MXPA99007025A - Immunization against endogenous molecules - Google Patents

Abstract

A method is described for immunoneutralization of endogenous molecules in mammalian subjects, wherein an immunogen is administered via injection to the ear. The method is used to elicit an efficient and uniform immune response sufficient to block or suppress the activity of an endogenous hormone in a vaccinated subject, or to target a diseased cell for an immune response.

Description

IMMUNIZATION AGAINST ENDOGENOUS MOLECULES TECHNICAL FIELD The present invention relates generally to active immunization against endogenous molecules. More particularly, the invention relates to methods for the immunoneutralization of endogenous molecules in mammalian subjects, wherein the immunogen is administered via injection to the ear.

BACKGROUND OF THE INVENTION A number of vaccination methods have been suggested for the use of fertility control or reproductive function in mammals. These vaccines operate by producing an immune response against an endogenous hormone in the vaccinated subject that is effective in neutralizing the activity of the hormone. For example, immunological methods have been used to produce an immune response against the reproductive hormone or human chorionic gonadotropin (Matsuura et al. (1979) Endocrinol. 101: 396-401). Other targets include two gonadotropic hormones known to be involved in the control of the estrus cycle, particularly luteinizing hormone (LH) and follicle stimulating hormone (FSH). In vertebrates, the synthesis and release of these two hormones is regulated by a polypeptide referred to as the gonadotropin releasing hormone (GNRH) (formerly designated LHRH). Accordingly, an approach to controlling fertility in an animal population is to reduce the levels of GnRH, such as by immunization against endogenous GnRH, which effect a reduction in the levels of LH and FSH and the concomitant breaking of the estrus cycle and the spermatogenesis. See, for example, Adams et al. (1990) J. Kani. Sci. 68: 2793-2802. Recent studies of the GnRH molecule have shown that it is possible to promote antisera in response to repeated injections of synthetic GnRH peptides (Ariura et al. (1973) Endocrinology _93 (_5): 1092-1103). In addition, antibodies to GnRH have been promoted in a number of species by chemical conjugation of GnRH to a suitable carrier and administration of the conjugate in an appropriate adjuvant (Careli et al. (1982) Proc. Nati. Acad. Sci. 79: 5392 -5395). Protein conjugates, and / or recombinant fusion proteins, comprising GnRH or analogues of GnRH have also been described for use in vaccines with peptides for castration or immunological inhibition of the reproductive function of several domestic and farm animals (Meloen et al. (1994) Vaccine 1_2 (8): 741-756: Hoskinson et al. (1 ° 990) Aust. J. Biotechnol. 4_: 166-170; and International Publication Nos. WO 96/24675, published August 15, 1996, WO 92/19746, published November 12, 1992; WO 91/02799, published March 7, 1991, WO 90/112989, published October 4, 1990 and WO 86/07383, published December 18, 1986). However, a need remains for a method of vaccination against these and other endogenous molecules, wherein the method provides improved uniformity and efficiency in the immune response directed against the target molecule. The need also remains for a method that can be practiced safely in a field setting, thereby reducing the incidence of inappropriate or accidental administration of the vaccine to the person administering the vaccine.

DESCRIPTION OF THE INVENTION The present invention is based on the discovery that vaccination against endogenous molecules can be carried out in a highly uniform and efficient manner by the distribution of immunogens to a mammalian subject via injection into the ear. In one embodiment, the invention relates to a method for presenting an endogenous immunogen, selected from a mammalian subject by administering to the ear of the subject a vaccine composition containing the immunogen. The administration can be carried out using conventional needle and syringe devices, needleless dispensing devices, or preferably, using a jet injector device. In another embodiment, the invention is directed to a method for inducing an immune response, uniform against an endogenous hormone in a mammalian subject by administering to the ear of the subject a vaccine composition containing an immunogen derived from the hormone. The vaccine composition is capable of inducing an immune response against the endogenous hormone of the subject.

In yet another embodiment, the invention is directed to a method of inducing a uniform immune response against an endogenous hormone receptor in an animal subject by administering to the ear of the subject a vaccine composition containing an immunogen derived from the hormone receptor. The vaccine composition is capable of inducing an immune response against the endogenous hormone receptor of the subject, thus neutralizing the biological activity, for example, the ligand binding activity, of that molecule. Thus, in one aspect of the invention, methods are provided for the immunoneutralization of endogenous hormones and / or hormone receptors by vaccines that are distributed to the ear. The vaccines contain an endogenous immunogen derived from the target molecule, either alone, or in combination with a suitable carrier molecule, and injected either subcutaneously, subdermally, or intradermally in the outer ear pinna. In a particular embodiment, the invention involves the distribution of an immunogen selected from GnRH to a mammalian subject to immunocastrate the vaccinated animal. The methods can be practiced in any suitable mammalian subject, however, commercially significant domestic animals are especially contemplated. For example, the methods of the present invention can be practiced in porcine subjects to reduce spots in boars, or as an alternative to surgical castration in cattle. These and other embodiments of the present invention will readily come to mind to those skilled in the art in view of the description herein.

I BRIEF DESCRIPTION OF THE FIGURES Figures IA (SEQ ID NOS: 1 and 2) and IB (SEQ ID NOS: 3 and 4) show the nucleotide sequences and amino acid sequences of the GnRH constructs used in the gene fusions of the polypeptides of leukotoxin-GnRH, chimeric. Figure IA represents GnRH-1 including an individual copy of a decapeptide of GnRH; Figure IB represents GnRH-2 which includes four copies of a decapeptide of GnRH when n = 1, and eight copies of GnRH when n = 2, etc.

