign to the immunized subject
Which enhances the associated GnRH immunogen
Spacer groups can be constructed to confer enhanced immunogenicity. Further
In addition, spacer sequences can be constructed to provide T cell antigenicity.
Examples of such spacer sequences provide epitopes for immunogenic helper T cells
Amphiphilic and / or α-
Sequence encoding a helical peptide sequence. With such a spacer sequence
The selection of a particular T cell epitope to be provided depends on the specific
May vary depending on the vertebrate species. Specific Gn containing spacer sequence
The RH moiety is exemplified, but also comprises one directly adjacent GnRH sequence.
Or more GnRH multimers (without intervening spacer sequences)
It is also an object of the present invention. The GnRH multimer sequences thus produced are used in the compositions of the invention.
Make the GnRH antigen highly immunogenic. GnRH polypeptides, immune complexes and multimers use the methods described below.
And nucleic acid immunization, gene therapy, protein based
Immunization methods, and others.Nucleic acid-based immunization methods Generally, the nucleic acid-based vaccines used in the present invention are GnRH-immunized.
Includes relevant regions encoding the epidermis, appropriate control sequences, and
Depending on the adjunctive therapy, it will have a nucleotide sequence. Nucleic acid molecules
Vector forms containing necessary elements that direct transcription and translation in Pient cells
Manufactured in a state. To enhance the immune response in the immunized subject, the nucleic acid molecule is an auxiliary substance, e.g.,
For example, in combination with pharmacological agents, adjuvants, or altered biological response.
Delivery and combination of vectors encoding additional factors such as cytokines and others
They can be administered together. Other auxiliary substances include, but are not limited to:
Without limitation: substances that increase weight gain, muscle mass or muscle strength, such as
Growth hormones, growth promoting factors, beta antagonists, partitioning factors and antibiotics
material. The nucleotide sequence selected for use in the present invention may be from a known source,
For example, cells containing the desired gene or nucleotide sequence according to standard techniques.
Or isolating them from tissues or deriving them through recombinant or synthetic techniques
can do. Once the coding sequence for the GnRH immunogen has been produced or isolated,
Such a sequence can be cloned into any suitable vector or replicon.
Can be. Many cloning vectors are known in the art, and
The selection of an appropriate cloning vector is then a matter of choice. Other arrays, for example,
The attachment to the auxiliary or carrier molecule can be achieved by techniques known in the art.
Is executed. The one or more GnRH immunogenic portions of the chimera
It can be fused 5 'and / or 3' to the sequence or carrier molecule.
Alternatively, one or more GnRH immunogen moieties are internalized with respect to the carrier molecule.
Or such a portion can be located at the end of the chimera or
It can be located at both locations inside. Alternatively, the DNA sequence encoding the GnRH immunogen in question is cloned.
Rather, it can be produced synthetically. Use appropriate codons for specific sequences
Thus, a DNA sequence can be designed. The complete sequence of the immunogen is then
Assembled from overlapping oligonucleotides produced by the
Build the complete coding sequence. For example, see the following document: Edge
(1981) Nature292: 756; ambair et al. (1984) Sc
ence223: 1299; and Jay et al. Biol. C
hem.259: 6311. Then, an appropriate control factor for in vivo expression in an appropriate host tissue.
Place the coding sequence under the child's control. The choice of control factor
Will depend on the subject to be treated and the type of preparation used. Thus,
If the immunogen is expressed using the sample's endogenous transcription and translation mechanisms, certain
A control factor compatible with the analyte will be used. In this regard
Thus, some promoters for use in mammalian systems are known in the art.
Have been. For example, a typical promoter for mammalian cell expression is
Still, include: SV40 early promoter, CMV promoter
-For example, CMV immediate-early promoter, mouse mammary tumor virus LTR promoter
The major late promoter of the adenovirus (Ad MLP), and
Pure herpesvirus promoter. Also, other non-viral promoters, even if
For example, the promoter derived from the murine metallothionein gene is
Will be used for Typically, transcription termination and polyadenylation sequences are also present, resulting in translation termination
It will be located 3 'to the codon. 5 'to coding sequence
Preferably, there are also sequences that optimize translation initiation. Transcription terminator
An example of a promoter / polyadenylation signal is given in Sambrook et al., Supra.
SV40-derived as well as bovine growth hormone
Includes a minator sequence. In addition, a splice donor site and an acceptor
Designing introns containing position in the construct used in the present invention
Can be. Also, enhancer elements may be used in the present invention to increase expression levels of the construct.
Can be added. Examples of these are as follows: SV40 early gene engine
Hanser (Dijkema et al. (1985) EMBO J. Clin.4: 761), Rous
Enhancer / promoter from sarcoma virus long terminal repeat (LTR)
(Gorman et al. (1982) Proc. Natl. Acad. Sci. USA.79: 6777) and a factor derived from human CMV (Boshart et al. (19).
85) Cell41: 521), for example, included in the CMV intron A sequence.
Factor. Once manufactured, the nucleic acid vaccine composition is delivered to the subject according to known methods.
Can be In this regard, various techniques using DNA encoding the antigen
Has been described. See, for example, US Pat.
9,466 (Felgner et al.); Tang et al. (1992) Nature.3
58: 152; Davis et al. (1993) Hum. Mol. Genet.2: 1
847; Ulmer et al. (1993) Science258: 1745; Wan
g et al. (1993) Proc. Notl. Acad. Sci. USA90: 41
56; Eisenbraun et al. (1993) DNA Cell Biol.12 : 791; Fynan et al. (1993) Proc. Notl. Acad. Sci.
USA90: 12476; Fuller et al. (1994) AIDS Res. H
um. Retrovir.10: 1433; and Raz et al. (1994) Pro.
c. Notl. Acad. Sci. USA91: 9519. In the continuing host
General method for delivering nucleic acid molecules into cells in vitro for reintroduction into cells
For example, liposome-mediated gene transfer can also be performed. For example, the following document
See Hazinski et al. (1991) Am. J. Respir. Ce
11 Mol. Biol.4: 206-209; Brightham et al. (1989).
Am. J. Med. Sci.298: 278-281; Canonico et al. (1
991) Clin. Res.39: 219A; and Nabel et al. (1990)
Science249: 1285-1288. Thus, nucleic acid vaccine compositions
Can be delivered in liquid or particulate form according to various known techniques. Scripture
A typical vaccine composition is described more fully below.Protein-based delivery method Protein-based compositions may also include species known in the art.
It can be manufactured according to various methods. In particular, GnRH is produced by standard purification techniques.
Polypeptides can be isolated directly from natural sources. Alternatively, in this area
And are well known, for example, as described in Sambrook et al., Supra.
Polypeptides can be produced recombinantly using nucleic acid expression systems
. Also, using chemical polymer synthesis, eg, solid phase peptide synthesis, GnRH
Polypeptides can also be synthesized. Such methods are known in the art.
Have been. For solid phase peptide synthesis techniques, see, for example, the following documents:
J. M. Stewart and J.M. D. Young, Solid Phase
Peptide Synthesis, 2nd edition, Pierce Chemica
l Co. Rockford, Illinois (1984); Barany O
And R. B. Merrifield, The Peptides: Analys
is, Synthesis, Biology, E .; Gross and J.M. Mei
Enhofer, Vol. 2. Academic Press, New Yo
rk, (1980), pp. 3-254. GnRH polypeptides for use in the compositions described herein also include
Cloning the coding sequence into any suitable expression vector or replicon.
Can be manufactured by cleaning. Many cloning vectors
Are known in the art, and the selection of a suitable cloning vector
It is a matter of choice. DNA vectors for cloning and their transformation
Examples of interchangeable host cells are as follows: bacteriophage lambda (E.co.
li), pBR322 (E. coli), pACYC177 (E. coli),
pKT320 (Gram negative bacteria), pGV1106 (Gram negative bacteria), pLA
HR1 (Gram negative bacteria), pME290 (Non-E. Coli Gram negative bacteria),
pHV14 (E. coli and Bacillus subtilis), pB
D9 (Bacillus), pIJ61 (Streptomyces), pUC
6 (Streptomyces), YIp5 (Saccharomyces),
YCp19 (Saccharomyces) and bovine papilloma virus (mammalian
Cells). See generally, the following references: DNA Cloning: Vol.
. I and II, supra; Sambrook et al., Supra; Perb
al, supra. For example, the coding sequences of pig, cow and sheep GnRH have been determined (M
urad et al. (1980) Hormones and Hormone Anta
gonists,The Pharmacological Basis of
Therapeutics, 6th edition), and the cDNA of human GnRH
The sequence is well established (Seeburg et al. (198)
4) Nature311: 666-668). Additional GnRH ports of known sequence
GnRH molecules present in polypeptides such as salmon and chicken are disclosed.
(International Publication No. WO86 / 07383, issued on December 18, 1986)
. The GnRH coding sequence used in the present invention is referred to herein as the second
This is shown in FIG. A and FIG. 2B. GnRH coding sequences are vertebrate,
Are highly conserved in mammals, and include pigs, cows, sheep and
GnRH sequences are identical to each other. The portion of these sequences that encode the desired GnRH polypeptide, and
Accordingly, sequences encoding carrier proteins are generally known in the art.
It can be cloned, isolated and ligated according to some recombinant techniques. For example
See, Sambrook et al., Supra. The gene contains a promoter, a ribosome binding site (for bacterial expression) and
If necessary, it is located under operator control,
The NA sequence is translated into RNA by a suitable transformant. The coding sequence is
With or without a signal peptide or leader sequence
Wear. The polypeptide may be, for example, an E. coli tac promoter or the like.
Or using a protein A gene (spa) promoter and signal sequence.
Can be manifested. Leader sequence is a bacterial host in post-translational processing
Can be removed. For example, US Pat. No. 4,431,739;
See U.S. Patent No. 4,425,437; U.S. Patent No. 4,338,397. Also,
Auxiliary sequences, such as those described above, can be present. Expression of polypeptide sequences with respect to growth of the host cell in addition to control sequences
It may be desirable to add a regulatory sequence that allows for the regulation of The regulatory sequence is
And examples thereof include the presence of a modulating compound.
Turn gene expression on or off in response to chemical or physical stimuli
Regulatory sequences. Also, other types of regulatory elements, such as enhancer sequences, may be used.
Can be present in the reactor. Specific coding sequences are located in the vector along with the appropriate regulatory sequences.
Thus, the expression vector was constructed as follows,
The positioning and orientation of the coding sequence are determined by the coding sequence of the control sequence.
Control sequence) (ie, the control sequence
RNA polymerase that binds to DNA molecules transcribes the coding sequence in
). To this end, a sequence encoding a particular GnRH polypeptide
Is desirable. For example, in some cases, the array is
So that the reading frame can be linked to the
It may be necessary to modify the sequence to carry it. Vector, for example
Before inserting into the coding vector described above,
Can be linked to a coding sequence. Or the control
In an expression vector that already contains the coding sequence and appropriate restriction sites,
Can be cloned directly. In some cases, sequences that secrete the polypeptide from the host organism are added and
It may be desirable to subsequently cleave the secretion signal. Also, polypepti
It may be desirable to produce mutants or analogs of the drug. Reference port
The portion of the sequence encoding the repeptide, or, if present, the desired carrier molecule.
By deleting parts of the coding sequence, by inserting the sequence
And / or by replacing one or more nucleotides in the sequence.
Thus, mutants or analogs can be produced. Nucleotide sequence
Techniques for modifying, for example, site-directed mutagenesis, and others are known to those of skill in the art.
Well known. See, for example, the following document: Sambrook et al., Su
pr; DNA Cloning, Vol. I and II, supra; Nuc
leic Acid Hybridization, supra; Kunkel
, T .; A. Proc. Notl. Acad. Sci. USA (1985)82 : 448; Geisselsider et al., BioTechniques (198
7)5: 786; Zoller and Smith, Methods Enzym.
ol. (1983)100: 468; Dalbie-McFarland et al., P.
rc. Natl. Acad. Sci. USA (1982)79: 6409. GnRH polypeptides can be expressed in a wide variety of systems;
Such systems include insect, mammalian, bacterial, viral and yeast expression systems, and include all.
Are well known in the art. For example, an insect cell expression system, for example,
Baculovirus expression systems are known in the art and include, for example,
Ummers and Smith, Texas Agricultural Ex
period Station Bulletin No. 1555 (19
87). Materials and Materials for Expression Systems of Baculovirus Insect Cells
And methods are, among others, Invitrogen, California
Commercially available in kit form (“MaxBac”) from San Diego, Calif.
is there. Similarly, bacterial and mammalian cell expression systems are well known in the art.
And are described, for example, in Sambrook et al., Supra. Yeast
Maternal expression systems are also known in the art, and include, for example, Yeas
t Genetic Engineering (Barr et al., Ed., 1989)
Ndon, Batterworth. Also, many suitable host cells for use with the above systems are known.
For example, mammalian cell lines are known in the art and
・ Type Culture Collection (American Type Cult)
ure collection (ATCC)
Cell lines, including, but not limited to:
Chinese hamster ovary (CHO) cells, HeLa cells, baby hamsters
-Kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, H
ep G2), Madin-Darby bovine kidney ("MD
BK ') cells, and others. Similarly, bacterial hosts such as E. coli (E. col.
i), Bacillus subtilis, and
Streptococcus species are included in the expression constructs of the present invention.
Will be used in Yeast hosts useful in the present invention are, among others,
Including: Saccharomyces
 cerevisiae), Candida alb
icans), Candida maltosa, Han
Hansenula polymorpha, Kruy
Kluyveromyces fragilis
, Kluyveromyces lactis
), Pichia guilermondiii
), Pichia pastoris, Schizosaccharomi
Seth Pombe (Schizosaccharo myces pombe) and
And Yarrowia lipolytica (Yarrowia lipolytica). Baki
Insect cells for use with the urovirus expression system include, among others:
Including: Aedes aegypti, autograph
Auto californica, Bo
Bombyx mori, Drosophila melanogaster (
Drosophila melanogaster), Shigella flexneri (S)
higella flexneri) and Trichoderma ni (Tricho)
derma ni). Depending on the chosen expression system and host,
Growing the isolated host cells under conditions in which the polypeptide is expressed,
An nRH polypeptide is produced. The expressed polypeptide is then
And purified. If the expression system secretes the polypeptide into the growth medium,
The product can be purified directly from the medium. If not secreted, it is cell lysate
Can be isolated from the site. Those skilled in the art can select appropriate growth conditions and methods for recovery.
Within the skill of A GnRH polypeptide with or without an associated carrier,
Once obtained, the composition, e.g.,
It can be formulated into a vaccine composition as described.Antibody production To generate antibodies for use in passive immunization methods, the subject GnR
H-immunogens can be used. Typically, peptides useful for producing antibodies
Is usually at least about 3-5 amino acids in length, preferably 7-10 amino acids in length
Would. Antibodies to the subject immunogens include polyclonal and monoclonal antibodies
And monospecific antisera, as well as preparations that include:
Hybrid antibody, modified antibody, F (ab ')₂Fragment, F (ab)
Fragment, Fv fragment, single domain antibody, chimeric antibody, humanized antibody
And their functional flags, which retain their specificity for the target molecule in question.
Gment. For example, the antibody may comprise a variable region, or a fragment of a variable region
And their regions and fragments retain specificity for the molecule in question.
You. The remainder of the antibody can be derived from the species using the antibody. Thus, the anti
If the body is to be used in humans, reduce immunogenicity and still retain activity
As such, antibodies can be "humanized." For an explanation of chimeric antibodies, see Example
See, for example, Winter, G .; And Milstein,
C. (1991) Nature349: 293-299; Jones, P .; T
. Other (1986) Nature321: 522-525; Riechman,
L. Other (1988)332: 323-327; and Carter, P .; Other (1
992) Proc. Natl. Acad. Sci. USA89: 4285-4
289. Such a chimeric antibody not only has a binding site for the target molecule,
And also contains binding sites for other proteins. In this way, external
Bifunctional reagents with targeted specificity for both antigens and internal antigens
Can be generated. If a polyclonal antibody is desired, select an animal (eg, mouse, rabbit, yaw).
Giant, horse, and other), as described above, with the desired antigen, or a flag thereof.
Immunization with a modified or suddenly altered antigen. Before immunization, a specific immunogen
It may be desirable to further increase immunogenicity. This is in this field
This can be achieved by one of several methods known in the art. For example, immunization for the production of antibodies can be achieved by combining the protein with a suitable excipient, for example,
Saline, preferably an adjuvant, such as Freund's complete adjuvant,
Or mixed or emulsified in any adjuvant described below, and the mixture or
By injecting the emulsion parenterally (generally subcutaneously or intramuscularly), the antibody
Immunization for production is generally performed. Generally, after two to six weeks,
Protein, preferably using Freund's incomplete adjuvant or other
Animals are boosted with one or more injections. Antibodies can also
By in vitro immunization using methods known in the art
Can be generated. Then a polyclonal antiserum from animals immunized
Acquire and process according to known techniques. For example, Jurgens et al. (1985) J.
. Chrom.348: 363-370. Blood containing polyclonal antibodies
When using CHI, the polyclonal antibody can be immunoaffinized according to known procedures.
Purify by chromatography. Monoclonal antibodies are generally known as Kohler and Milstein, Na.
cure (1975)256: Manufactured by the method of 495-96 or a modification thereof.
Built. Typically, a mouse or lad is immunized as described above. Only
Alternatively, rather than exsanguinating the animal to extract serum, the spleen (and
Some large lymph nodes) are removed and dissociated into single cells. If desired
Applying cell suspension to plates or wells coated with protein antigen
Allows screening of splenocytes (removing non-specific adherent cells)
After). B cells expressing membrane-bound immunoglobulin specific for the antigen
And does not rinse off from the rest of the suspension. The resulting B cells or everything
The dissociated splenocytes are then induced to fuse with myeloma cells and hybridoma
To form a selective medium (eg, hypoxanthine, aminopterin, thymidine medium)
(HAT, ground). The resulting hybridoma is screened by the limiting dilution method.
Antibodies that bind specifically to the immunizing antigen (and do not bind to unrelated antigens)
Assay for body production. Then secretes the selected monoclonal antibody
Hybridomas can be cultured in vitro (eg, in a tissue culture bottle or hollow fiber).
Culture in an incubator) or in vivo (as ascites in mice). An example
See, for example, the following: Schreier et al., Hybridoma
 Techniques (1980); Hammerling et al., Monocl.
onal Antibodies and T-cell Hybridoma
s (1981); Kennet et al., Monoclonal Antibody.
s (1980); see also U.S. Pat.
No. 761, No. 4,399,121; No. 4,427,783; No. 4,444.
No. 887; No. 4,452,570; No. 4,466,917; No. 4,472.
No. 500; 4,491,632; and 4,493,890. Problem G
Monoclonal antibodies raised against nRH immunogens, or fragments thereof
Panel of different properties, ie, isoforms, epitopes, affinity
, And others. Also, a functional fragment of the antibody is generated against the GnRH immunogen in question and the antigen
Cleavage of the constant region unrelated to binding from the antibody molecule, for example, using pepsin
And F (ab ')₂Fragments can be produced. These fragments
Contains two antigen binding sites but lacks a portion of the constant region from each of the heavy chains.
It will be. Similarly, if desired, for example, polyclonal antibodies with papain
Or contains a single antibody binding site by digesting a monoclonal antibody
Can be produced. Heavy and light chain variable regions
Functional fragments, which can also be produced according to standard techniques.
You. These fragments are known as Fv. Also, producing chimeric or humanized antibodies using the subject immunogens
Can be. Typically present in monoclonal and polyclonal antibodies
Contributing to heterologous constant regions and the variable regions of a species-specific framework
These antibodies can be designed to minimize unwanted immunological reactions.
You. For example, if antibodies are to be used in human subjects,
The non-human constant regions in both the heavy and light chains are
By substituting with human constant regions, chimeric antibodies can be generated.
See, for example, the following document: Winter, G .; And Milstein
, C.I. (1991) Nature349: 293-299; Jones, P .;
T. Other (1986) Nature321: 522-525; Riechman
, L .; Other (1988)332: 323-327; and Carter, P .; other(
1992) Proc. Natl. Acad. Sci. USA89: 4285
4289.GnRH composition Once the GnRH polypeptides or antibodies are produced, they are
Formulated into a composition for delivery to an animal subject. The GnRH molecules involved are cast alone.
Or mixed with a pharmaceutically acceptable vehicle or excipient. Appropriate
Vehicles include, for example, water, saline, dextrose, glycerol, ethanol
Or other, and combinations thereof. In addition, a small amount of vehicle
Auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or vaccine compositions
Contains adjuvants in the case of products, which enhance the effectiveness of the vaccine
I do. Suitable adjuvants are described further below. The composition of the present invention
And also auxiliary substances, such as pharmacological agents, cytokines or other biology
Response modifiers. As explained above, the vaccine composition of the present invention provides for the immunogenicity of a GnRH immunogen.
An adjuvant may be included to further increase the Adjuvants are
For example, emulsifiers, muramyl dipeptide, aviridine, aqueous adjuvants, such as
, Ammonium hydroxide and any of a variety of saponins, chitosan-based
Including adjuvants, oils, and other substances known in the art
Can be. For example, compounds that can act here as emulsifiers are natural
And synthetic emulsifiers, and anionic, cationic and nonionic compounds
Is included. Among the synthetic compounds, anionic emulsifiers include, for example, lauric acid and
Potassium, sodium and ammonium salts of oleic acid and
Salt, magnesium and aluminum salts (ie, metal soaps),
And organic sulfonates such as sodium lauryl sulfate. Synthetic power
Thionic agents include, for example, cetyltrimethylammonium bromide,
Synthetic nonionics include glyceryl esters (eg, glyceryl monostearate).
), Polyoxyethylene glycol esters and ethers, and sorbitan
Fatty acid esters (eg, sorbitan monopalmitate) and their polio
Xylene derivatives (eg, polyoxyethylene sorbitan monopalmitate)
). Natural emulsifiers include acacia gum, gelatin, lecithin and
And cholesterol. Other suitable adjuvants are oil components, such as a single oil, a mixture of oils, water-in-oil.
Emulsions, or oil-in-water emulsions.
. The oil can be a mineral, vegetable, or animal oil. Mineral oil or oil component
Oil-in-water emulsions that are mineral oils are preferred. In this regard, "mineral oil" is here
As a mixture of liquid hydrocarbons obtained from petrolatum by distillation techniques
The terms "liquid paraffin", "liquid petrolatum" and "white"
Mineral oil ". The term also refers to "light mineral oil", that is, petrolata.
Oil, which is also obtained by distillation of oil, but has a slightly lower specific gravity than white mineral oil
It is intended to encompass For example, Remington's Pharmac
See, Eugene Sciences, supra. Particularly preferred oil components are commercial
EMULSIGEN PLUS^TMOil-in-water emulsion sold at
(Light mineral oil and 0.05% formalin as preservative, and 30 μg / m
1 Gentamicin) (MVP Laboratories, Neb
(Available from Ralston, LA). Suitable animal oils include, for example,
Cod liver oil, halibut oil, menhaden oil, orange roughy
Includes oil and shark liver oil, all of which are commercially available. Appropriate
Vegetable oils include, but are not limited to, canola oil, almond oil, cottonseed oil
, Corn oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil
, And others. Alternatively, a number of aliphatic nitrogen-based bases may be used with the vaccine formulation.
Can be used as an adjuvant. For example, known immunogenic adjuvants
Are amines, quaternary ammonium compounds, guanidine, benzamidine and
Thiouronium compounds (Gall, D. (1966) Immunol).
ogy11: 369-386). Certain compounds are dimethyldioctadecyl
Ammonium bromide (DDA) (available from Kodak) and N, N
-Dioctadecyl-N, N-bis (2-hydroxyethyl) propanediamine (
"Aviridin"). Note the use of DDA as an immunological adjuvant
See, for example, the following article: the Kodak Labor
attory Chemicals Bulletin56(1): 1-5 (1
986); Adv. Drug Deliv. Rev ..5(3): 163-187
(1990); Controlled Release7: 123-13
2 (1988); Clin. Exp. Imunol.78(2): 256-2
62 (1989); Immunol. Methods97(2): 159
-164 (1987); Immunology 58 (2): 245-250 (
1986); and Int. Arch. Allergy Appl. Immun
ol.68(3): 201-208 (1982). Abilidine is well known
Adjuvant. For example, U.S. Pat. No. 4,310,550 (Wo)
lff, III et al.), which generally include N, N- as a vaccine adjuvant.
Higher alkyl-N ', N'-(2-hydroxyethyl) propanediamine, especially
Aviridine is described. No. 5,151,267 (Bab)
iuk), and Babiuk et al. (1986) Virology.159: 57
-66 relates to the use of aviridine as a vaccine adjuvant. EMULSIGEN PLUS including DDA^TMAdjuvant modified
An adjuvant, known as "VSA-3", is a modified form of the present invention.
(US Patent Application No. 08 / 463,837).
reference). Actual methods of preparing such dosage forms are known, or will be apparent to those skilled in the art.
It will be clear. See, for example, the following document: Remington
'S Pharmaceutical Sciences, I, Pennsylvania
Stoneton, 18th edition, 1990. The composition or formulation to be administered is treated
An appropriate amount of a GnRH polypeptide to achieve the desired condition in the subject.
Will contain. The compositions of the present invention will usually be administered as a liquid solution or suspension or prior to injection.
Manufactured as a solid form suitable for dissolution or suspension in a liquid vehicle
. Alternatively, the preparation may be emulsified, or the liposome vehicle or other
The active ingredient can be encapsulated in a particulate carrier. The composition can also be manufactured in solid form. For example, solid granular processing
The product may be manufactured for delivery from commercially available needleless injection devices.
Can be. Alternatively, provide implants in solid form for implantation into the patient
can do. Use control or sustained release formulations
And they carry the GnRH polypeptide in a carrier or vehicle.
Manufactured by taking in. Such a carrier or vehicle can be, for example,
Liposomes, non-absorbable impermeable polymers, such as ethyl vinyl acetate copolymer
ー and Hytrel^RCopolymers, swellable polymers such as hydrogels,
Or absorbent polymers such as collagen and certain polyacids or polyesters.
Such as those used to make absorbable sutures. In addition, polypeptides can be formulated in a neutral or salt form. Pharmaceutical
Acceptable salts include acid addition salts (formed with the free amino groups of the active polypeptide).
And these are inorganic acids, such as hydrochloric acid, phosphoric acid, or organic acids, such as
Formed with acetic acid, oxalic acid, tartaric acid, mandelic acid, and others. Free carbo
Salts formed from xyl groups also include inorganic bases such as sodium, potassium
, Ammonium, calcium, or ferric hydroxides, and organic bases, such as
For example, isopropylamine, trimethylamine, 2-ethylaminoethanol,
It can be derived from stidine, procaine, and others. The composition is formulated to contain an effective amount of a GnRH polypeptide, wherein the exact amount is
Is readily determined by one skilled in the art, where the amount is, in the case of a vaccine composition,
The animal to be treated, the ability of the animal's immune system to synthesize antibodies, and the desired GnRH
Depends on the degree of immune neutralization. For the purposes of the present invention, from about 1 μg to about 2 mg,
Generally from about 5 μg to about 800 μg, even more particularly from 10 μg to about 400 μg of Gn.
Formulations containing an injection solution of RH polypeptide / ml give rise to immunological
Would be appropriate to generate a dynamic response. When using peptide-carrier chimeras
In such cases, the immunogen / carrier ratio in the vaccine formulation will be selected to construct such a molecule.
It will vary based on the particular carrier and immunogen chosen. For example, if a leukotoxin-GnRH chimera is used,
The immunogen / leukotoxin ratio of
Will vary based on the leukotoxin and GnRH polypeptide components of
. One preferred vaccine composition has about 1 to about 90% GnRH, preferably about
3 to 80%, most preferably about 10 to 70% GnRH polypeptide / fusion molecule
Containing a leukotoxin-GnRH chimera having LKT-GnRH fusion
The increase in the percentage of GnRH present in
Total amount of antigen that must be administered to a subject to elicit an objective response
. A subject is exposed to one of the aforementioned compositions during a fattening period, for example, in a primary immunization.
At least one dose and, if necessary, two or more doses.
Give. Same or different after one or more primary immunizations and shortly before sacrifice
The GnRH composition is boosted one or more times to provide one or more boosters.
Circulating levels of androgenic and / or non-androgenic steroids
Is substantially reduced. The composition is delivered to the vertebrate subject using any suitable pharmaceutical delivery means.
be able to. For example, conventional needle syringes, spring or compressed gas (air) syringes
(U.S. Pat. No. 1,505,763 (Smoot); U.S. Pat. No. 3,788,3)
No. 15 (Laurens); U.S. Pat. No. 3,853,125 (Clark et al.)
U.S. Pat. No. 4,596,556 (Morrow et al.); And U.S. Pat.
062,830 (Dunlap)), a liquid injection syringe (U.S. Pat. No. 2,754).
U.S. Patent No. 3,330,276 (Gudo).
n); and U.S. Pat. No. 4,518,385 (Lindmayer et al.))
And particle syringes (US Pat. No. 5,149,655 (McCabe et al.) And US
No. 5,204,253 (Sanford et al.) All disclose the delivery of a composition.
Suitable for getting out. The composition can be administered intramuscularly, subcutaneously, intravenously, subcutaneously, intradermally, transdermally, or transmucosally.
Is administered. When using injection syringes, a single injection of the liquid vaccine composition is
Injection at a high pressure and speed, for example, at 1200 to 1400 psi.
An opening is formed in the skin more deeply and penetrates to a depth suitable for immunization. Examples of specific embodiments for practicing the invention are described below. These examples are for illustration only.
And is not intended to limit the scope of the invention.3. Experiment Example 1 Construction of pCB122 and pCB130 The plasmids pCB122 and pCB130 were used in the examples described below.
Produced a GnRH fusion protein for use. Both plasmids are the same
Produces a protein with one amino acid sequence. The GnRH construct in both plasmids
The construct is the 5 'end of the DNA sequence encoding the carrier leukotoxin polypeptide.
And 8 tandem repeats of the GnRH sequence fused to the 3 'end. Each alternate
GnRH identity has a fourth base change in the sequence from cysteine to guanosine.
I do. This allows a single amino acid of the second amino acid of the GnRH molecule from His to Asp.
Amino acid changes occur. See FIGS. 2A and 2B. Construct leukotoxin
The part encodes a shortened version of leukotoxin, which is
Removal of the internal fragment approximately 1300 bp long resulted in plasmid pAA352 (
ATCC Accession No. 68283, described in U.S. Pat. No. 5,476,657.
(Listed)). Leuco
The toxin polypeptide has a predicted molecular weight of 52 kDa and
It contains convenient restriction sites for use in producing the fusion protein. Ki
The mela construct is under the control of the Tac promoter and induction is Lac
Controlled by use of I. Plasmids pCB122 and pCB13
GnRH-leukotoxin fusion protein produced by
This is shown in FIG. 3F. Plasmid pCB122 was produced as follows. U.S. Pat.
No. 657 and U.S. Pat. No. 5,837,268.
The cotoxin gene was isolated. In particular, to isolate the leukotoxin gene,
Pasteurella haemolytica (P. haemolytica) A1 (B122 strain)
Gene libraries were constructed according to standard techniques. See the following document: L
o et al., Infect. Immun. , Supra; DNA CLONING: V
ol. I and II, supra; and Sambrook et al., Supra. Get
A nome library was constructed in plasmid pUC13 and a DNA library
Was constructed in bacteriophage lambda gt11. Using the resulting clone
To transform E. coli, pool individual colonies, and
Survive and pass P. haemolytica infection
The concentrated culture supernatant of P. haemolytica (P. haemolytica) was
With serum from calves with increased anti-leukotoxin antibody levels after star administration
Screened for reaction. Incubate cell lysate with bovine neutrophils.
And subsequently measuring the release of lactate dehydrogenase from the latter.
Positive colonies were screened for their ability to produce leukotoxin.
. Several positive colonies were identified and these recombinants were
Analyzed by creatase mapping. One clone was cloned earlier
Appeared to be identical to the selected leukotoxin gene. Lo et al., Infect
. Immun. Supra. To confirm this, a small fragment
Was recloned and the restriction maps were compared. Almost 4 kilobase pairs of DNA
It was decided that it was done. Isolate a full-length recombinant that is approximately 8 kb long
Chromosome walking (5 '→ 3' direction)
Clones were isolated. The last construct was named pAA114. This construct
It contained the body leukotoxin gene sequence. lktA, ie pAA114 containing the entire leukotoxin gene
The MaeI restriction endonuclease fragment was used to cleave DNA polymerase I.
Treatment with a Renault fragment + nucleotide triphosphate, the cloning vector pUC13
In the SmaI site. This plasmid was designated as pAA179. This
The vector pGH432: l based on ptac digested with SmaI
Two expression constructs were made in acl. On the other hand, pAA342 of the lktA gene
The 5'-AhaIII fragment consisted of the other pAA345
Of the entire MaeI fragment. Clone pAA342 is truncated
Although the traucotoxin peptide was expressed at high levels, pAA345
Icotoxin was expressed at very low levels. Therefore, 3 of the lktA gene
'End (StyI BamHI fragment from pAA345) to StyI
Ligation to BamHI digested pAA342 generated plasmid pAA352.
Pasteurella haemolytica produced from the pAA352 construct (P. haemol)
ytica) leukotoxin is hereafter referred to as LKT352. The plasmid pAA352 is then used to produce the recombinant leukotoxin polypeptide.
Made a shortened version of the code. Almost 1 from recombinant LKT gene as follows
By deleting a 300 bp long internal DNA fragment, the shortened LKT
The gene was produced. Plasmid pCB113 containing LKT 352 polypeptide
(ATCC Accession No. 69497, US Pat. No. 5,837,268)
Described) with the restriction enzyme BstB1 (New England Biol)
abs). The resulting linearized plasmid is then transferred to the yeast nuclease (
Single-strand protrusions generated by BstB1 digestion, digested with Pharmacia)
Ends were removed. Then, the blunt DNA was subjected to restriction enzyme NdeI (New Engl).
and Biolabs) and the digested DNA is placed on a 1% agarose gel.
Loading, where the DNA fragments were separated by electrophoresis. Almost 6190b
The large p DNA fragment was isolated and Gene Clean was used.
n) Purify from agarose gel using kit (Bio 101)
The resulting fragment was ligated to bacteriophage T4 DNA ligase (Pharmaci
Attached to itself using a). Competent using the resulting binding mixture
Transform E. coli JM105 and aggregate with appropriate molecular weight
Positive clones were identified by their ability to produce proteins. Formed in this way
The recombinant plasmid was designated as pCB111 (ATCC Accession No. 69748).
As shown, this is a truncated leukotoxin fused to four copies of the GnRH polypeptide.
Produces a polypeptide (hereinafter referred to as LKT 111). Plasmid pCB11
4 is a multiple-copy GnRH sequence inserted twice (corresponding to the oligomer in FIG. 2B)
). Both of these plasmids are described in US Pat. No. 5,837,268.
Abbreviations designated LKT 111 and LKT 114, respectively.
Produces a leukotoxin polypeptide. The multiple copy GnRH sequence (corresponding to the oligomer in FIG. 2B) was added to LKT 11
By binding twice to the 5 'end of the 4 coding sequence, the pCB114
From the LKT-GnRH fusion sequence obtained from the sumid, one of the LKT114 N
2 located at the '-terminus and the other at the C'-terminus of LKT114.
Construct a recombinant LKT-GnRH fusion molecule with two 8-copy GnRH multimers
I built it. Synthetic nucleic acid molecule having the following nucleotide sequence: 5'-ATGGCTAC
TGTTATAGATCGATCT-3 '(SEQ ID NO: ) For multiple copy GnR
The 5 'end of the H sequence was attached. The synthetic nucleic acid molecule has an eight amino acid sequence (Met-Ala
-Thr-Val-Ile-Asp-Arg-Ser) (SEQ ID NO: ) Code
I do. The resulting recombinant molecule thus has the following in the order described in the 5 '→ 3' direction:
Contains: a synthetic nucleic acid molecule; encodes the first 8 copy GnRH multimer
Nucleotide sequence; nucleotide encoding truncated LKT peptide (LKT114)
Otide sequence; and nucleotides encoding the 28th copy GnRH multimer
Array. The recombinant molecule is circularized and the resulting molecule is used to competent E. coli (E.co.
li) JM105 cells were transformed. Aggregate having a molecular weight of approximately 74 kDa
Positive clones were identified by their ability to produce structures. The thus-formed recombinant
The rasmid is designated pCB122, which represents 16 copies of the GnRH polypeptide.
A fused LKT114 polypeptide is produced. For plasmid pCB130, Amp^rGene or pCB122 to Te
t^RThe gene was replaced. Thus, the plasmid is under tetracycline selection
. Recombinant LKT-GnRH fusion of plasmids pCB122 and pCB130
Are shown in FIGS. 3A to 3F.Example 2 Purification of LKT-antigen fusion The recombinant LKT-GnRH fusion from Example 1 was purified according to the following procedure. 1
Transformation into 10 ml TB broth supplemented with 00 μg / ml ampicillin
5-10 colonies of the transformed E. coli strain were inoculated and 220 rpm
For 6 hours at 37 ° C. on a G10 shaker incubator. 400ml
Baffle Fernbach flas containing TB broth + ampicillin
Dilute 4 ml of this culture into each of the cells and incubate overnight as described above.
I did it. In a polypropylene bottle with a volume of 500 ml, Sorva
11) Centrifuge for 10 minutes at 4,000 rpm using a GS3 rotator.
And collected the cells. Containing ampicillin preheated to 37 ° C
Resuspend the pellet in a small volume of TB broth (ie 2 x 400 ml)
Cells were incubated for 2 hours as described above. 3.2 ml of 500 mM isopropyl-B, D-thiogalactopyrano in water
Sid (IPTG, Gibco / BRL) (final concentration = 4 mM) added to each culture
To induce the synthesis of the recombinant fusion protein. Incubate culture for 2 hours
did. Cells are harvested by centrifugation as before and 30 ml of 50 mM Tris
-Resuspended in hydrochloride, 25% (w / v) sucrose, pH 8.0, -7
Frozen at 0 ° C. After 60 minutes at -70 ° C, the frozen cells are brought to room temperature.
And 5 ml of lysozyme (Sigma, 250 mM Tris-HCl
, PH 8.0, 20 mg / ml). Mix this mixture at high speed for 10 seconds
Vortexed and then placed on ice for 15 minutes. The cells are then transferred to a 1000 ml beaker.
By adding to 500 ml of lysis buffer and stirring with a 2 ml pipette.
Was mixed. The beaker containing the lysed cell suspension was placed on ice and 100
Braun ultrasonic device set to watts power, large probe
Sonication for a total of 2.5 minutes (burst of 5-30 seconds, 1 minute cooling between each)
. An equal volume of the solution was placed in a Teflon SS34 centrifuge tube and the Soval SS34
Centrifuge at 10,000 rpm for 20 minutes in a rotator. 10 pellets in total
Resuspended in 0 ml of sterile double distilled water by vortexing at high speed;
The centrifugation step was repeated. The supernatant is discarded and the pellet is washed with 20 ml of 10 mM Tris.
In HCl, 150 mM NaCl, pH 8.0 (Tris buffered saline)
And the suspension was frozen at -20 ° C overnight. The recombinant suspension is thawed at room temperature and 100m in Tris buffered saline.
1 M of guanidine HCl (Sigma) and mixed vigorously. Magnetic stirring
The stir bar was placed in the bottle and the solubilized sample was mixed for 30 minutes at room temperature. this
The solution was transferred to a 2000 ml Erlenmeyer flask and 1200 ml of Tri
S-buffered saline was added rapidly. The mixture is left at room temperature for a further 2 hours
Stirred. Aliquot a 500 ml aliquot into a dialysis bag (Spectrum, diameter 63)
. 7 mm, 6,000-8,000 molecular weight cutoff, # 132670, Fis
her Scientific) and add them to 3,500 ml of T
4,000 m containing ris buffered saline + 0.5 M guanidine HCl
1 in a beaker. Place the beaker overnight on a magnetic stirrer in a room at 4 ° C
The dialysis buffer was then replaced with Tris buffered saline + 0.1 M guanidine HCl.
And dialysis was continued for 12 hours. The buffer was then added to Tris buffered saline +
Replacement with 0.05M guanidine HCl and dialysis continued overnight. Tris buffer
Replaced with buffered saline (without guanidine) and dialysis continued for 12 hours.
This was repeated three more times. Put the final solution in a 2000ml plastic roller
Pour into Corning and add 13 ml of 100 mM PMSF (Ethanor
Was added to inhibit protease activity. This solution is heated at -20 ° C for 1 hour.
Stored in 00 ml aliquots. Aliquots of each preparation were used to confirm that the fusion protein was isolated.
Dilute 20-fold in double-distilled water, equal volumes of SDS-PAGE sample buffer
And place in a boiling water bath for 5 minutes to remove the 12% polyacrylamide gel.
Deployed through. A recombinant leukotoxin control was also developed. Fusion proteins
It was expressed at high levels as inclusion bodies.Example 3 Antibody titers after GnRH immunization of pigs This test consists of a second injection secondary immunization and primary vaccination on volume, first
It was designed to evaluate a variable that included the number of injections (1 to 2) for the the study
160 pigs 28 days old and weighing 3-4 kg for 8 treatment groups
Group (see Table 1). 10 females and 10 in each group
There were castrated boars. Animals housed at 10 / cage
In Center Incorporated (Prairie Spine Cen)
tre, Inc. [The University of Saskatchewan (University of Sa
Skatchewan), Canadian Council on Animal
・ Care (Canadian Council on Animal Care)
Laboratory equipment censored by)) using standard operating procedures developed by
did. For groups 1 to 7, GnRH immunization from plasmid pCB122 as described above.
A GnRH vaccine was made using the original. In particular, the GnRH immunogen was dissolved in 8M urea.
At a concentration of 20 mg / ml. Used to prescribe the GnRH vaccine
Adjuvant is VAS-3 (EMULSIGEN PLUS including DDA)^TM
(A modified form of an adjuvant) (Allowed US Patent Application Serial No. 08/4)
63, 837). A stock of GnRH immunogen was combined with phosphate buffer and combined with VAS-3 1: 1 (
(v / v) to form a stable emulsion. Groups 1, 2, 3, 4, and
The dose of GnRH immunogens of 6 and 6 was 40 μg, but the volume was
Was different. Details for each treatment group are listed in Table 1. glue
The GnRH vaccines of steps 5 and 7 contain 30 μg of GnRH immunogen / 0.25 ml.
Group 8, but contained plasmid pCB13 in 0.4 ml of adjuvant.
0 to 40 μg of GnRH immunogen was administered. In all cases, VAS
-3 adjuvant / aqueous phase (phosphate buffer) ratio remains only 1: 1 (v / v)
Was. Adjustments were made by changing the volume of the immunogen stock. Needleless Biojector 2000 injection development (manufactured by
Manufacturer: Bioject Inc. In Portland, Oregon, USA)
Vaccine was administered. This device uses a gas cylinder to pass through a small opening
Inject the vaccine under high pressure. The vaccine penetrates the skin and is deposited subcutaneously. each
In the treatment group, when the pig was 38 days old, the first injection was performed and 35 days
A second injection was performed later. The injection is performed on the outer surface of the pinna, except that the second injection in group 7 is behind the head
The test was performed at the center of the back of 10 to 15 cm. Days 35, 49 and 63 of the experiment (
Blood was collected by jugular vein puncture at the start of the study (day 0). Cells
The serum is collected by coagulation at room temperature and then centrifuged to collect the serum and the GnRH antibody titer.
Stored at -20 ° C until analyzed for GnRH antibody titers were determined by a modified radioimmunoassay procedure. Combination
GnRH (Bachem., Inc.)¹²⁵(Amersham) ON
(Oakville, Tario). Add serum dilution to test tube, then
Standard amount of I¹²⁵Add the labeled GnRH to bring the final incubation volume to 0.1.
It was made up to 7 ml. Incubate the charcoal suspension in assay buffer for 24 hours.
Added at 2-6 ° C at the end of the¹²⁵-Absorbs GnRH
did. After centrifugation, the radioactivity in the charcoal fraction was measured. Present the data as numbers,
The value is the I bound to the antibody at a particular serum dilution.¹²⁵-Standard dose of GnRH (
(Approximately 12,000 cpm). Student Version of St
atistix, Version 1, Copyright 1996)
And performed descriptive statistics, variance and "t" test. The volumes of 0.15, 0.25 and 0.35 given to a single injection site were each
And evaluated in groups 1, 2 and 3. When the animal was 63 days old,
Antibody titers before booster vaccination on day 35 of study and animals on day 77
At age 14 days post-boost vaccination on day 49 of the study
Table 1 shows the relationship between the body titers. 10 at 1: 5000 on day 35
Animals with titers greater than% binding had a weak response to primary immunization
The response to the booster vaccination was better than the animals. For other experiments
Approximately 20% binding at a 1: 5000 dilution
We know that it will be partially suppressed. These results show that a strong response to the primary immunization provided there
Utilization has no effect on growth and feed utilization efficiency.Example 4 Effect of GnRH immunogen on testosterone levels In the experiments described below, testosterone levels were reduced in early adolescent animals.
An immunological approach is used to demonstrate lack of efficacy. 60 full
Male boars were divided into three treatment groups. Surgical castration of Group 1 at birth
And groups 2 and 3 were left intact. Almost 21 days old, natural
An interior comprising 38-378 amino acids and 815-951 of leukotoxin
Comprising eight copies of GnRH bound to a leukotoxin molecule that has been genetically deleted
Group 3 was immunized with the GnRH immunogen. As described above, the GnRH immunogen
Was formulated in a VAS-3 adjuvant. Immunization produces testosterone secretion
Antibody production increased sufficiently to cause a detectable decrease. Group 2 is real
It remained intact throughout the experiment and was not immunized. In previous studies, animals immunized with this adjuvant had moderate, persistent
GnRH antibody titer increased, the GnRH antibody titer
Low but reduced to detectable levels. Feed consumption and carcass composition during the experiment
To compare their parameters at various ages. Animals treated with this GnRH vaccine [immuno-castrated pigs (immunoca
strates)] were castrated to approximately 90 days of age.
And behaved similarly to non-castrated boars. Further, as shown in FIG.
As such, all three groups had similar weight gains.Example 5 Immunocastration of sexually mature boars by GnRH vaccination The purpose of this study was to assess serum testosterone and
Fat androstenone levels equal to those found in surgically castrated pigs
Determine whether the value reduces GnRH vaccination and determine the primary immunization and
Of GnRH antibody after serum and secondary immunization, serum testosterone concentration and
The purpose was to determine the kinetics of fat androstenone levels. As shown in Table 2, 24 intact boars were treated on day 0 with three treatment groups (groups).
Assigned to one of loops 1, 2 and 3). Surgical castration younger than one week old
Six litters of age-matched litters were placed in a fourth treatment group (Group 4-Early
Castrated boar). Keep one pig until the pig weighs approximately 60 kg.
Housed with 0 animals / cage and then housed with 2 animals / cage. Feed the pig
Access to water and water, Prairie S.
wine Center) [University of Saskatchewan (University of
 Saskatchewan) and the Canadian Council on A
Nimal Care (Canadian Council on Animal C)
animal facilities censored by are)).
Managed. Plasmid p dissolved at a concentration of 28 mg / ml in 4 M guanidine HCl
A GnRH vaccine was made using the GnRH immunogen from CB122. Gn
The adjuvant used to formulate the RH vaccine was VAS-3. GnRH free
The epidermis was combined with phosphate buffer and mixed with VAS-3 in a 1: 1 (v / v) ratio.
The vaccine was produced by forming a stable emulsion. This vaccine is
Contains 40 μg of GnRH immunogen / 0.5 ml dose and was administered intramuscularly. Step
The placebo contained phosphate buffer and VAS-3. Pigs received two intramuscular injections of vaccine or placebo into the neck. The first injection
Performed on day 0 of the experiment, at which time the pigs were 21 days old. Pigs approach sexual maturity
A second injection was performed, at which time the pig weighed almost 100 kg (
(Days 110 to 120) (Table 2). Pigs in Group 2 (late castrations
When they reached sexual maturity, they were surgically castrated. Sexual maturity depends on weight
More strongly affected and occurs when the body weight is approximately 110 kg (115-125
Day). Almost one week before the pigs in Group 2 were surgically castrated, Group 1
Pigs were secondly immunized. This is because pigs in Group 2 were surgically removed.
Approximately at the same time when the GnRH antibody titer generated by the secondary immunization is
This was done to reach a chemically effective level. To simplify the analysis and presentation of the data, day 120 is an "event"
The animals are given a second injection (all groups) or
It meant the time of castration (Group 2). "Events"
All data are “events”, ie 7 days after “event” means day 127
, On the 14th day after the "event" means the 134th day, and on any other "event"
Is described. Blood tests were performed by jugular vein puncture at approximately 28 day intervals between days 28 and 120.
I got the fee. Thereafter, the animals were killed on day 162 of the experiment (42 days after the "event").
) Blood was collected from all pigs at weekly intervals. Blood is allowed to clot at room temperature,
Centrifuged and serum frozen within 24 hours after collection. Individual weight gain by weighing all animals from day 0 to "event"
The animals were weighed weekly until sacrifice. Topical from alternate sides of neck of all pigs every week until "sacrifice" and sacrifice
Under anesthesia, a sample of subcutaneous fat (approximately 5 g) was taken for androstenone measurement.
Was. The fat sample was immediately cooled and frozen within 4 hours after biopsy. Measurements at slaughter were: carcass weight, back fat depth at the 10th rib level,
Round weight and urethral gland length were included. GnRH antibody titers were measured by a modified radioimmunoassay procedure. Synthetic Gn
RH (Bachem, Inc.)¹²⁵(Amersham, Ontario
Oakville). The serum dilution is added to the test tube and then a standard amount of
I¹²⁵Add labeled GnRH to bring the final incubation volume to 0.7 ml
did. At the end of the 24 hour incubation of the charcoal suspension in assay buffer
At 2 to 6 ° C. to give non-antibody-bound I¹²⁵-Absorbed GnRH. Far
Afterwards, the radioactivity in the charcoal fraction was measured. Present data as numerical values and specify numerical values
Bound to antibody at serum dilution of¹²⁵A standard dose of GnRH (approximately 12
, 000 cpm). Coat-A-Count Total Testosterone Kit
(DPS, Los Angeles, CA) using serum testosterone
Was measured. This assay is¹²⁵-For testosterone and testosterone
It is based on antibodies with high affinity for it. Fat androstenone concentration was measured by a colorimetric method. The measurement of the primary result was a serum dilution of 1: 5000 in GnRH antibody titer group 1 and groups 2, 3 and 4
GnRH antibody titer, measured as% binding at 1: 100 in the assay.
Also, serum testosterone concentration, fat androstenone, body weight, back fat, testicle weight
The amount and urethral gland length were measured. All of the pigs in Group 1 are at 1: 100 serum dilution after primary immunization
A readily detectable GnRH antibody titer was generated (see Table 3). In addition, on the 21st
Immunization of pigs at age and near 140 days of age occurs early and late in life.
Testosterone and fat, equivalent to those found in castrated pigs
GnRH antibody titers were generated which resulted in a decrease in androstenone concentration. Also, nothing
There was a significant decrease in the size of testes and urethral glands compared to wounded males. Example 6 GnRH immunization of bulls This experiment was performed using 58 early adolescent calves. group
28 in I. plasmid pCB in 200 μg VAS-3 adjuvant
Subcutaneous twice with a vaccine composition comprising a GnRH immunogen from C.122
Kuching inoculated (day 0 and day 56) and 30 pairs in group 2
Terrier cattle were vaccinated with a placebo. Group I vaccination is 42nd
A significant titer for GnRH was developed by day 84 and around the scrotum by day 84.
Significantly reduced testosterone levels by day 98
(Table 4). Involved in these significant anti-GnRH titers and reduced testosterone
The daily increase and the difference in feed efficiency were not observed from day 0 to day 84
(Table 5).  As these novel findings indicate, there is significant utility for GnRH vaccines.
, GnRH vaccines give a sufficiently strong immune response after primary immunization and this immune response
Reduces detectable serum testosterone but significantly increases growth or feed efficiency
This produces an antibody titer that does not decrease. This finding is linked to Andro's early growth
Shows that temporary suppression of gen secretion does not reduce body growth or feed efficiency
So it is especially new. This is after a lifetime, with the aim of achieving a strong second order response.
When using a vaccination protocol that requires subsequent immunization in the phase
, Have a specific utility.Contracting of useful strains when practicing the present invention Deposits of biologically pure cultures of the following strains are available from American Type Culture:
・ Collection (American Type Culture Collection)
Tion) (ATCC), Manassas, VA, University Boulevard
Mode 1081. The acceptance number given after a successful viability test
No. was assigned and the required fee was paid. Deposits are for patent purposes only
Of the Budapest Treaty on the International Recognition of Deposits of Microorganisms
Made about). This is the structure of the sample deposited by the depository for 30 years from the date of deposit.
Ensure the maintenance of a viable culture for at least 5 years after the most recent request for growth.
Organisms have been made available by the ATCC in accordance with the provisions of the Budapest Treaty,
35U. S. C. §122 and the rules of the Commissioner pursuant thereto (37 USC §1
. 12 in accordance with the United States Patent and Trademark Office.
However, the permanent, unrestricted availability of cultures is guaranteed. Patent granted
All restrictions on the availability of the deposited culture to the public
Will be removed. These deposits are provided merely for convenience to those skilled in the art, and the deposits are 35 U.S.M. S
. C. It is not an admission that it is required under §122. The amino acids of these plasmids
No acid sequence and the amino acid sequence of the polypeptide encoded thereby are quoted
And is hereby incorporated by reference into this specification, and conflicts with the description herein.
If you are controlled. To manufacture, use, or sell a contract,
A license is required and no such license is granted here. Thus, a method of immunizing against GnRH has been disclosed. Preferred of the present invention
The embodiments have been described in some detail, but are defined by the appended claims.
Obviously various changes and modifications can be made without departing from the spirit and scope of the invention.
It should be understood that

[Brief description of the drawings]

FIG. 1. Antibody titers before booster vaccination on day 35 of the study when pigs are 63 days old and pigs are 77 days old, as described in the Examples. Sometimes the relationship between the booster vaccine injection on day 49 of the study and the antibody titer 14 days after is depicted.

FIG. 2A shows the nucleotide sequence and amino acid sequence of a GnRH construct used in the chimeric leukotoxin-GnRH polypeptide of the present invention. FIG. 2A depicts a single copy of the GnRH decapeptide.

FIG. 2B shows the nucleotide and amino acid sequences of the GnRH construct used in the chimeric leukotoxin-GnRH polypeptide of the present invention. FIG. 2B shows four copies of the GRH decapeptide when n = 1, and n =
2 depicts a molecule with eight copies of the GnRH decapeptide when 2, and others.

FIG. 3A shows LKT-GnR from plasmids pCB122 and pCB130.
1 shows the nucleotide sequence and predicted amino acid sequence of the H chimeric protein.

FIG. 3B shows LKT-GnR from plasmids pCB122 and pCB130.
1 shows the nucleotide sequence and predicted amino acid sequence of the H chimeric protein.

FIG. 3C shows LKT-GnR from plasmids pCB122 and pCB130.
1 shows the nucleotide sequence and predicted amino acid sequence of the H chimeric protein.

FIG. 3D shows LKT-GnR from plasmids pCB122 and pCB130.
1 shows the nucleotide sequence and predicted amino acid sequence of the H chimeric protein.

FIG. 3E shows LKT-GnR from plasmids pCB122 and pCB130.
1 shows the nucleotide sequence and predicted amino acid sequence of the H chimeric protein.

FIG. 3F shows LKT-GnR from plasmids pCB122 and pCB130.
1 shows the nucleotide sequence and predicted amino acid sequence of the H chimeric protein.

FIG. 4. FIG. 4 shows castrated boars and non-castrated boars (boa).
2 shows the body weight as a function of the age of pigs treated with the GnRH vaccine according to the invention (immunocastrates) compared to rs).

[Procedure amendment]

[Submission date] November 16, 2000 (2000.11.16)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Correction contents]

Title of the Invention Breeding Animals for Edible Meat Production

[Claims]

DETAILED DESCRIPTION OF THE INVENTION

[0001] Technical Field The present invention generally relates to a method for rearing animals for the meat production. More particularly, the present invention relates to primary vaccination of GnRH immunogens to reduce circulating gonadal steroid levels, and to GnRH just before sacrifice to substantially reduce the levels of one or more androgenic and / or non-androgenic steroids A method for immunizing an animal by re-vaccination of an immunogen.

[0002] BACKGROUND male invention cattle, pigs, and sheep have been more highly muscles of typically,
Has a larger mature body than females. According to conventional explanations, when male animals reach sexual maturation, the secretion of testosterone, an anabolic steroid produced by the testes, increases muscle protein deposition and reduces fat. Also, a number of studies have established that males use feed more efficiently than females during the growth period (Field, RA, Journal Animal Soc. (1971) 32 : 849-858).
. These differences are most apparent near puberty, and testosterone has an important role in these changes.

[0003] In males, androgenic steroids, testosterone and androsterone are involved in the regulation of two different biological processes. One effect is physiological, increasing muscle deposition, decreasing fat synthesis, and increasing feed utilization efficiency. Testosterone has a similar direct effect on the growth of gonads, such as the seminal vesicles, prostate and testes. These effects include the direct interaction of androgens with receptors on target tissues.

[0004] A second common effect of androgens is to cause male sexual and other behavioral changes. Their effects are transmitted through the central nervous system. Because these different effects occur in different tissues by different molecular mechanisms, some of them are maintained at low plasma concentrations of androgens, while others may require higher levels.

Although it is generally assumed that androgens are required to maintain optimal growth, muscle deposition and feed efficiency in life, even if measurable amounts of androgens are present, that assumption is not true There is. Alternatively, androgen-sensitive tissues, such as muscle, may be much more responsive to lower androgens earlier in life than in puberty or later. Allrich et al., Journal Animal Soc. (1982) 55 : 1139-1146 compared the rate of increase in body weight and testosterone-sensitive tissue weight in pigs as a function of age and testosterone concentration.

[0006] In particular, a comparison of the growth rates of different tissues at different ages indicated that tissues susceptible to testosterone, as well as testosterone levels, changed with age. For example, at days 40-100, the authors showed that serum testosterone increased by 3.1 fold, but body weight increased by 3.2 fold, testis weight increased by 3.6 fold, and seminal vesicles increased by 4.0 fold. .

[0007] Also, these data indicate that early adolescent testosterone levels are low, but readily detectable, and that body weight increases in the presence of low levels of testosterone at approximately the same rate as other tissues. Pigs begin to reach sexual maturity at approximately 130-150 days of age (65-85 kg body weight), and testosterone levels increase significantly at that time. On days 100-190, serum testosterone increases by 4.1 fold, but body weight, testes weight, and seminal vesicle weights increase by 2.6 fold, 13.5 fold, respectively.
And increased 9.2 times.

[0008] In addition, Knudson et al., Journal Animal Soc. (1985) 61 : 789-796 show that castrated male pigs gained weight at a similar rate up to about 90 days of age compared to intact males,
Beyond that age, full males have shown to grow more efficiently than castrated males. This feature is particularly important for the infertility of the herd of animals, particularly when the boars are castrated and produced by sex steroids in the normally functioning testes of the boars, "boar taint" Is particularly important where it is desirable to prevent See, for example, the following reference: Meloen et al., Vaccine (199).
4) 12 (8): 741-746.

[0009] Numerous studies have been performed in pigs and cattle to investigate the use of GnRH immunogens as a way to improve growth rate and feed efficiency in animals. See, for example, the following references: Adams and Adams, Journal Animal Soc.
. (1992) 70 : 1691-198; Caraty and Bonneau, CR, Acad. Sc. Paris.
(1986) 303 : 673-676; Chaffaux et al., Recueil de Medecine Veterinaire (1
985) 161 : 133-145; Finnerty et al., J. Repro. Fertil. (1994) 101 : 333-343.
. The purpose of many of these studies was to grow animals as full males until near the end of the fattening period, and then immunologically castrate them.

[0010] Towards the end of the fattening period and to achieve immunological castration just prior to slaughter, the animals are primed with the immune system by vaccinating one or more early in their lifespan and thus at the end of the fattening period The animals are strongly responsive to the given re-vaccination. The first vaccination primes the immune system against the GnRH immunogen, but avoids the induction of high anti-GnRH antibody titers that reduce serum testosterone levels or increase serum testosterone levels when animals approach puberty Designed to prevent This was based on the belief that reducing serum testosterone reduced growth rate or feed efficiency in young animals.

For example, Meloen et al., Vaccine (1994) 12 : 741-746, describe the use of a GnRH-associated vaccine administered in two doses to reduce boar rot in pigs. Testicle size did not decrease until after the second immunization. See also International Publication No. WO 90/11298, issued October 4, 1990. Falvo et al., Journal Animal Soc. (1986) 63 : 986-994 report the use of a GnRH vaccine to study various effects in pigs, including the presence of boar spoilage and carcass characteristics.

[0012] The authors reported that plasma testosterone levels were significantly reduced two weeks after the first booster injection. Similarly, US Pat. No. 5,573,767 relates to a method of improving the organoleptic quality of edible meat using GnRH immunization. This method involves two immunizations, one designed to have no effect on gonadal steroids, and the second to eliminate the effects of androgenic and non-androgenic steroids before slaughter. Designed.

[0013] In addition, previous attempts at immunoinfertility have shown that the immunogenicity of GnRH peptides and / or related carrier systems is inadequate, and that the various previous GnRH-based vaccines that are available The inability to elicit a sufficient immune response against them did not produce uniform results. See, for example, the following references:
Robertson, Vet. Reccord (1981) 108 : 381-382. Therefore, a reliable method for immuno-sterility of food producing animals would be desirable.

[0014] Disclosure of the Invention The present invention rearing food Bussan Ikuo animals for the meat production, reliable, based on a reproducible manner. In particular, contrary to the prevailing belief, based on the tests described herein, a substantial reduction in testosterone early in life to produce a commercially acceptable amount of edible meat. Avoidance is unnecessary,
And primary GnRH immunization, which induces antibodies with a measurable effect on gonadal steroid secretion during the fattening period, appears to be achievable without significantly reducing growth rate or feed efficiency. After the primary immunization, a secondary immunization can be performed later in life to abolish the effects of androgenic and / or non-androgenic steroids.

Accordingly, in one embodiment, the present invention provides a method of vaccinating a non-castrated male food-producing animal for edible meat production before or during a fattening period with said first vaccine composition. The testosterone level is reduced and about 2 to about 8 weeks prior to sacrifice of the animal, the animal is vaccinated with a second vaccine composition to reduce the level of one or more androgenic and / or non-androgenic steroids. A method of breeding a non-castrated male food producing animal for edible meat production comprising substantially reducing. In a particularly preferred embodiment, the first
And / or the second vaccine composition comprises an immunological adjuvant, for example, an adjuvant comprising oil and dimethyldioctadecyl ammonium bromide.

In addition, the GnRH immunogen in the first and / or second vaccine composition has the general formula (Gn
(RH-X-GnRH) y, where GnRH is a GnRH immunogen and X is selected from the group consisting of a peptide bond, an amino acid spacer group, a carrier molecule and [GnRH] n Is one or more molecules, where n is an integer greater than or equal to 1 and y is an integer greater than or equal to 1.

In some embodiments, administration of the first vaccine composition produces antibodies that cross-react with endogenous GnRH in the animal, and the second vaccine composition is administered after the antibody levels are reduced. In another embodiment, the present invention relates to a method of vaccinating a non-castrated male bovine, ovine or porcine animal for edible meat production before or during a fattening period with said first vaccine composition to circulate testosterone. About 2 to about 8 weeks prior to sacrifice of the animal, the animal is vaccinated with a second vaccine composition to reduce the level of one or more androgenic and / or non-androgenic steroids. A method of breeding a non-castrated male cow, sheep or pig animal for edible meat production, comprising reducing the amount of edible meat. 1st and / or
Alternatively, the second vaccine composition can further include an immunological adjuvant.

In still other embodiments, the invention provides: (a) a first vaccine composition in a non-castrated male bovine, ovine or porcine animal prior to or during the fattening period for the production of edible meat; To reduce circulating testosterone levels, wherein the first vaccine composition comprises an immunological adjuvant and a GnRH multimer, wherein the GnRH multimer has the general formula (GnRH-X
-GnRH) y, wherein GnRH is a GnRH immunogen, and X is one or more molecules selected from the group consisting of peptide bonds, amino acid spacer groups, leukotoxin polypeptides and [GnRH] n. There, where n
Is an integer greater than or equal to 1, and y is an integer greater than or equal to 1, and

(B) about 2 to about 8 weeks prior to sacrifice of said animal, vaccinating said animal with a second vaccine composition to substantially reduce the level of one or more androgenic steroids and / or non-androgenic steroids; Wherein the second vaccine composition comprises an adjuvant and a GnRH multimer, wherein the GnRH multimer has the general formula (GnRH
RH-X-GnRH) y, where GnRH is a GnRH immunogen, and X is one or more selected from the group consisting of peptide bonds, amino acid spacer groups, leukotoxin polypeptides and [GnRH] n Where n is
Is an integer greater than or equal to one and y is an integer greater than or equal to one.

In still other embodiments, the present invention provides: (a) a first vaccine composition in a non-castrated male bovine, ovine or porcine animal prior to or during the fattening period for the production of edible meat; To reduce circulating testosterone levels, wherein the first vaccine composition comprises an immunological adjuvant and a GnRH multimer, wherein the GnRH multimer is comprised of FIGS.
3F diagram (SEQ ID NO: ) Or an amino acid sequence having at least about 75% sequence identity thereto, and

(B) about 2 to about 8 weeks before sacrifice of the animal, wherein the animal is vaccinated with a second vaccine composition to substantially reduce the level of one or more androgenic and / or non-androgenic steroids. Wherein the second vaccine composition comprises an immunological adjuvant and a GnRH multimer, wherein the GnRH multimer is represented in FIGS. 3A-3F (SEQ ID NO: Breeding a non-castrated male cow, sheep or pig animal for production of edible meat, comprising the amino acid sequence depicted in) or an amino acid sequence having at least about 75% sequence identity thereto. On how to do it. The immunological adjuvant in the first and / or second vaccine composition can comprise light mineral oil and dimethyldioctadecyl ammonium bromide. These and other aspects of the invention will be readily apparent to one of skill in the art in light of the disclosure herein.

[0022] In the practice of the Detailed Description of the Invention, unless otherwise indicated, molecular biology, microbiology, virology, using conventional techniques of recombinant DNA technology, and immunology, within these of skill in the art It is. Such techniques are explained fully in the literature. For example, see the following documents: Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual; DNA Cloning, Vol. I and II (ed. By DN Clover)
Oligonucleotide Synthesis (MJ Gait); Nucleic Acid Hybridizati
on (ed. by BD Hames & SJ Higins); B. Perbal, A Practical Guid
e to Molecular Cloning; the Series, Methods in Enzymology (S. Col.
owick and N. Kaplan eds., Academic Press, Inc.); and Handbook of Exp
erimental Immunology, Vol. I-IV (DM Weir and CC Blackwell
Ed., Blackwell Scientific Publications).

Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or process parameters, which can, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments of the present invention and is not intended to be limiting.

1. Definitions When describing the present invention, the following terms will be used, and are intended to be defined as indicated below. The term "conadotropin-releasing hormone" or "GnRH" refers to a decapeptide secreted by the hypothalamus that controls the release of both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in vertebrates (Fink, G.
, British Medical Bulletin (1979) 35 : 155-160).

The amino acid sequence of GnRH is highly conserved among vertebrates, especially mammals.
I have. In this regard, most of the human, bovine, porcine and ovine GnRHs, including
GnRH from mammals (formally designated as LHRH) has the amino acid sequence pyroGl
u-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH_Two(SEQ ID NO: 1)
(Murad et al., Hormones and Hormones Antagonists, inThe Pharmacologica l Basis of Therapeutics , 6th edition (1980) and Seeburg et al., Nature (1984). 311 : 666-668).

As used herein, a “GnRH polypeptide” is a molecule derived from a native GnRH sequence, as described below, that is substantially homologous to and retains immunogenicity to native GnRH. GnRH polypeptides that are recombinantly produced or chemically synthesized having the amino acid sequence of Thus, the term
Includes derivatives or analogs of GnRH that include additions, substitutions and / or deletions of any single or multiple amino acids present within the peptide or at the amino or carboxy terminus. Therefore, in the present invention, the “GnRH polypeptide” includes a molecule having a natural sequence as well as an analog of GnRH.

Representative GnRH analogs include the following analogs: N-terminal rather than pyroGlu
Analogue with Gln or Glu residue at the end, Asp in place of His at amino acid position 2
Analogs (see Figures 2A and 2B); GnRH analogs with N-terminal additions
For example, Cys-Gly-GnRH (see, for example, the following document: Prendivill)
e et al., Journal Animal Soc. (1995)73: 3030-3037); containing carboxyl
GnRH analogs (see, eg, Jago et al., Journal Ani
mal Soc. (1997)75: 2609-2619; Brown et al., J. Reproduc. Fertil. (1994). 101 : 15-21); GnRH analog (D-Trp6-Pro9-ethylamide) GnRH (for example,
See Tilbrook et al., Hormoes and Behavior (1993).27:
5-28) or (D-Trp6) GnRH (see, for example, the following literature: Chaffaux et al.)
, Recueil de Medecine Veterinaire (1985)161: 133-145);

GnRH analogs having amino acids substituted with the first, sixth and / or tenth commonly occurring Cys and / or N-terminally acylated and / or C-terminally amidated ( See, for example, US Pat. No. 4,608,25.
No. 1 and U.S. Pat. No. 4,975,420); GnRH analog pyroGlu-His-Trp-Ser-
Tyr-X-Leu-Arg-Pro-Gly-YZ (SEQ ID NO: X is a Gly or D-amino acid, Y is one or more amino acid residues which can be the same or different, preferably 1-3 Gly residues, and Z is Cys or Tyr. Is (
UK Patent Publication No. GB2196969);

The GnRH analog described in US Pat. No. 5,688,506, the GnRH analog Cys
-Pro-Pro-Pro-Pro-Ser-Ser-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-
Pro-Gly (SEQ ID NO: ), PyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro
-Gly-Ser-Ser-Pro-Pro-Pro-Pro-Cys (SEQ ID NO: ), PyroGlu-His-Tr
p-Ser-Tyr-Gly-Leu-Arg-Pro-Arg-Pro-Pro-Pro-Pro-Cys (SEQ ID NO:
From Deslorelin, Apeptech, Australia
Commercially available, and Ovuplant^TMGnRH analog, also known as;
Gn with additions, substitutions and / or deletions of other amino acids and
A molecule that retains the ability to trigger the formation of antibodies that cross-react with RH.

Thus, the term “GnRH polypeptide” differs from a reference sequence by having one or more amino acid substitutions, deletions and / or additions, and is at least about 50 amino acids identical to the reference molecule. More preferably about 75-85% identity to the relevant portion of the natural polynucleotide sequence of interest, most preferably about 90%
Include GnRH molecules with 95% identity. The amino acid sequence will have no more than about 1-5 amino acid substitutions, or no more than about 1-3 amino acid substitutions. Particularly preferred substitutions will generally in fact be conserved, ie, substitutions that occur within a family of amino acids. In this regard, 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, cysteine,
Serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.

For example, isolated substitution of leucine by isoleucine or valine, or vice versa; substitution of aspartate by glutamate or vice versa; substitution of threonine by serine or vice versa; or structurally related amino acids Similar conservative substitutions with amino acids can reasonably be expected to have no major effect on activity. Proteins having substantially the same amino acid sequence as the reference molecule, but having small amino acid substitutions that retain the desired activity, are therefore within the definition of a GnRH polypeptide.

“GnRH polypeptide” also refers to a molecule as long as the molecule retains the desired activity.
Includes peptide fragments of the GnRH molecule. Also, the GnRH epitope is captured by definition. A multimer of GnRH comprising a repeat sequence of a GnRH polypeptide, e.g., a multimer comprising 2, 4, 8, 16, 35, and more copies of one or more GnRH polypeptides and optionally a spacer sequence, e.g., Those described in International Publication Nos. WO 98/06848 and WO 96/24675 and shown in FIG. 2B therein are particularly contemplated in the present invention. Such multimers are described more fully below.

For the purposes of the present invention, a GnRH polypeptide can be derived from any of the various known GnRH sequences described above, including but not limited to: human, bovine,
Pigs, sheep, dogs, cats, deer subjects, rodents, eg, hamsters, guinea pigs, gerbils, aardvarks, gophers, lagomorphs, rabbits, ferrets, squirrels, reptiles and birds GnRH polypeptide from which it is derived.

A “GnRH peptide” is a GnRH polypeptide as described herein having a length less than the full length of the reference GnRH molecule in question and comprising at least one epitope as defined below. Thus, a vaccine composition comprising a GnRH peptide will include a portion of the full length molecule, but will not include the entire GnRH molecule in question.
Particular GnRH peptides for use in the present invention are, for example, GnRH peptides having 5, 6 or 7 amino acids, in particular peptides containing an amino or carboxy terminus, for example amino acids 1-5, 1-6 of the native sequence, 1-7, 2-8, 3-8, 3-10, 4-
GnRH peptides, including 10 and 5-10 (eg, International Publication No. WO88 / 0530).
No. 8).

“GnRH multimer” refers to a molecule having two or more copies of a selected GnRH polypeptide, GnRH immunogen, GnRH peptide or epitope, or a multimer of a selected GnRH polypeptide, GnRH immunogen, GnRH peptide or epitope. Means tandem repetition. A GnRH multimer can correspond to a molecule having a repeating unit of the general formula (GnRH-X-GnRH) y, where GnRH is a GnRH polypeptide, X is a peptide bond, an amino acid spacer group, a carrier molecule and [GnRH N is one or more molecules selected from the group consisting of n, wherein n is an integer greater than or equal to 1, y is an integer greater than or equal to 1, and "GnRH" can comprise any GnRH polypeptide.

Thus, y can define 1 to 40 or more repeat units, more preferably 1 to 30 repeat units, and most preferably 1 to 20 repeat units. In addition, the selected G
All of the nRH sequences can be identical, or different derivatives of GnRH,
They can correspond to analogs, variants or epitopes, but they retain the ability to elicit an immune response. Furthermore, if the GnRH unit is chemically or recombinantly attached to the carrier, the GnRH molecule can be attached at the 5'-end, 3'-end, or flank the carrier in question. Furthermore, the GnRH multimer can be located at a site internal to the support. GnRH multimers are discussed in further detail below.

The term “GnRH immunogen” means a GnRH polypeptide, as described above, that elicits an immune response in the absence of an associated immunological carrier, adjuvant or immunostimulator, and comprises a carrier molecule, Immunogenicity, by association with an adjuvant or immunostimulant, or by mutation of the native sequence and / or by incorporation of at least one epitope of the GnRH molecule into a molecule containing multiple repetitive units, or A GnRH polypeptide that can be made more immunogenic is meant.
The term can be used to refer to a solid macromolecule or a homogeneous or heterogeneous population of antigenic macromolecules derived from GnRH.

In general, GnRH immunogens elicit antibodies that cross-react with naturally occurring endogenous GnRH of the vertebrate species to which such immunogens are delivered. The term “GnRH immunogen” also refers to in vitro when administered using the nucleic acid delivery techniques described further below.
A nucleic acid molecule encoding a GnRH polypeptide that can be expressed in vo, e.g.,
DNA and RNA molecules are meant.

“Homology” means the percentage of identity between two nucleotides or two polypeptide moieties. The two DNA or two polypeptide sequences are at least about 75% to 85%, preferably at least about 90%, over a defined length of the molecule.
%, Most preferably at least about 95% to 98%, are "substantially homologous" to each other. As used herein, substantially homologous also refers to sequences that exhibit complete identity to the specified DNA or polynucleotide sequence.

The “identity” percentage between two amino acid or polynucleotide sequences is used to align the sequences, count the exact number of matches between the two aligned sequences, and divide by the length of the shorter sequence. By multiplying the obtained value by 100, it can be determined by directly comparing the sequence information between the two molecules. Analyzes can be facilitated using readily available computer programs, such as: ALIGN, Dayhoff, MO, Atlas of Protein Sequence and Struc.
ture, MO Dayhoff, 5 Suppl. 3 : 353-358, National Research Fou
ndation, Washington D.C. C. This employs the local homology algorithm of Smith and Waterman (1981) Advance in Appl. Math. 2 : 482-489 for peptide analysis.

Programs that determine nucleotide sequence identity, such as the BESTFIT, FASTA and GAP programs, which also rely on the algorithm of Smith and Waterman, are available from the Wisconsin Sequence Analysis Package.
ackage), version 8 (available from the Genetics Computer Group, Madison, WI).

These programs are readily utilized with the default parameters recommended by the manufacturer and described in the aforementioned Wisconsin Sequence Analysis Package. For example, the percent identity of a particular nucleotide sequence to a reference sequence can be determined using the Smith and Waterman homology algorithm with a default score table and a penalty for gaps of 6 nucleotide positions.

“Epitope” means any portion or region of a molecule that has the ability or potential to elicit and bind to a GnRH-specific antibody. For the purposes of the present invention, an epitope of a polypeptide will usually comprise at least about 3 amino acids, preferably at least about 5 amino acids of the reference molecule. There is no critical upper limit on the length of the fragment, and the fragment is
It can comprise approximately the entire length of a fusion protein comprising two or more epitopes.

Since GnRH is a very small molecule, identification of its epitope that can elicit an antibody response can be easily achieved using techniques well known in the art. For example, epitopes in a polypeptide molecule can be identified using a number of epitope mapping techniques that are well known in the art. For example, see the following document: Epitope Mapping Prot
ocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris
Ed., 1996) Humana Press, Totowa, NJ.

For example, a linear epitope synthesizes a number of peptides simultaneously, for example, on a solid support, where the peptides correspond to a portion of a protein molecule and the peptide is attached while the peptide is still attached to the support. Is reacted with an antibody. Such techniques are known in the art and include, for example,
No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81 : 3998-4002; Geysen et al. (1986) Molec. Immun.
ol. 23 : 709-715. Similarly, conformational epitopes are readily identified by determining the spatial conformation of amino acids, for example, by X-ray crystallography and two-dimensional nuclear magnetic resonance. For example, Epitope Mapping Protoc
See ols, supra.

Also, a computer program for formulating a hydropathic scale from the amino acid sequence of a protein utilizing the hydrophobicity and hydrophilicity of each of the 20 amino acids (eg, Kyte et al., J. Mol. Biol. (1982) 157 : 105 −132; and Hopp and Wood
Natl. Acad. Sci. USA (1981) 78 : 3824-3828) can be used to determine the antigenic portion of a given molecule. For example, the Hopp and Woods technique assigns each amino acid to a number of hydrophilicity values and then averages these values repeatedly along the peptide chain. The highest local average hydrophilicity points indicate the antigenic portion of the molecule.

“Immunological carrier” refers to any molecule that, when associated with the GnRH immunogen in question, confers or enhances the molecule's immunogenicity. Examples of suitable carriers are: large, slowly metabolizing macromolecules, eg, proteins; polysaccharides, eg, Sepharose, agarose, cellulose, cellulose beads and others; polymeric amino acids, eg, polyglutamic acid, polystyrene Amino acid copolymers; inactivated virus particles;
Bacterial toxins such as diphtheria, tetanus, cholera, toxoids from leukotoxin molecules, and others. Carriers are described further below.

When a GnRH immunogen is chemically coupled to or associated with a carrier, or when the immunogen is expressed from a chemical DNA molecule encoding the immunogen and the carrier in question, the GnRH immunogen is "Bound" to a particular carrier molecule. "Immune complex" is a GnRH attached to a carrier molecule, as defined above.
An immunogen, such as a GnRH peptide or multimer.

The term “leukotoxin polypeptide” or “LKT polypeptide” refers to carbohydrate
Characterized by the X-terminal consensus amino acid sequence Gly-Gly-X-Gly-X-Asp
Family of molecules (Highlander et al. (1989) DNA8: 15-28)
A polypeptide derived from protein is intended, where X is Lys, Asp, Val or Asn.
is there. Such proteins include, among others: Pasteurella
Haemolytica (P. haemolytica) and Actinobacillus priperum pneumoniae
Leukotoxin from Actinobacillus pleuropneumoniae
Enterobacteriaceae (E. coli) alpha hemolysin (Strathdee et al. (1987) Infect. Immun. 55 : 3233-3236; Lo (1990) Can. J. Vet. Res.54: S33-S35; Welch (199
1) Mol. Microbiol.Five: 521-528).

This family of toxins is known as the “RTX” family of toxins (Lo (1990) Can. J. Vet. Res. 54 : S33-S35). Further, the term "leukotoxin polypeptide" means a leukotoxin polypeptide that has been chemically synthesized, isolated from an organism that expresses it, or produced recombinantly.
Further, the term refers to an immunogenic protein having an amino acid sequence that is substantially homologous to adjacent amino acid sequences found in a particular native leukotoxin molecule. Thus, the term encompasses both full-length and partial sequences, as well as analogs. Whereas natural full-length leukotoxin displays cytotoxic activity, the term "leukotoxin" also refers to molecules that remain immunogenic and that lack the cytotoxic properties of native leukotoxin.

The nucleotide sequences and the corresponding amino acid sequences of some leukotoxins are known. See, for example, U.S. Patent Nos. 4,957,739 and 5,055,400; Lo et al. (1985) Infect. Immun. 50 : 667-67; Lo et al.
987) Infect. Immun. 55 : 1987-1996; Strathdee et al. (1987) Infect. Immun. 55 : 3233-3236; Highlander et al. (1989) DNA 8 : 15-28; and Welch (1991).
) Mol. Microbiol. 5 : 521-528. In a preferred embodiment of the invention, leukotoxin chimeras are provided, wherein the chimeras are selected leukotoxin poly, which confers enhanced immunogenicity to one or more GnRH multimers fused thereto. Has a peptide sequence.

A specific example of an immunogenic leukotoxin polypeptide for use in the present invention is
Truncated leukotoxin molecules described in US Pat. Nos. 5,476,657 and 5,837,268. These truncated molecules include LKT 352, LKT 111 and LKT 114. LKT 352 is derived from the lktA gene present in plasmid pAA352 (ATCC Accession No. 68283). The nucleotide sequence and the corresponding amino acid sequence of this gene are described in US Pat. No. 5,476,657.
This gene encodes a truncated leukotoxin with 914 amino acids and an estimated molecular weight of about 99 kDa.

LKT111 is the lk present in plasmid pCB111 (ATCC Accession No. 69748).
Leukotoxin polypeptide derived from the tA gene. The nucleotide sequence and the corresponding amino acid sequence of this gene are described in US Pat. No. 5,837,268. This gene is a shortened version of leukotoxin generated from the recombinant leukotoxin gene present in plasmid pAA352 (ATCC Accession No. 68283) by removing an internal DNA fragment approximately 1300 bp in length. .

The LKT111 polypeptide has a predicted molecular weight of 52 kDa (compared to a 99 kDa LKT 352 polypeptide), but contains a T cell epitope required for sufficient T cell immunogenicity. And the C-terminal part of LKT 352, which contains the usual restriction sites used for the production of the fusion proteins used in the present invention. LKT114 is derived from a gene present in plasmid pAA114 (described in US Pat. No. 5,837,268). LKT114 differs from LKT111 by having additional amino acids deleted from an internal portion of the LKT111 molecule.

An “immunological adjuvant” acts to increase the immune response to a particular antigen in a non-specific manner, thus reducing the amount of antigen required in any given vaccine, and / or Is a factor that reduces the frequency of injections required to generate an appropriate immune response to the antigen of interest. See, for example, AC Allison, J. Reticuloendothel. Soc. (1979) 26 : 619-630.

A “native” protein, polypeptide or peptide is a protein, polypeptide or peptide isolated from the source in which the protein occurs in nature. "Recombinant" polypeptide refers to a polypeptide produced by recombinant DNA technology; that is, produced from a cell transformed with an exogenous DNA construct encoding the desired polypeptide. "Synthetic" polypeptides are those produced by chemical synthesis.

“Polynucleotide” refers to a sequence of nucleotides including, but not limited to, RNA, eg, mRNA, cDNA, genomic DNA sequences and even synthetic DNA sequences. The term also refers to sequences that include the known base analogs of DNA and RNA.

The term “derived from” as used herein refers to the actual or theoretical source or source of the subject molecule or immunogen. For example, an immunogen "derived from" a particular GnRH molecule will support contiguous sequences similar to the relevant portion of the reference molecule. Thus, an immunogen "derived from" a particular GnRH molecule can include all of the wild-type GnRH sequence or, as long as the derived sequence provides an immunogen corresponding to the targeted GnRH molecule, the amino acid residue It can be modified by insertion, deletion or substitution of groups. An immunogen derived from the indicated molecule will contain at least one epitope specific for the indicated molecule.

“Food producing animal” means an animal intended for consumption by humans or domestic pets, such as cats or dogs. Such animals include, without limitation,
Includes mammalian, eg, sheep, cow, pig, and deer subjects, and includes sheep, cattle, pigs, and deer. "Enhancing the organoleptic quality of edible meat" refers to the edible meat from an animal treated according to the present invention as compared to edible meat from a typical non-castrated member of the same species that is not so treated. It means improving odor, taste and / or fragility.

In general, edible meat from non-castrated males suffers from several disadvantages. In this regard, edible meat from non-castrated boars and sheep often has an unpleasant taste and odor. For example, "boar spoilage" means a urine-like odor found in cooked edible meat of non-castrated pigs. Boar spoilage is produced by stored steroids in tissues in boars with normally functioning testes.
See, for example, Brooks et al., Journal Animal Soc. (1986) 62 : 1279.

The presence of androstenone in non-castrated boars is one of boar spoilage
Two measures, and a measure of gonadal steroid production. In addition, skatole can contribute to boar spoilage. For example, see the following document: Mort
ensen and Sorensen, Proc. 30th European Meeting of Meat Research Workers, Chent Belgium (1986), pp. 394-396, for a method to assay for skatole in fat. Similarly, bulls that do not castrate often produce lean but tough edible meat due to increased muscle mass. Thus, edible meat with improved organoleptic properties has a more desirable odor, fragility and / or
Or edible meat with taste.

“Reduced circulating testosterone” refers to the typical non-castrated, untreated males of the same age and species as measured using a standard assay, for example, the RIA described herein. A statistically significant decrease in serum testosterone levels when compared to expected serum testosterone levels.

“Androgenic” steroids include androstenone, androstenedione,
Androstenediol and / or testosterone. Androgenic steroids can be measured by well-known techniques. For example, testosterone and other androgenic steroids can be measured using ELISAs and RIAs well known in the art.

A particularly advantageous measurement is the use of commercially available test kits such as Coat-A-
Coat-A-Count Total Testesteron Kit
TM (Diagnostic Products Corporation, Los Angeles, CA). This kit is a solid-phase RIA designed for the quantitative measurement of testosterone in serum, based on a testosterone-specific antibody immobilized on a polypropylene tube wall. For a description of a direct RIA for measuring testosterone levels, see the following document: Schanbacher
And D'Occhio, J. Andrology (1982) 3 : 45-51.

An ELISA for measuring androstenone levels is described, for example, in Abouzied et al., J. Agric
Food Chem. (1990) 38 : 331-335. See also: Meloen et al., Vaccine (1994) 12 (8): 741-746; and Booth et al., Anim. P., which describe an ELISA performed on androstenone extracted from fat.
rod. (1986) 42 : 145-152. RIAs that measure androstenone levels are also known. See, for example, Andersen, O., Acta. Vet. Scand. (1979) 20 : 343-350.

“Non-androgenic” steroids are 5α-androstenone (5α-androst-
16-en-3-one) and 16-androstene derivatives. Non-androgenic steroids are well known in the art, for example, EL
It can be measured using ISA and RIA. For example, Claus et al., Archiv fue
r See Lebensmittelhyginene (1988) 38 : 87-90.

A “substantially reduced” level of one or more androgen steroids and / or non-androgenic steroids is defined as a level at which at least one androgen steroid or non-androgen steroid is at the same age and typical for a species. Not castrated, at least less than about 50%, preferably at least less than about 75%, more preferably at least about 80% of the expected level in untreated males
Means 90% or less.

The term “fat period” means the period from weaning to slaughter and thus includes before, around and after the puberty. Typical fattening periods vary from species to species, and even within one species will vary depending on the preferences of the food producing animal and the country in which the animal is raised. Thus, fattening period is primarily a matter of choice, and one of ordinary skill in the art can readily determine an appropriate fattening period for a given animal.

2. General Methods Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulation or process parameters, which can, of course, vary.
It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the present invention only and is not meant to be limiting. Although many compositions and methods similar or equivalent to those described herein can be used in the practice of the present invention, preferred materials and methods are described herein.

The present invention is based on the discovery of a method for improving the quality of edible meat by modulating the secretion of gonadal steroids. This method performs one or more primary immunizations with a GnRH formulation prior to or during the animal's fattening period to measurably reduce circulating testosterone levels, but generally does not result in complete immune castration. Is included. After the primary vaccination, the same or a different composition is boosted just prior to sacrifice to substantially reduce the level of one or more androgenic and / or non-androgenic steroids.

Although GnRH is generally recognized as “self,” and is therefore non-immunogenic, the compositions described herein surprisingly survive appropriate immunological studies in subjects immunized therewith. To provide a means for producing an objective response. The timing of vaccination depends on the animal in question, and such animals are generally sheep, cows or pigs, as well as favorite food-producing animals. However,
The first vaccination will be performed before or during the fattening period of the animal. For example, in pigs and sheep, the primary immunization is generally performed at a time between the birth of the animal and about 15 weeks of age, preferably at a time between the birth of the animal and about 10 weeks of age. In cows, primary immunization is generally performed at a time between the birth of the animal and about 48 weeks of age.

One or more booster treatments are given before sacrifice. The timing of booster administration also depends on the animal in question. For example, in pigs and sheep, booster administration is generally about 1 to about 12 weeks before slaughter, preferably about 2 to about 12 weeks before slaughter.
Eight weeks before, most preferably about 4 to about 6 weeks before slaughter, and even about 3 weeks before slaughter. In cows, it may be preferable to administer the second vaccine several months before slaughter.

In some embodiments, one or more subsequent immunizations are performed after the GnRH antibodies raised against the primary immunization are reduced, ie, at levels below at least about 50% of the maximum antibody level detected. And preferably at least below about 75% of the maximum antibody level detected. The vaccine composition of the present invention uses a GnRH polypeptide, as defined above, optionally linked to a carrier molecule to enhance its immunogenicity.

GnRH Immune Complex As described above, GnRH is an endogenous molecule and as such, by binding GnRH to a carrier to form a GnRH immune complex, a GnRH polypeptide (or a multimer It may be desirable to increase the immunogenicity of This is particularly necessary when administering the GnRH immunogen to the same species from which it is derived.

Suitable carriers are generally proteins derived from infectious agents, for example, an antigenic region of a virus surface protein, or a polypeptide that includes the sequence of a carrier peptide. These carriers serve to non-specifically stimulate the activity of T helper cells, and associate with the molecules of the major histocompatibility complex (MHC) for processing and presentation at the cell surface, which is of interest. The immunogen is converted to antigen-presenting cells (APC
).

[0077] Several carrier systems have been developed for this purpose. For example, a small peptide hapten may be a protein carrier, such as keyhole limpet hemocyanin (Bittle et al. (1982) Nature 298 : 30-33), a bacterial toxin such as a tetanus toxoid (Muller et al. (1982) Proc. Natl. Acad. Sci. USA 79 : 569
-573), often coupled to ovalbumin, leukotoxin polypeptide, and sperm whale myoglobin to generate an immune response. Typically, these coupling reactions result in the incorporation of several moles of peptide hapten / mole of carrier protein.

Other suitable carriers for use with the present invention include rotavirus VP6 polypeptides, or functional fragments thereof, as described in US Pat. No. 5,071,651. Also useful are fusion products of viral proteins with one or more epitopes from GnRH, and these fusion products are described in U.S. Pat.
It is made by the method disclosed in US Pat. No. 4,722,840. Still other suitable carriers include cells, for example, lymphocytes. This is because this form of presentation simulates the natural mode of presentation in the subject, resulting in an immunized state. Alternatively, the GnRH immunogen can be coupled to red blood cells, preferably to the subject's own red blood cells. Methods for coupling peptides to proteins or cells are known in the art.

Delivery systems useful in the practice of the present invention also employ particulate carriers. For example,
Preformed particles have been used as platforms on which immunogens can be coupled and incorporated. Proteosome-based systems (Lowell et al. (1988) Science 240 : 800-802) and immunostimulatory complexes (Morein et al. (1984) Nature 308 : 457-460) are also known in the art.

Also, carrier systems can be used in the present invention using recombinantly produced chimeric proteins that self-assemble into particles. For example, the yeast retrotransposon Ty encodes a series of proteins that assemble like a virus (Ty-V
LP; Kingsman et al. (1988) Vaccine 6 : 304-306). Thus, the gene encoding the GnRH immunogen in question, or a fragment thereof, can be inserted into the TyA gene and expressed as a fusion protein in yeast. This fusion protein retains the ability to self-assemble into uniformly sized particles.

[0081] Other useful virus-like carrier systems are based on: HBsAg (Valenzuela)
(1985) Bio / Technol. 3 : 323-326; U.S. Patent No. 4,722,840; Delpeyroux.
(1986) Science 233 : 472-475), hepatitis B core antigen (Clarke et al. (1988) Vac
cine 88 (editor: H. Ginsberg et al., pp. 127-131), poliovirus (Burke
(1988) Nature 332 : 81-82) and tobacco mosaic virus (Haynes et al. (1986) Bio / Technol. 4 : 637-641).

[0082] Particularly preferred carriers include serum albumin, keyhole limpet hemocyanin, ovalbumin, sperm whale myoglobin, leukotoxin molecules, and other proteins well known to those skilled in the art, as described above. For example, a chimeric system using a leukotoxin polypeptide as defined above, eg, Pasteurella haemlytica fused to the antigen of interest.
) Leukotoxin (LKT) polypeptides can also be used. In this regard, the nucleotide sequences and corresponding amino acid sequences of some leukotoxin carriers are known.

For example, US Pat. No. 5,422,110, US Pat. No. 5,708,155, US Pat.
No. 129 and International Publication No. WO98 / 24675. Particular examples of immunogenic leukotoxin polypeptides for use in the present invention include LKT 342, LKT 352, LKT 111, LKT 326 and LKT 101, which are described in the patents and publications cited above. Have been. LKT 111 and LKT 114 are particularly preferred. The gene encoding LKT111 was generated by removing an internal DNA fragment approximately 1300 bp in length from the recombinant leukotoxin present in pAA352 (ATCC Accession No. 68283).

The LKT111 polypeptide has an estimated molecular weight of 52 kDa (compared to a 99 kDa LKT 352 polypeptide), but contains a T cell epitope required for sufficient T cell immunogenicity. Retains a portion of the N-terminus and the C-terminus of LKT 352, which contains convenient restriction sites for use in producing the fusion proteins of the invention. LKT
114 differs from LKT 111 due to an additional amino acid sequence deletion from an internal portion of the molecule. For a description of these molecules, see, for example, US Patent No. 5,837,268 and International Publication Nos. WO98 / 06848 and WO96 / 24675.

The protein carriers can be used in their natural form, or their functional group content can be modified, for example, by succinylation of lysine residues or by reaction with Cys-thiolactone. Sulfhydryl groups can also be used, for example, by converting the amino function to 2-iminothiolane or the N-
By reacting with a hydroxysuccinimide ester, it can be incorporated into a carrier (or antigen). Suitable carriers can also be modified to incorporate a spacer arm (eg, hexamethylene diamine or other bifunctional molecule of similar size) to attach the peptide immunogen.

The carrier can be physically conjugated to the GnRH immunogen in question using standard coupling reactions. Alternatively, for use in the invention, for example, by fusing a gene encoding a suitable polypeptide carrier to one or more copies of a gene encoding a selected GnRH immunogen, or a fragment thereof, Chimeric molecules can be produced recombinantly. A portion of GnRH can be fused 5 'or 3' to the carrier portion of the molecule, or a portion of GnRH can be located at a site internal to the carrier molecule.

[0087] The GnRH immunogen can also be administered via a carrier virus that expresses the GnRH immunogen. Carrier viruses used in the present invention include, but are not limited to, vaccinia and other poxviruses, adenoviruses, and herpes viruses. As an example, a vaccinia virus recombinant expressing a protein can be constructed as follows. First, it is inserted into a suitable vector such that the DNA encoding the particular protein is adjacent to the vaccinia promoter and flanks the vaccinia DNA sequence, eg, the sequence encoding thymidine kinase (TK).

The vector is then used to transfect cells while simultaneously infecting these cells with vaccinia. Homologous recombination serves to insert the vaccinia promoter and 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 up viral plaques resistant to it.

GnRH Multimers The immunogenicity of a GnRH immunogen can also be significantly increased by producing an immunogenic form of the molecule comprising multiple copies of the selected epitope. In this way, the endogenous radiation hybrid can be an effective autoantigen.

Thus, in one aspect of the present invention, a vaccine composition comprising a multimer of a GnRH immunogen is provided in the form of a nucleic acid or peptide. GnRH Multimer
As noted above, one will have more than one copy of the selected GnRH immunogen, peptide or epitope, or multiple tandem repeats of the selected GnRH immunogen, peptide or epitope. Thus, a GnRH multimer can comprise multiple or tandem repeats of a selected GnRH sequence, multiple or tandem repeats of a selected GnRH epitope, or any possible combination thereof. GnRH epitopes can be identified using the techniques described in detail above.

For example, a GnRH multimer can correspond to a molecule having a repeating unit of the general formula (GnRH-X-GnRH) y, where GnRH is a GnRH immunogen, X is a peptide bond, an amino acid spacer group, One or more molecules selected from the group consisting of 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 And then "Gn
"RH" can comprise any GnRH immunogen. Thus, a GnRH multimer may have from 2 to 64 or more GnRH immunogens, more preferably from 2 to 32 or 2 to 16 GnRH immunogens.
An immunogen can be included.

In addition, all of the selected GnRH immunogenic sequences can be identical or can correspond to different derivatives, analogs, variants or epitopes of GnRH, as long as they retain the ability to elicit an immune response. it can. Furthermore, when the GnRH immunogen is chemically or recombinantly conjugated to a carrier, the GnRH immunogen is 5'-terminal, 3 '
It can be attached to the end or flank the carrier in question. Furthermore, the GnRH multimer can be located at a site internal to the support. One particular carrier for use with the GGnRH multimers of the present invention is a leukotoxin polypeptide described above.

As explained above, a spacer sequence can be present between GnRH molecules.
For example, Ser-Gly-Ser trimers and Gly-Ser dimers are present in the GnRH multimers exemplified herein, which provide spacers between the repetitive sequences of the GnRH immunogen. See, for example, FIG. 2B. Various spacers can be strategically placed between the selected GnRH immunogens to increase immunogenicity to the subject construct.

Thus, in the present invention, the selected spacer sequence can encode a wide variety of components, for example, a single amino acid linker or a sequence of two to more than one amino acid. Since the selected spacer group can preferably provide an enzyme cleavage site, the multimer expressed is processed in vivo by a proteolytic enzyme (APC, or other) to yield a number of polypeptides, each of which is It contains at least one T cell epitope from a carrier moiety and is preferably fused to a substantially complete GnRH polypeptide sequence.

[0095] The binding region between the selected GnRH moieties comprises distinctly foreign sequences to the immunized subject, thereby conferring enhanced immunogenicity to the associated GnRH immunogen As such, a spacer group can be constructed. In addition, spacer sequences can be constructed to provide T cell antigenicity. Examples of such spacer sequences are those encoding amphipathic and / or α-helical peptide sequences that are generally recognized in the art as providing epitopes for immunogenic helper T cells.

The selection of a particular T cell epitope to be provided by such a spacer sequence can vary depending on the particular vertebrate species to be vaccinated.
Specific GnRH moieties including spacer sequences are exemplified, but also directly adjacent GnRH
It is also an object of the present invention to provide one or more GnRH multimers (without intervening spacer sequences) comprising an RH sequence.

[0097] The GnRH multimer sequences thus produced render the GnRH antigen highly immunogenic for use in the compositions of the invention. GnRH polypeptides, immune complexes and multimers can be produced using the methods described below, and can be used for nucleic acid immunization, gene therapy, protein-based immunization methods, and others. it can.

Nucleic Acid-Based Immunization Methods In general, the nucleic acid-based vaccines used in the present invention will contain the relevant regions encoding the GnRH immunogen, appropriate control sequences and, if necessary, Would have a therapeutic nucleotide sequence. The nucleic acid molecule is produced in the form of a vector containing the necessary elements that direct transcription and translation in the recipient cell.

[0099] To enhance the immune response in the immunized subject, the nucleic acid molecule may be combined with ancillary substances, such as pharmacological agents, adjuvants, or may encode biological response modifiers, such as cytokines and the like. Can be administered in combination with delivery of the vector. Other auxiliary substances include, but are not limited to, substances that increase weight gain, muscle mass or muscle strength, such as growth hormones, growth promoting factors, beta antagonists, partitioning factors and antibiotics .

The nucleotide sequence selected for use in the present invention may be from known sources,
For example, they can be isolated from cells or tissues containing the desired gene or nucleotide sequence according to standard techniques, or can be derived by recombinant or synthetic techniques.

[0101] Once the coding sequence for the GnRH immunogen has been produced or isolated, such a sequence can be cloned into any suitable vector or replicon. Numerous cloning vectors are known in the art, and the selection of an appropriate cloning vector is a matter of choice. The attachment to other sequences, such as auxiliary molecules or carrier molecules, is performed by techniques known in the art. One or more GnRH immunogenic portions of the chimera can be fused 5 'and / or 3' to the desired auxiliary sequence or carrier molecule. Or 1
Alternatively, more GnRH immunogen moieties can be located at sites internal to the carrier molecule, or such moieties can be located at both terminal or internal locations of the chimera.

Alternatively, the DNA sequence encoding the GnRH immunogen in question can be produced synthetically, rather than cloned. Using the appropriate codons for a particular sequence,
A sequence can be designed. The complete sequence of the immunogen is then assembled from overlapping oligonucleotides produced by standard methods to construct a complete coding sequence. For example, see the following reference: Edge (1981) Natu
re 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 a control element suitable for in vivo expression in a suitable host tissue. The choice of control factor will depend on the subject to be treated and the type of preparation used. Thus, where the immunogen is expressed using the subject's endogenous transcription and translation machinery, a control factor that is compatible with the particular subject will be utilized. In this regard, several promoters for use in mammalian systems are known in the art.

For example, typical promoters for expression in mammalian cells include, among others, the following: SV40 early promoter, CMV promoter such as CMV immediate early promoter, mouse mammary tumor virus LTR promoter, Adenovirus major late promoter (Ad MLP), and herpes simplex virus promoter. Also, other non-viral promoters, such as those derived from the murine metallothionein gene, will be used for mammalian expression.

Typically, transcription termination and polyadenylation sequences will also be present and will be located 3 ′ to the translation stop codon. Preferably, there is also a sequence that optimizes translation initiation, located 5 'to the coding sequence. Examples of transcription terminator / polyadenylation signals include those derived from SV40, as described in Sambrook et al., Supra, as well as bovine growth hormone terminator sequences. Also, introns containing splice donor and acceptor sites can be designed into the constructs used in the present invention.

[0106] Enhancer elements can also be used in the present invention to increase the expression level of the construct. Examples of these are: SV40 early gene enhancer (Dijkema et al. (1985) EMBO J. 4 : 761), enhancer / promoter from the Rous sarcoma virus long terminal repeat (LTR) (Gorman et al. (1982)). Proc.
Natl. Acad. Sci. USA 79 : 6777) and a factor derived from human CMV (Boshart
(1985) Cell 41 : 521), for example, factors contained in the CMV intron A sequence.

Once manufactured, the nucleic acid vaccine composition can be delivered to a subject according to known methods. In this regard, various techniques using DNA encoding the antigen have been described. See, for example, US Pat. No. 5,589,466.
No. (Felgner et al.); Tang et al. (1992) Nature 358 : 152; Davis et al. (1993) Hum. Mo
l. Genet. 2 : 1847; Ulmer et al. (1993) Science 258 : 1745; Wang et al. (1993) Pr.
USA 90 : 4156; Eisenbraun et al. (1993) DNA Cell B. oc. Natl. Acad. Sci.
iol. 12 : 791; Fynan et al. (1993) Proc. Natl. Acad. Sci. USA 90 : 12476.
Fuller et al. (1994) AIDS Res. Hum. Retrovir. 10 : 1433; and Raz et al.
94) Proc. Natl. Acad. Sci. USA 91 : 9519.

General methods for delivering nucleic acid molecules into cells in vitro for subsequent reintroduction into a host can also be used, for example, liposome-mediated gene transfer. See, for example, Hazinski et al. (1991) Am. J. Respir. Cell Mol. Biol. 4 : 206-209; Brigham et al. (1989) Am. J. Med. Sci. 298 : 278-28.
1; Canonico et al. (1991) Clin. Res. 39 : 219A; and Nabel et al. (1990) Science 249 : 1285-1288. Thus, nucleic acid vaccine compositions can be delivered in liquid or particulate form according to various known techniques. Exemplary vaccine compositions are described more fully below.

Protein-Based Delivery Methods Protein-based compositions can also be prepared according to various methods known in the art. In particular, GnRH polypeptides can be isolated directly from natural sources by standard purification techniques. Alternatively, the polypeptides can be produced recombinantly using nucleic acid expression systems well known in the art and described, for example, in Sambrook et al., Supra. GnRH polypeptides can also be synthesized using chemical polymer synthesis, for example, solid phase peptide synthesis.

[0110] Such methods are known in the art. For solid phase peptide synthesis techniques, see, for example, the following references: JM Stewart and JD Young
Solid Phase Peptide Synthesis, Second Edition, Pierce Chemical Co., Rockford, Ill. (1984) and G. Barany and RB Merrifield, The Peptides: Analysis, Synthesis, Biology, E. Gross and J. Meienhofer, Vol. 2, Academic Press, New York, (1980), pp. 3-254.

[0111] GnRH polypeptides for use in the compositions described herein can also be produced by cloning the coding sequence into any suitable expression vector or replicon. Numerous cloning vectors are known in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of DNA vectors for cloning and host cells that can transform them are as follows:

Bacteriophage lambda (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT320 (Gram negative bacteria), pGV1106 (Gram negative bacteria), pLAHR1 (
Gram-negative bacteria), pME290 (non-E. Coli Gram-negative bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6
(Streptomyces), YIp5 (Saccharomyces), YCp19 (Saccharomyces) and bovine papillomavirus (mammalian cells). See generally, the following references: DNA C
loning: Vol. I and II, supra; Sambrook et al., supra; B. Perbal, supra.

For example, the coding sequences of porcine, bovine and ovine GnRH have been determined (Murad
(1980) Hormones and Hormone Antagonists, The Pharmaceutical Basis of Therapeutics , 6th ed.), And the cDNA of human GnRH has been cloned and its sequence is well established (Seeburg et al. (1984) Nature 311 : 666). −668
).

Additional GnRH polypeptides of known sequence, such as GnRH molecules present in salmon and chicken, have been disclosed (WO No. WO86 / 07383, published December 18, 1986). The GnRH coding sequence used in the present invention is shown herein in FIGS. 2A and 2B. The GnRH coding sequence is highly conserved in vertebrates, especially mammals, and the porcine, bovine, ovine and human GnRH sequences are identical to each other.

[0115] Portions of these sequences encoding the desired GnRH polypeptide, and optionally, the sequence encoding the carrier protein, may be cloned, isolated, and ligated, generally according to recombinant techniques known in the art. can do. For example, Sa
See mbrook et al., supra.

The gene comprises a promoter, a ribosome binding site (for bacterial expression) and
If necessary, the DN in question is located under operator control
The A sequence is translated into RNA by a suitable transformant. The coding sequence may or may not contain a signal peptide or leader sequence.
The polypeptide may be, for example, an E. coli tac promoter or protein A.
Expression can be achieved using a gene (spa) promoter and signal sequence. The leader sequence can be removed by the bacterial host during post-translational processing. See, for example, U.S. Pat. No. 4,431,739; U.S. Pat. No. 4,425,437; U.S. Pat. No. 4,338,397. Also, auxiliary sequences, such as those described above, can be present.

[0117] In addition to control sequences, it may be desirable to add regulatory sequences that allow for regulation of the expression of the polypeptide sequence with respect to growth of the host cell. Regulatory sequences are known in the art, and examples thereof include regulatory sequences that turn on or off expression of a gene in response to a chemical or physical stimulus, including the presence of a regulatory compound. Also, other types of regulatory elements, such as enhancer sequences, can be present in the vector.

The expression vector is constructed as follows so that the particular coding sequence is located in the vector along with the appropriate regulatory sequences, and the positioning and orientation of the coding sequence relative to the control sequence is such that the coding sequence is It is such that it is transcribed under "control" (ie, RNA polymerase that binds to a DNA molecule at the control sequence transcribes the coding sequence). To this end, modification of the sequence encoding a particular GnRH polypeptide is desirable.

For example, in some cases, it may be necessary to modify the sequence so that it can be linked to the control sequence in the proper orientation, ie, maintain the reading frame. Control and other regulatory sequences can be ligated to the coding sequence prior to insertion into the vector, eg, a coding vector as described above. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and appropriate restriction sites.

In some cases, it may be desirable to add a sequence that secretes the polypeptide from the host organism and subsequently cleave the secretion signal. It may also be desirable to produce mutants or analogs of the polypeptide. By deleting portions of the sequence encoding the reference polypeptide, or, if present, portions of the sequence encoding the desired carrier molecule, by inserting the sequence, and / or by removing one or more of the sequences within the sequence. By substituting nucleotides, mutants or analogs can be produced.

Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, and others, are well known to those skilled in the art. See, for example, Sambro
ok et al., supra; DNA Cloning, Vol. I and II, supra; Nucleic Acid Hybrid
Kunkel, TA, Proc. Natl. Acad. Sci. USA (1985) 82 :
448; Geisselsoder et al., BioTechniques (1987) 5 : 786; Zoller and Smith, Met.
Hods Enzymol. (1983) 100 : 468; Dalbie-McFarland et al., Proc. Natl. Acad. Sci. USA (1982) 79 : 6409.

GnRH polypeptides can be expressed in a wide variety of systems, including insect, mammalian, bacterial, viral and yeast expression systems, all of which are well known in the art. I have. For example, insect cell expression systems, such as baculovirus expression systems, are known in the art, and include, for example, Summer
s and Smith, Texas Agricultural Experiment Station Bulletin No. 1
555 (1987).

Materials and methods for baculovirus insect cell expression systems are commercially available, inter alia, from Invitrogen, San Diego, Calif. In kit form (“MaxBac”). Similarly, bacterial and mammalian cell expression systems are well known in the art and are described, for example, in Sambrook et al., Supr.
It is described in a. Yeast expression systems are also known in the art,
And for example, Yeast Genetic Engineering (Barr et al., Ed., 1989) London,
Butterworth.

[0124] Also, a number of suitable host cells for use with the above systems are known.
For example, mammalian cell lines are known in the art, and are available from the American Type Culture Collection (ATCC).
Including the following, including but not limited to: Chinese hamster ovary (CHO) cells, HeL
a cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, Hep G2), Madin-Darby bovine kidney (“M
DBK ') cells, and others.

Similarly, bacterial hosts such as E. coli, Bacill subtilis (Bacill)
us subtilis) and Streptococcus species will be used in the expression constructs of the present invention. Yeast hosts useful in the present invention include, among others, the following: Saccharomyces ces
revisiae), Candida albicans (Candida albicans), Candida maltosa (Candida maltosa), Hansenula polymorpha (Hansenula polymorpha),
Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillermondii, Pichia pastoris, Schizosaccharomyces pombi and Schizosaccharomy ants Polika (Yarr)
owia lipolytica).

Insect cells for use with the baculovirus expression system include, among others, the following: Aedes aegypti, Autographa californica, Bombyx mori,
Drosophila melanogaster, Shigella flexneri, and Trichoderma ni.

Depending on the expression system and host selected, Gn is grown by growing host cells transformed with the aforementioned expression vectors under conditions in which the polypeptide is expressed.
An RH polypeptide is produced. The expressed polypeptide is then isolated from the host cell and purified. If the expression system secretes the polypeptide into the growth medium, the product can be purified directly from the medium. If not secreted, it can be isolated from cellular lysates. Selection of appropriate growth conditions and methods of recovery are within the skill of those in the art.

Once obtained, the GnRH polypeptide, with or without an associated carrier, can be formulated into a composition, eg, a vaccine composition, as further described below, to elicit the production of antibodies. it can.

Production of Antibodies The subject GnRH immunogen can be used to generate antibodies for use in passive immunization methods. Typically, peptides useful for the production of antibodies will usually be at least about 3-5 amino acids in length, preferably 7-10 amino acids in length.

Antibodies to the subject immunogens include polyclonal and monoclonal antibody preparations, monospecific antisera, and preparations that include the following: hybrid antibodies, modified antibodies, F (ab ') ₂ fragments, F (ab) fragment,
Fv fragments, single domain antibodies, chimeric antibodies, humanized antibodies, and functional fragments thereof that retain specificity for the target molecule of interest. For example, antibodies can include variable regions, or fragments of variable regions, that retain specificity for the molecule in question. The remainder of the antibody can be derived from the species using the antibody.

Thus, if the antibody is to be used in humans, the antibody can be "humanized" so as to reduce immunogenicity and still retain activity. For a description of chimeric antibodies, see, for example, the following references: Winter, G. and Milstein, C .;
(1991) Nature 349 : 293-299; Jones, PT et al. (1986) Nature 321 : 522-52.
5; Riechman, L. et al. (1988) 332 : 323-327; and Carter, P. et al. (1992) Proc.
Natl. Acad. Sci. USA 89 : 4285-4289. Such chimeric antibodies contain not only binding sites for the target molecule, but also binding sites for other proteins. In this way, bifunctional reagents with targeted specificity for both external and internal antigens can be generated.

If a polyclonal antibody is desired, a selected animal (eg, mouse, rabbit, goat, horse, and others) is immunized with the desired antigen, or a fragment thereof, or a mutated antigen, as described above. . Prior to immunization, it may be desirable to further increase the immunogenicity of the particular immunogen. This can be achieved by one of several methods known in the art.

For example, immunization for the production of antibodies may be achieved by combining the protein with a suitable excipient, for example,
Saline, preferably an adjuvant, such as Freund's complete adjuvant,
Alternatively, immunization for the production of antibodies is generally performed by mixing or emulsifying in any of the adjuvants described below and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly).

In general, animals are boosted 2-6 weeks later with one or more injections of the protein in saline, preferably using Freund's incomplete adjuvant or otherwise. Antibodies can also be generated by in vitro immunization, using methods known in the art. A polyclonal antiserum is then obtained from the immunized animal and processed according to known procedures. For example, Ju
See rgens et al. (1985) J. Chrom. 348 : 363-370. When using a serum containing a polyclonal antibody, the polyclonal antibody is purified by immunoaffinity chromatography according to a known method.

[0135] Monoclonal antibodies are generally described in Kohler and Milstein, Nature (1975) 256
: Produced by the method of 495-96, or a modification thereof. Typically, a mouse or rat is immunized as described above. However, rather than exsanguinating the animal to extract serum, the spleen (and optionally some large lymph nodes)
Remove and dissociate into single cells. If desired, splenocytes can be screened (after removing non-specific adherent cells) by applying the cell suspension to a plate or well coated with the protein antigen.

B cells expressing membrane-bound immunoglobulin specific for the antigen bind to the plate and are not rinsed away from the rest of the suspension. The resulting B cells or all dissociated splenocytes are then induced to fuse with the myeloma cells to form hybridomas, and to select media (eg, hypoxanthine, aminopterin, thymidine media, "HA
T "). The resulting hybridomas are plated by limiting dilution and assayed for the production of antibodies that bind specifically to the immunizing antigen (and not to unrelated antigens). The hybridoma secreting the selected monoclonal antibody is then cultured in vitro (eg, in a tissue culture bottle or hollow fiber reactor) or in vivo (as ascites in mice).

See, for example, M. Schreier et al., Hybridoma Techniques.
(1980); Hammerling et al., Monoclonal Antibodies and T-cell Hybrido
mas (1981); Kennet et al., Monoclonal Antibodies (1980); see also the following U.S. Patents: U.S. Patent Nos. 4,341,761; 4,399,121; 4,427,783;
No. 4,444,887; No. 4,452,570; No. 4,466,917; No. 4,472,500; No. 4,491,632
No .; and 4,493,890. Panels of monoclonal antibodies produced against the GnRH immunogen in question, or fragments thereof, can be screened for various properties, ie, for isoforms, epitopes, affinity, and the like.

Also, functional fragments of the antibody are generated against the GnRH immunogen in question, and the constant regions unrelated to antigen binding are cleaved from the antibody molecule, eg, using pepsin.
F (ab ') ₂ fragments can be produced. These fragments contain two antigen binding sites, but will lack a portion of the constant region from each of the heavy chains. Similarly, if desired, Fab fragments comprising a single antibody binding site can be produced, for example, by digestion of the polyclonal or monoclonal antibodies with papain. Functional fragments containing only the variable regions of the heavy and light chains can also be produced according to standard techniques. These fragments are known as Fv.

The subject immunogens can also be used to produce chimeric or humanized antibodies. Design these antibodies to minimize undesired immunological reactions that contribute to the heterologous constant regions and species-specific framework variable regions typically present in monoclonal and polyclonal antibodies. be able to.

For example, if the antibody is to be used in a human subject, the non-human constant regions in both the heavy and light chains, both heavy and light chains, are replaced with human constant regions, generally using techniques known in the art. By doing so, a chimeric antibody can be generated. See, for example, Winter, G. and Milstein, C. (1991) Nature 349 : 293-299; Jones, PT et al. (1986) Nature 321 : 522-525; Riechman, L.
Et al. (1988) 332 : 323-327; and Carter, P. et al. (1992) Proc. Natl. Acad. Sci. USA 89 : 4285-4289.

GnRH Compositions Once the GnRH polypeptides or antibodies have been produced, they are formulated into a composition for delivery to a vertebrate subject. The GnRH molecule of interest 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.

In addition, vehicles contain small amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants in the case of vaccine compositions, which will enhance the effectiveness of the vaccine. Suitable adjuvants are described further below. The compositions of the present invention can also include auxiliary substances, such as pharmacological agents, cytokines, or other biological response modifiers.

As explained above, the vaccine compositions of the present invention can include an adjuvant to further increase the immunogenicity of the GnRH immunogen. Adjuvants include, for example, emulsifiers, muramyl dipeptides, avidin, aqueous adjuvants such as ammonium hydroxide and any of a variety of saponins, chitosan-based adjuvants, oils, and other materials known in the art. can do. For example, compounds that can serve as emulsifiers herein include natural and synthetic emulsifiers, as well as anionic, cationic, and nonionic compounds.

Among the synthetic compounds, anionic emulsifiers include, for example, potassium, sodium and ammonium salts of lauric and oleic acids, calcium salts of fatty acids,
Includes magnesium and aluminum salts (ie, metal soaps), and organic sulfonates, such as sodium lauryl sulfate. Synthetic cationic agents include, for example, cetyltrimethylammonium bromide, while synthetic nonionic agents include glyceryl esters (eg, glyceryl monostearate), polyoxyethylene glycol esters and ethers, and sorbitan fatty acid esters (eg, sorbitan) Monopalmitates) and their polyoxyethylene derivatives (eg, polyoxyethylene sorbitan monopalmitate). Natural emulsifiers include gum acacia, gelatin, lecithin and cholesterol.

Other suitable adjuvants can be formed using an oil component, for example, a single oil, a mixture of oils, a water-in-oil emulsion, or an oil-in-water emulsion. The oil can be a mineral, vegetable, or animal oil. Mineral oils or oil-in-water emulsions in which the oil component is a mineral oil are preferred. In this context, "mineral oil" is defined herein as a mixture of liquid hydrocarbons obtained from petrolatum by distillation techniques; the term is synonymous with "liquid paraffin", "liquid petrolatum" and "white mineral oil".

The term is also intended to encompass "light mineral oils", ie, oils which are also obtained by distillation of petrolatum, but have a slightly lower specific gravity than white mineral oil. See, for example, Remington's Pharmaceutical Sciences, supra. A particularly preferred oil component is an oil-in-water emulsion sold under the trade name EMULSIGEN PLUS ^™ , comprising light mineral oil and 0.05% formalin as preservative, and 30 μg / ml gentamicin (MVP Laboratories, Ralston, Nebraska, Available from

Suitable animal oils include, for example, cod liver oil, halibut oil, menhaden oil, orange loughy oil and shark liver oil, all of which are commercially available. Suitable vegetable oils include, but are not limited to, canola oil, almond oil, cottonseed oil, corn oil, olive oil, peanut oil, safflower oil,
Includes sesame oil, soybean oil, and others.

Alternatively, a number of aliphatic nitrogen-based bases can be used as adjuvants with the vaccine formulation. For example, known immunogenic adjuvants include amines, quaternary ammonium compounds, guanidine, benzamidine and thiouronium compounds (Gall, D. (1966) Immunology 11 : 369-386).
). Certain compounds are identified as dimethyldioctadecyl ammonium bromide (DDA) (K
odak) and N, N-dioctadecyl-N, N-bis (2-hydroxyethyl) propanediamine ("aviridin").

The use of DDA as an immunological adjuvant has been described; for example, see the following: the Kodak Laboratory Chemicals Bulletin 56 (1): 1-5.
(1986); Adv. Drug Deliv. Rev. 5 (3): 163-187 (1990); J. Contro
lled Release 7 : 123-132 (1988); Clin. Exp. Imuunol. 78 (2): 256.
-262 (1989); J. Immunol. Methods 97 (2): 159-164 (1987); Immunol
ogy 58 (2): 245-250 (1986); and Int. Arch. Allergy Appl. Immun.
ol. 68 (3): 201-208 (1982).

[0150] Aviridine is also a well-known adjuvant. See, for example, U.S. Pat. No. 4,310,550 (Wolff, III et al.), Which generally comprises N, N-higher alkyl-N ', N'-(2-hydroxyethyl) propanediamine as a vaccine adjuvant.
In particular, aviridine is described. Also, US Pat. No. 5,151,267 (Babiuk),
And Babiuk et al. (1986) Virology 159 : 57-66 relate to the use of aviridine as a vaccine adjuvant.

[0151] A modified form of the EMULSIGEN PLUS ^™ adjuvant, including DDA, "VSA
An adjuvant known as "-3" is particularly preferred for use in the present invention (see, granted US Patent Application No. 08 / 463,837). The actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art. See, for example, Remington's Pharm
aceutical Sciences, Easton, PA, 18th edition, 1990. The composition or formulation to be administered will contain an appropriate amount of GnRH polypeptide to achieve the desired condition in the subject being treated.

The compositions of the present invention are usually prepared as a liquid solution or suspension, or as a solid form suitable for solution or suspension in a liquid vehicle prior to injection. The preparation can also be emulsified or the active component encapsulated in a liposomal vehicle or other particulate carrier for use.

[0153] The compositions can also be manufactured in solid form. For example, solid particulate formulations can be manufactured for delivery from commercially available needleless injection devices. Alternatively, a solid form of the implant can be provided for implantation into a patient. Alternatively, control or sustained release formulations can be used, and these are prepared by incorporating the GnRH polypeptide into a carrier or vehicle. Such carriers or vehicles are, for example, liposomes, non-absorbent impermeable polymers such as vinyl ethyl acetate copolymers and Hytrel ^R copolymers, swellable polymers such as hydrogels or absorbent polymers, such as collagen and certain Polyacids or polyesters, such as those used to make absorbable sutures.

In addition, polypeptides can be formulated in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino group of the active polypeptide), and these include inorganic acids such as hydrochloric acid, phosphoric acid, or organic acids such as, for example,
Formed with acetic acid, oxalic acid, tartaric acid, mandelic acid, and others. Salts formed from free carboxyl groups also include inorganic bases, such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, 2-ethylaminoethanol , Histidine, procaine, and others.

The composition is formulated to contain an effective amount of a GnRH polypeptide, the exact amount being readily determined by one of skill in the art, where the amount is the amount of the animal to be treated, the antibody, in the case of a vaccine composition. Depends on the ability of the animal's immune system to synthesize GnRH and the desired degree of immune neutralization of GnRH. For purposes of the present invention, from about 1 μg to about 2 mg, more usually about 5 μg
Formulations containing from g to about 800 μg, and even more particularly from 10 μg to about 400 μg, of an injection solution of GnRH polypeptide / ml will be suitable for generating an immunological response when administered. When using peptide-carrier chimeras, the immunogen / carrier ratio in the vaccine formulation will vary based on the particular carrier and immunogen chosen to construct such a molecule.

For example, when using a leukotoxin-GnRH chimera, the immunogen / leukotoxin ratio in the vaccine formulation is based on the particular leukotoxin and GnRH polypeptide components selected to construct such a molecule. Will change. One preferred vaccine composition comprises leukotoxin-Gn having about 1 to about 90% GnRH, preferably about 3 to 80%, and most preferably about 10 to 70% GnRH polypeptide / fusion molecule.
Contains RH chimera. As the percentage of GnRH present in the LKT-GnRH fusion increases, the total amount of antigen that must be administered to the subject to elicit a sufficient immunological response to GnRH decreases.

A subject is administered one of the foregoing compositions, eg, in a primary immunization, during the fattening period, in at least one dose, and optionally two or more doses. . After one or more primary immunizations, shortly before sacrifice, the same or different GnRs
The H composition is boosted one or more times to substantially reduce the circulating levels of one or more androgenic steroids and / or non-androgenic steroids.

The composition can be delivered to the vertebrate subject using any suitable pharmaceutical delivery means. For example, conventional needle syringes, spring or compressed gas (air) syringes (US Pat. No. 1,505,763 (Smoot); US Pat. No. 3,788,315 (Laurens); US Pat. No. 3,853,125 (Clark et al.); US Pat. No. 4,596,556 (Morrow et al.); And U.S. Pat. No. 5,062,830 (Dunlap); liquid injection syringes (U.S. Pat. No. 2,754,818 (Scherer); U.S. Pat. No. 3,330,276 (Goudon); and U.S. Pat. No. 4,518,385.
(Lindmayer et al.), And a particle syringe (U.S. Pat. No. 5,149,655 (McCabe et al.)
And US Patent No. 5,204,253 (Sanford et al.) Are all suitable for delivery of the composition.

The compositions are administered to a subject intramuscularly, subcutaneously, intravenously, subcutaneously, intradermally, transdermally, or transmucosally. When using a jet syringe, a single jet of the liquid vaccine composition is injected under high pressure and speed, e.g., under 1200-1400 psi, thereby creating an opening in the skin and a suitable depth for immunization. Let it penetrate. Examples of specific embodiments for practicing the invention are described below. These examples are for illustrative purposes only and are not intended to limit the scope of the invention.

3. Experimental Example 1 Construction of pCB122 and pCB130 Plasmids pCB122 and pCB130 were used to produce GnRH fusion proteins for use in the Examples below. Both plasmids produce proteins with identical amino acid sequences. The GnRH construct in both plasmids contains the GnRH fused to the 5 'and 3' ends of the DNA sequence encoding the carrier leukotoxin polypeptide.
Contains eight tandem repeats of the sequence. Each alternate GnRH identity has a fourth base change in the sequence from cysteine to guanosine. This allows GnR from His to Asp
A single amino acid change of the second amino acid of the H molecule occurs.

See FIG. 2A and FIG. 2B. The leukotoxin portion of the construct encodes a truncated version of the leukotoxin, which is obtained by removing an internal fragment approximately 1300 bp in length by the removal of plasmid pAA352 (ATCC Accession No. 68283, US Pat.
No. 6,657) (as described in US Pat. No. 6,657). Leukotoxin polypeptides have a predicted molecular weight of 52 kDa and contain convenient restriction sites for use in producing fusion proteins of the invention. The chimeric construct is under control of the Tac promoter and induction is L
Controlled by use of acI. The GnRH-leukotoxin fusion proteins produced by plasmids pCB122 and pCB130 are shown in FIGS. 3A-3F.

[0162] Plasmid pCB122 was produced as follows. The leukotoxin gene was isolated as described in US Pat. Nos. 5,476,657 and 5,837,268. In particular, to isolate the leukotoxin gene, Pasteurella haemolytica (
A gene library of P. haemolytica) A1 (B122 strain) was constructed according to standard techniques. See the following references: Lo et al., Infect. Immun., Supra; DNA CLONING.
Vol. I and II, supra; and Sambrook et al., Supra. A genomic library was constructed in plasmid pUC13 and a DNA library was constructed in bacteriophage lambda gt11.

The resulting clones are used to transform E. coli, individual colonies are pooled, and survive P. haemolytica infection and survive P. haemolytica. The concentrated culture supernatant was boosted and screened for reaction with serum from calves that had increased anti-leukotoxin antibody levels. By incubating the cell lysate with bovine neutrophils and subsequently measuring the release of lactate dehydrogenase from the latter,
Positive colonies were screened for the ability to produce leukotoxin.

Several positive colonies were identified and these recombinants were analyzed by restriction endonuclease mapping. One clone appeared to be identical to the previously cloned leukotoxin gene. Lo et al., Infect. Immun
., See supra. To confirm this, a small fragment was recloned and the restriction maps were compared. It was determined that approximately 4 kilobase pairs of DNA had been cloned. Chromosome walking (5 '→ 3) to isolate a full-length recombinant, approximately 8 kb long
'Direction), progressively larger clones were isolated. The last construct was named pAA114. This construct contained the entire leukotoxin gene sequence.

LktA, a MaeI restriction endonuclease fragment from pAA114 containing the entire leukotoxin gene, was treated with the Klenow fragment of DNA polymerase I + nucleotide triphosphate and ligated into the SmaI site of the cloning vector pUC13. . This plasmid was designated as pAA179. From this, two expression constructs were made in the ptac-based vector pGH432: lacI digested with SmaI. One pAA342 is l
It consisted of the 5'-AhaIII fragment of the ktA gene, whereas the other pAA345 contained the entire MaeI fragment described above.

Clone pAA342 expressed high levels of truncated leukotoxin peptide, while pAA345 expressed very low levels of full length leukotoxin. Therefore, the 3 'end of the lktA gene (StyI BamHI fragment from pAA345) was replaced with StyI BamHI.
Ligation to HI digested pAA342 generated plasmid pAA352. Pasteurella haemolytica (P. haemolytica) leukotoxin produced from the pAA352 construct was subsequently transformed into LKT 35
Call it 2.

[0167] A shortened version of the recombinant leukotoxin polypeptide was then produced using plasmid pAA352. A truncated LKT gene was produced by deleting an internal DNA fragment of approximately 1300 bp length from the recombinant LKT gene as follows. LK
Plasmid pCB113 containing the T352 polypeptide (ATCC Accession No. 69749, described in US Pat. No. 5,837,268) was replaced with the restriction enzyme BstB1 (New England Biol.
abs).

The resulting linearized plasmid was then digested with Mung bean nuclease (Pharmacia) to remove the single-stranded protruding ends generated by BstB1 digestion. Then smooth D
NA was digested with the restriction enzyme NdeI (New England Biolabs) and the digested DNA was loaded on 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 an agarose gel using the Gene Clean kit (Bio 101) and the purified fragment itself was purified using bacteriophage T4 DNA ligase (Pharmacia). Was bound.

The resulting ligation mixture was used to transform competent E. coli JM105 and positive clones were identified by their ability to produce aggregated proteins of appropriate molecular weight. The recombinant plasmid thus formed was replaced with pCB111 (ATCC accession number).
No. 69748), which produces a truncated leukotoxin polypeptide (hereinafter LKT111) fused to four copies of the GnRH polypeptide. Plasmid pCB114
It has a multiple copy GnRH sequence inserted twice (corresponding to the oligomer in FIG. 2B). Both of these plasmids are described in US Pat. No. 5,837,268 and produce truncated leukotoxin polypeptides designated LKT111 and LKT114, respectively.

The LKT obtained from the pCB114 plasmid was obtained by ligating the multiple copy GnRH sequence (corresponding to the oligomer in FIG. 2B) twice to the 5 ′ end of the LKT114 coding sequence.
From the GnRH fusion sequence, one is located at the N'-terminus of LKT114 and the other is LKT1
Recombinant LKT-G with two 8-copy GnRH multimers located at the 14 C'-terminus
An nRH fusion molecule was constructed. Synthetic nucleic acid molecule having the following nucleotide sequence: 5'-ATG
GCTACTGTTATAGATCGATCT-3 '(SEQ ID NO: ) Was attached to the 5 'end of the multiple copy GnRH sequence.

The synthetic nucleic acid molecule has an eight amino acid sequence (Met-Ala-Thr-Val-Ile-Asp-Arg-Ser)
(Sequence number Code). The resulting recombinant molecule thus contains, in the order described in the 5 ′ → 3 ′ direction: a synthetic nucleic acid molecule; a nucleotide sequence encoding the first eight copies of the GnRH multimer; encoding a truncated LKT peptide (LKT114) And a nucleotide sequence encoding the 28th copy GnRH multimer.

The recombinant molecule is circularized and the resulting molecule is used to competent E. coli.
) JM105 cells were transformed. Positive clones were identified by their ability to produce an aggregate production having a molecular weight of approximately 74 kDa. The recombinant plasmid thus formed was replaced with pCB1
Designated as 22, which produces an LKT114 polypeptide fused to 16 copies of the GnRH polypeptide. For plasmid pCB130, the Ampr gene or pCB122 was replaced with the TetR gene. Thus, the plasmid is under tetracycline selection. The nucleotide sequences of the recombinant LKT-GnRH fusions of plasmids pCB122 and pCB130 are shown in FIGS. 3A-3F.

Embodiment 2 FIG. Purification of LKT- antigen fusion The recombinant LKT-GnRH fusion from Example 1 was purified by the following procedure. 100μg / ml
In 10 ml of TB broth supplemented with Ampicillin, transformed E. coli (E.
coli) strain and inoculate at 37 ° C. on a G10 shaking incubator at 220 rpm.
Incubated for hours. 4 ml of this culture was diluted into each of two baffle Fernbach flasks containing 400 ml of TB broth + ampicillin and incubated overnight as described above.

Cells were collected by centrifugation at 4,000 rpm for 10 minutes in a 500 ml polypropylene bottle using a Sorvall GS3 rotator. 37 ℃
An equal volume of TB broth containing pre-warmed ampicillin (ie, 2 × 400
The pellet was resuspended and the cells were incubated for 2 hours as described above.

3.2 ml of 500 mM isopropyl-B, D-thiogalactopyranoside in water (IPTG,
Gibco / BRL) (final concentration = 4 mM) was added to each culture to induce synthesis of the recombinant fusion protein. The culture was incubated for 2 hours. Cells are harvested by centrifugation as before and 30 ml of 50 mM Tris-hydrochloride, 25% (w / v) sucrose, pH
Resuspended in 8.0 and frozen at -70 ° C. After 60 minutes at −70 ° C., the frozen cells were thawed at room temperature and 5 ml of lysozyme (Sigma, 250 mM Tris-HCl,
(20 mg / ml in 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 set with a power of 100 watts with a Braun sonicator, large probe for a total of 2.5 minutes (5-3
(0 second burst, 1 minute cooling between each). An equal volume of the solution was placed in a Teflon SS34 centrifuge tube and centrifuged at 10,000 rpm for 20 minutes in a Soval SS34 rotator. The pellet was resuspended by vortexing at high speed in a total of 100 ml of sterile double distilled water and the centrifugation step was repeated. Discard the supernatant and pellet the 20 ml
Combine in 10 mM Tris-HCl, 150 mM NaCl, pH 8.0 (Tris buffered saline) and freeze the suspension at -20 ° C overnight.

The recombinant suspension was thawed at room temperature, added to 100 ml of 8M guanidine HCl (Sigma) in Tris buffered saline and mixed vigorously. The magnetic stir bar was placed in the bottle and the solubilized sample was mixed for 30 minutes at room temperature. This solution was transferred to a 2000 ml Erlenmeyer flask and 1200 ml of Tris buffered saline was added rapidly.
The mixture was stirred at room temperature for another 2 hours. Aliquots of 500 ml were placed in dialysis bags (Spectrum, 63.7 mm diameter, 6,000-8,000 molecular weight cutoff, # 132670, from Fisher Scientific) and these were placed in 3,500 ml of Tris-buffered saline + 0.5
Placed in a 4,000 ml beaker containing M guanidine HCl.

Place the beaker on a magnetic stirrer in a 4 ° C. room overnight and then add dialysis buffer to Tris
Replacement with buffered saline + 0.1 M guanidine HCl and dialysis continued for 12 hours. The buffer was then replaced with Tris-buffered saline + 0.05 M guanidine HCl and dialysis continued overnight. The buffer was replaced with Tris-buffered saline (without 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. This solution was stored in 100 ml aliquots at -20 ° C.

To confirm that the fusion protein was isolated, aliquots of each preparation were
Dilute 20-fold in double distilled water, mix equal volumes of SDS-PAGE sample buffer,
Placed in a boiling water bath for 5 minutes and developed through a 12% polyacrylamide gel. A recombinant leukotoxin control was also developed. The fusion protein was expressed at high levels as inclusion bodies.

Embodiment 3 FIG. Antibody Titers After GnRH Immunization of Pigs This study was designed to evaluate variables including volume, number of injections (1 vs. 2) for the second injection secondary immunization and primary vaccination on the first. Was done. For the study, 160 pigs aged 28 days and weighing 3-4 kg were assigned to one of eight treatment groups (see Table 1). There were 10 females and 10 castrated male pigs in each group. Animals are housed at 10 / cage and are sold to Prairie Swine Center, Inc. [University of Saskatchewan (Universi)
ty of Saskatchewan) and controlled by standard operating procedures developed by a laboratory facility censored by the Canadian Council on Animal Care.

For groups 1-7, using the GnRH immunogen from plasmid pCB122 as described above
GnRH vaccine was made. In particular, the GnRH immunogen was dissolved in 8 M urea at a concentration of 20 mg / ml. The adjuvant used to formulate the GnRH vaccine was VAS-3 (a modified form of the EMULSIGEN PLUS ^™ adjuvant, including DDA) (see granted US patent application Ser. No. 08 / 463,837).

The stock solution of GnRH immunogen was combined with phosphate buffer and mixed with VAS-3 at a ratio of 1: 1 (v / v) to form a stable emulsion. The dose of the GnRH immunogens for Groups 1, 2, 3, 4, and 6 was 40 μg, but the volumes varied in some of the formulations. Details for each treatment group are provided in Table 1. Group 5 and 7 GnRH vaccines contained 30 μg GnRH immunogen / 0.25 ml, whereas Group 8 received 40 μg GnRH immunogen from plasmid pCB130 in 0.4 ml adjuvant. In all cases, the ratio of VAS-3 adjuvant / aqueous phase (phosphate buffer) remained at 1: 1 (v / v). Adjustments were made by changing the volume of the immunogen stock.

[0183]

[Table 1]

Development of Biojector 2000 injection without needle (Manufacturer: Bioje
ct Inc., Portland, Oregon, USA). This device utilizes a gas cylinder to inject the vaccine under high pressure through a small opening. The vaccine penetrates the skin and is deposited subcutaneously. In each treatment group, the first injection was performed when the pigs were 38 days old, and a second injection was performed 35 days later.

The injections were performed on the outer surface of the pinna, except that the second injection in Group 7 was 10 hours behind the head
Performed in the center of the dorsal の 15 cm. Days 35, 49 and 63 of the experiment (start of study (day
On day 0)), blood was collected by jugular vein puncture. Cells were allowed to clot at room temperature, then centrifuged to collect serum, and the serum was stored at -20 ° C until analyzed for GnRH antibody titer.

GnRH antibody titers were measured by a modified radioimmunoassay procedure. Synthetic Gn
RH (Bachem., Inc.) was iodinated with I ¹²⁵ (Amersham, Oakville, Ontario). Serum dilutions were added to the tubes, then a standard amount of I ¹²⁵ labeled GnRH was added to bring the final incubation volume to 0.7 ml. A suspension of charcoal in assay buffer was added at the end of the 24-hour incubation at 2-6 ° C. to absorb non-antibody bound I ¹²⁵ -GnRH. After centrifugation, the radioactivity in the charcoal fraction was measured. The data are presented as numerical values, which are the% of the standard dose (approximately 12,000 cpm) of I ¹²⁵ -GnRH bound to the antibody at a particular serum dilution.

[0187] Student Version (Student Version of Statistix, Version 1
Descriptive statistics, variance, and "t" test were performed using the method described in U.S. Pat. Groups of 0.15, 0.25 and 0.35 volumes given to a single injection site, respectively
Evaluations were made at 1, 2 and 3. Antibody titers before booster vaccination on day 35 of study when animals were 63 days old and antibody 14 days after booster vaccination on day 49 of study when animals were 77 days old Table 1 shows the relationship between titers
Shown in

Animals with a titer greater than 10% binding at 1: 5000 on day 35
Better responses were given to booster vaccinations than animals that had a weaker response to primary immunization. Almost 20% at 1: 5000 dilution based on other experiments
We know that the binding of will partially suppress testosterone secretion. As these results show, the utilization of the strong response to primary immunization provided there has no effect on growth and feed utilization efficiency.

Embodiment 4 FIG. Effect of GnRH Immunogen on Testosterone Levels In the experiments described below, an immunological approach is used to demonstrate the lack of effect of reducing testosterone levels in early adolescent animals. Sixty complete boars were divided into three treatment groups. Group 1 was surgically castrated at birth, and groups 2 and 3 were left intact.

At approximately 21 days of age, 38-378 amino acids and 815-951 of native leukotoxin
Group 3 was immunized with a GnRH immunogen comprising 8 copies of GnRH linked to an internally deleted leukotoxin molecule comprising GnRH immunogen was formulated in VAS-3 adjuvant as described above. Immunization increased antibody production sufficiently to cause a detectable decrease in testosterone secretion. Group 2 remained intact throughout the experiment and was not immunized.

In a previous study, animals immunized with this adjuvant had moderate and sustained increases in GnRH antibody titers, with GnRH antibody titers having lower testosterone concentrations,
Reduced to detectable levels. Feed consumption and carcass composition were measured during the experiment to compare their parameters at different ages. Animals treated with this GnRH vaccine [immunocastrates]
Are castrated (barrows) and non-castrated boars (boa) until approximately 90 days of age.
rs). Furthermore, as shown in FIG. 4, all three groups had similar weight gain.

Embodiment 5 FIG. The purpose of sexually mature immunocastration of boars this study by GnRH vaccination, sexually mature value equal to the value found in surgically castrated pigs serum testosterone and fat androstenone concentrations in male pigs It was to determine if GnRH vaccination was reduced and to determine the kinetics of GnRH antibody response, serum testosterone levels and fat androstenone levels after primary and secondary immunizations.

As shown in Table 2, 24 intact boars were assigned to one of three treatment groups (Groups 1, 2 and 3) on Day 0. Six age-matched litters of surgically castrated pigs younger than one week were assigned to the fourth treatment group (Group 4-early castrated boars). Pigs were housed with 10 animals / cage and then 2 animals / cage until the pigs weighed approximately 60 kg. Pigs have access to feed and water and are provided by the Prairie Swine Centre, an animal facility belonging to the University of Saskatchewan and censored by the Canadian Council on Animal Care. Controlled using standard operating procedures as described.

G from plasmid pCB122 dissolved at a concentration of 28 mg / ml in 4 M guanidine HCl
The GnRH vaccine was made using the nRH immunogen. The adjuvant used to formulate the GnRH vaccine was VAS-3. GnRH immunogen combined with phosphate buffer, VAS
The vaccine was prepared by mixing -3 with a 1: 1 (v / v) ratio to form a stable emulsion. This vaccine contains a dose of 40 μg GnRH immunogen / 0.5 ml,
Administered intramuscularly. Placebo contained phosphate buffer and VAS-3.

Pigs received two intramuscular injections of vaccine or placebo in the neck. The first injection was performed on day 0 of the experiment, when the pigs were 21 days old. When the pigs approached sexual maturity, a second injection was performed, at which time the pigs weighed approximately 100 kg (110th to 1st).
20th) (Table 2). When the pigs in Group 2 (late castrations) reached sexual maturity, they were surgically castrated. Sexual maturity is strongly influenced by weight,
Occurs when the body weight is approximately 110 kg (days 115-125). The pigs in Group 1 were sub-immunized approximately one week before the pigs in Group 2 were surgically castrated. This was done to bring the GnRH antibody titers generated by the secondary immunization to biologically effective levels at about the same time as pigs in Group 2 were surgically castrated.

[0196]

[Table 2]

To simplify analysis and presentation of the data, day 120 is an “event”, ie, the animals are given a second injection (all groups) or the animals are surgically castrated (group 2) meant time. All data subsequently collected for "event" is "event", i.e., 7 days after "event" means day 127, 14 days after "event" means day 134, and so on. Are described in terms of "events".

Blood samples were obtained by jugular vein puncture at approximately 28 day intervals between day 28 and day 120. Thereafter, blood was collected from all pigs at weekly intervals until the animals were killed on day 162 of the experiment (42 days after the "event"). Blood was allowed to clot at room temperature, centrifuged, and serum frozen within 24 hours after collection. Individual weight gain was determined by weighing all animals from day 0 to "event", after which the animals were weighed weekly until sacrifice.

Samples (approximately 5 g) of subcutaneous fat for androstenone measurement were taken under local anesthesia from alternating sides of the neck of all pigs every week until the time of the “event” and sacrifice. The fat sample was immediately cooled and frozen within 4 hours after biopsy. Measurements at sacrifice included carcass weight, back fat depth at the 10th rib level, testis weight and urethral gland length.

GnRH antibody titers were measured by a modified radioimmunoassay procedure. Synthetic GnRH (Ba
chem, Inc.) was iodinated with I ¹²⁵ (Amersham, Oakville, Ontario).
Serum dilutions were added to the tubes, then a standard amount of I ¹²⁵ labeled GnRH was added to bring the final incubation volume to 0.7 ml. A suspension of charcoal in assay buffer was added at 2 to 6 ° C. At the end of the 24 hour incubation, non-antibody binding I ¹²⁵ -
GnRH was absorbed. After centrifugation, the radioactivity in the charcoal fraction was measured. The data are presented as numerical values, which are the% of the standard dose (approximately 12,000 cpm) of I ¹²⁵ -GnRH bound to the antibody at a particular serum dilution.

Serum testosterone was measured using a Coat-A-Count total testosterone kit (DPS, Los Angeles, CA). This assay is based on I ¹²⁵ -testosterone and antibodies with high affinity for testosterone. Fat androstenone concentration was measured by a colorimetric method.

The measurement of primary results was: GnRH antibody titer 1: 5000 serum dilution in group 1 and 1: 2 in groups 2, 3 and 4.
GnRH antibody titers measured as% binding at 100 were included. In addition, serum testosterone concentration, fat androstenone, body weight, back fat, testes weight and urethral gland length were measured.

All of the pigs in Group 1 developed readily detectable GnRH antibody titers at 1: 100 serum dilution after primary immunization (see Table 3). In addition, immunization of pigs at 21 days and almost 140 days of age generates GnRH antibody titers resulting in reduced serum testosterone and fat androstenone levels, similar to those seen in castrated pigs early and late in life. I let it. There was also a significant decrease in the size of the testes and urethral glands compared to intact males.

[0204]

[Table 3]

Embodiment 6 FIG. GnRH Immunization of Bulls This experiment was performed using 58 early adolescent calves. Group I
Gn from plasmid pCB122 in 200 μg VAS-3 adjuvant
The vaccine composition comprising the RH immunogen was vaccinated twice subcutaneously (day 0 and day 56), and 30 control bulls in Group 2 were vaccinated with placebo. Group I vaccination produced significant titers against GnRH by day 42, significantly reduced scrotum circumference by day 84, and significantly reduced testosterone levels by day 98. (Table 4). Despite these significant anti-GnRH titers and testosterone decreases, no daily increase and no difference in feed efficiency was observed from day 0 to day 84 (Table 5).

[0206]

[Table 4]

[0207]

[Table 5]

As these novel findings indicate, there is significant utility for GnRH vaccines,
The RH vaccine provides a sufficiently strong immune response after primary immunization, which results in antibody titers that detectably reduce serum testosterone but do not significantly reduce growth or feed efficiency. This finding indicates that temporary suppression of androgen secretion during early growth does not reduce body growth or feed efficiency,
Especially new. This has particular utility when using vaccination protocols that require subsequent immunization later in life to achieve a strong secondary response.

Deposit of strains useful in practicing the present invention Deposit of biologically pure cultures of the following strains can be obtained from the American Type Culture Collection (ATCC), University Boulevard, Manassas, Virginia. 1081, made in. After a successful viability test, the indicated accession number was assigned and the required fee was paid. The deposit was made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedures (Budavest Treaty).

This ensures maintenance of a viable culture for at least 5 years after the most recent request for the composition of the deposited sample by the depository for 30 years from the date of deposit. Organisms are made available by the ATCC pursuant to the provisions of the Budapest Treaty and are thereby entitled by the Director of the U.S. Patents and Trademarks in accordance with 35 U.SC § 122 and the regulations of the Commissioner (including 37 U.SC § 1.12). Permanent, unrestricted availability of cultures obtained is assured. Once the patent is granted, all restrictions on the availability of the deposited culture to the public will eventually be removed.

[0211] These deposits are conveniently provided to those skilled in the art, and the deposits are
It is not an admission that is required under 122. The amino acid sequences of these plasmids,
As well as the amino acid sequence of the polypeptide encoded thereby, is incorporated herein by reference and is controlled where conflicting with the description herein. A license is required to manufacture, use, or sell the consignment, and such license is not granted herein.

[0212] strain No. date of deposit ATCC Escherichia coli (E. coli) pAA352 1990 March 30, 68283 E. coli in W1485 (E. coli) pCB113 in JM105 2 May 1, 1995 69,749 E. coli (E. coli) JM105 PCB111 in February 1, 1995 69748 Thus, a method of immunizing against GnRH was disclosed. While the preferred embodiment of the invention has been described in some detail, it is understood that various changes and modifications may be apparent without departing from the spirit and scope of the invention as defined by the appended claims. Should.

[Brief description of the drawings]

FIG. 1 shows that when pigs are 63 days old, as described in the Examples,
7 depicts the relationship between antibody titers 5 days prior to booster vaccination and when pigs are 77 days old, 14 days after booster vaccine injection on day 49 of the study.

FIG. 2A shows the nucleotide and amino acid sequences of the GnRH construct used in the chimeric leukotoxin-GnRH polypeptide of the present invention. FIG. 2A depicts a single copy of the GnRH decapeptide.

FIG. 2B shows the nucleotide and amino acid sequences of the GnRH construct used in the chimeric leukotoxin-GnRH polypeptide of the present invention. FIG. 2B shows that n = 1
7 depicts a molecule with 4 copies of the GRH decapeptide when n = 2 and 8 copies of the GnRH decapeptide when n = 2, and so on.

FIG. 3A shows the nucleotide sequence and predicted amino acid sequence of the LKT-GnRH chimeric protein from plasmids pCB122 and pCB130.

FIG. 3B shows the nucleotide and predicted amino acid sequence of the LKT-GnRH chimeric protein from plasmids pCB122 and pCB130.

FIG. 3C shows the nucleotide and predicted amino acid sequence of the LKT-GnRH chimeric protein from plasmids pCB122 and pCB130.

FIG. 3D shows the nucleotide and predicted amino acid sequence of the LKT-GnRH chimeric protein from plasmids pCB122 and pCB130.

FIG. 3E shows the nucleotide sequence and predicted amino acid sequence of the LKT-GnRH chimeric protein from plasmids pCB122 and pCB130.

FIG. 3F shows the nucleotide and predicted amino acid sequence of the LKT-GnRH chimeric protein from plasmids pCB122 and pCB130.

FIG. 4 shows pigs treated with the GnRH vaccine according to the invention compared to castrated boars (barrows) and non-castrated boars (boars).
strates)] as a function of age.

[Procedure amendment 2]

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──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR , BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS , JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Acres, Steven D. Saskatchewan S7N0J6, Canada 6, University Drive, Saskatoon, University 820 (72) Inventor Harland, Richard Saskatchewan S7H 3A7, Saskatoon, Beit Crescent 514 F-term, Canada (Reference) 4B024 AA01 AA10 BA31 BA38 CA01 DA06 EA04 FA02 GA11 GA19 4C085 BB23 CC21 FF11 FF12 FF13 4H045 AA30 CA11 DA83 EA20 FA74 GA01 GA10

Claims (28)

[Claims] Claims 1. An animal that is vaccinated with a first vaccine composition to reduce circulating testosterone levels prior to or during a fattening period of a non-castrated male food producing animal for edible meat production, and About 2 to about 8 weeks prior to the sacrifice of said animal, comprising vaccinating said animal with a second vaccine composition to substantially reduce one or more androgen steroid and / or non-androgen steroid levels. A first method for producing first and second vaccine compositions for use in a method of breeding a non-castrated male food producing animal for edible meat production.
And use of a second GnRH immunogen. 2. The use according to claim 1, wherein the vaccine composition is administered to the animal between the time of birth of the animal and about 10 weeks of age. 3. The use according to claim 1, wherein the first and second vaccine compositions further comprise an immunological adjuvant. 4. The use according to claim 3, wherein the immunological adjuvant in the first and / or second vaccine composition comprises oil and dimethyldioctadecyl ammonium bromide. 5. The method of claim 3, wherein the GnRH immunogen in the first and second vaccine compositions is the same. 6. The GnRH immunogens in the first and second vaccine compositions are different.
Use according to claim 3. 7. The method of claim 1, wherein administration of the first vaccine composition produces antibodies that cross-react with the animal's endogenous GnRH, and administering the second vaccine composition after the antibody levels are reduced. Use of the description. 8. The GnRH immunogen in the first and / or second vaccine composition is a GnRH multimer having 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 peptide bonds, amino acid spacer groups, carrier molecules and [GnRH] n, where n is greater than 1 Or an integer equal to, and y is an integer greater than or equal to 1. 9. Use according to claim 8, wherein the carrier molecule is a leukotoxin polypeptide. 10. The use according to claim 1, wherein said GnRH immunogen is a nucleic acid molecule. 11. The use according to claim 1, wherein the second vaccine composition is administered about 4 to about 6 weeks before sacrifice of the animal. 12. The use according to claim 11, wherein the immunological adjuvant in the second vaccine composition is an aqueous adjuvant. 13. A method of reducing circulating testosterone levels by vaccinating a non-castrated male bovine, ovine or porcine animal for edible meat production before or during a fattening period with said first vaccine composition. And about 2 to about 8 weeks before sacrifice of the animal, the animal is vaccinated with a second vaccine composition to substantially reduce the level of one or more androgenic and / or non-androgenic steroids. A first and a second vaccine composition for producing a first and second vaccine composition for use in a method of breeding a non-castrated male bovine, ovine or porcine animal for edible meat production. Use of GnRH immunogen. 14. The use according to claim 13, wherein the first and second vaccine compositions further comprise an immunological adjuvant. 15. The method of claim 14, wherein the GnRH immunogens in the first and second vaccine compositions are the same. 16. The use according to claim 14, wherein the GnRH immunogens in the first and second vaccine compositions are different. 17. The GnRH immunogen in the first and / or second vaccine composition is a GnRH multimer having 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 bond, an amino acid spacer group, a carrier molecule and [GnRH] n, where n is an integer greater than or equal to 1 and y 14. Use according to claim 13, wherein is an integer greater than or equal to one. 18. The use according to claim 17, wherein the carrier molecule is a leukotoxin polypeptide. 19. The use according to claim 13, wherein the second vaccine composition is administered about 4 to about 6 weeks before sacrifice of the animal. 20. A method for reducing circulating testosterone levels by vaccinating a non-castrated male cow, sheep or pig animal for edible meat production before or during the fattening period of said animal with a first vaccine composition. And about 2 to about 8 weeks before sacrifice of the animal, the animal is vaccinated with a second vaccine composition to substantially reduce the level of one or more androgenic and / or non-androgenic steroids. A first and a second vaccine composition for producing a first and second vaccine composition for use in a method of breeding a non-castrated male bovine, ovine or porcine animal for edible meat production. Use of a GnRH multimer, wherein said first and second vaccine compositions comprise an adjuvant, wherein said first and second G The nRH multimer has the general formula (GnRH-X-GnRH) y
Wherein GnRH is a GnRH immunogen, and X is one or more molecules selected from the group consisting of peptide bonds, amino acid spacer groups, leukotoxin polypeptides and [GnRH] n, wherein The above uses, wherein n is an integer greater than or equal to 1 and y is an integer greater than or equal to 1. 21. The use according to claim 20, wherein said first and second vaccine compositions comprise the same GnRH multimer. 22. The GnR in the first and / or second vaccine composition
The amino acid sequence wherein the H multimer is depicted in FIGS. 3A-3F (SEQ ID NO_);
21. The use according to claim 20, comprising an amino acid sequence having at least about 75% sequence identity thereto. 23. The use of claim 22, wherein the GnRH multimer comprises the amino acid sequence depicted in FIGS. 3A-3F (SEQ ID NO: _). 24. The use according to claim 20, wherein the second vaccine composition is administered about 4 to about 6 weeks before sacrifice of the animal. 25. A method for reducing circulating testosterone levels by vaccinating a non-castrated male bovine, ovine or porcine animal for edible meat production prior to or during the fattening period of the animal with a first vaccine composition. And about 2 to about 8 weeks before sacrifice of the animal, the animal is vaccinated with a second vaccine composition to substantially reduce the level of one or more androgenic and / or non-androgenic steroids. A first and a second vaccine composition for producing a first and second vaccine composition for use in a method of breeding a non-castrated male bovine, ovine or porcine animal for edible meat production. Use of a GnRH multimer, wherein said first and second vaccine compositions comprise an adjuvant, wherein said first and second G The use wherein the nRH multimer comprises the amino acid sequence depicted in FIGS. 3A-3F (SEQ ID NO_), or an amino acid sequence having at least about 75% sequence identity thereto. 26. The GnR in the first and / or second vaccine composition
26. The use according to claim 25, wherein the H multimer comprises the amino acid sequence depicted in Figures 3A-3F (SEQ ID NO_). 27. The use according to claim 25, wherein the second vaccine composition is administered about 4 to about 6 weeks before sacrifice of the animal. 28. The use according to claim 25, wherein the immunological adjuvant in the first and / or second vaccine composition comprises light mineral oil and dimethyldioctadecyl ammonium bromide.