DETAILED DESCRIPTION The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, and immunology, which are within the skill of the art. These techniques are fully exemplified in the literature. See for example, Sambrook,. Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual; DNA Cloning, Vols. I and II (D.N. Glover ed.); Oligonucleo t ide Synthesis (M.J. Gait ed.); Nucleic Acid Hybridization (B. D. Hames &S.J. Higgins eds.); animal Cell Culture (R.K. Freshneyh ed.); Immobilized Cells and Enzymes (IRL press); B. Perbal, A Practical Guide to Molecular Cloning; the series, Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D.M. eir and C.C. Blackwell eds., Black ell Scientific Publications).

A, Definitions In describing the present invention, the following terms will be employed, and are intended to be defined as follows: An "immunogen" refers to any agent, generally a macromolecule, that can produce an immune response in an individual. The immune response can be of B and / or T lymphocyte cells. The term can be used to refer to an individual macromolecule or a homogeneous or heterogeneous population of antigenic macromolecules. An "immune response" to an immunogen or vaccine is the development of the host of an immune response mediated by antibody and / or cell to the immunogen or vaccine of interest. Usually, this response includes, but is not limited to one or more of the following effects; the production of antibodies, B cells, T helper cells, suppressor T cells, and / or cytotoxic T cells and / or T? d cells, directed specifically to an immunogen or immunogens included in a composition or vaccine of interest.

An immune response can be detected using any of several immunoassays well known in the art. The phrase "endogenous immunogen", as used herein, refers to all, or a portion of, an endogenous, cellular component of an objective against which an immune response is to be promoted.

The term includes molecules in this way (immunogen) derivatives of peptide hormones and steroids, hormone receptors, hormone agonists, hormone antagonists, markers associated with cancer and / or antigens; and the like, molecules that are capable of being immunogenic, or more immunogenic, by association with a carrier molecule, by mutation of a native sequence, and / or by incorporation into a multimer containing multiple repeat units of at least an epitope of an immunogen, endogenous to the subject. The term includes peptide molecules that have substitutions, deletions and / or additions of amino acids and that have at least about 50% amino acid identity to the reference molecule used, more preferably about 75-85% identity and more preferably about 90-95% identity plus, the relevant portion of the native peptide sequence in question. Expressly excluded from the definition of "endogenous immunogen" are any portion derived from an infectious agent such as a bacterium or a virus. An "epitope" refers to any portion or region of a molecule with the ability or potential to produce, and be combined with, a specific antibody. For the purpose of the present invention, a polypeptide epitope will usually include at least about 3 amino acids, preferably at least about 5 amino acids, more preferably at least 10-15 amino acids, and more preferably 25 or more amino acids, of the molecule reference. There is no critical upper limit to the length of the fragment, which could comprise close to the full length of a protein sequence, or even a fusion protein comprising two or more epitopes of a protein in question. Epitopes on polypeptide molecules can be identified using any number of epitope correlation techniques, well known in the art. See, for example, Mapping Epitope Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes can be determined for example, by concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to the portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. These techniques are well known in the art and described in, for example, U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc. Nati Acad. Sci. USA 8jL_: 3998-4002; Geysen et al. (1986) Molec. Im unol. 2_3_: 709-715. Similarly, conformational epitopes are easily identified when determining the spatial conformation of amino acids such as, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, supra. By "carrier" is meant any molecule that, when associated with an endogenous immunogen of interest, imparts immunogenicity to that molecule. Examples of suitable carriers include large, slowly metabolized macromolecules such as: proteins; polysaccharides, such as sepharose, agarose, cellulose, cellulose beads and the like, polymeric amino acids such as polyglutamic acid, polylysine and the like; amino acid copolymers; inactive virus particles; bacterial toxins such as tetanus toxoid, leukotoxin molecules, and the like. The carriers are described in further detail below. The endogenous immunogen is "bound" to a specified carrier molecule when the immunogen is chemically coupled to the carrier, or when the immunogen is expressed from a chimeric DNA molecule encoding the immunogen and the carrier of interest. The "native" proteins or polypeptides refer to proteins or polypeptides that are isolated from the source in which the proteins naturally occur. "Recombinant" polypeptides refer to polypeptides produced by recombinant DNA techniques; that is, produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide. "Synthetic" polypeptides are those prepared by chemical synthesis. "A" vector "is a replicon, such as a plasmid, phage, or cosmid, to which another DNA fragment can be attached to give rise to replication of the attached segment. A "coding sequence" of DNA or a "nucleotide sequence encoding" a particular protein is a DNA sequence that is transcribed and translated into a polypeptide in vitro or in vivo when placed under the control of regulatory elements The limits of the coding sequence are determined by a start codon at the 5'-terminus (amino) and a translation-terminating codon at the 3'-terminus (carboxy) A coding sequence may include, but is not limited to, prokaryotic sequences, eukaryotic mRNA cDNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. ion will usually be located 3 'to the coding sequence. The term "control elements" of DNA refers collectively to promoters, ribosome binding sites, polyadenylation signals, transcription termination sequences, regulatory domains in the 5 'direction, enhancers, and the like, which collectively provide for transcription and translation of a coding sequence in a host cell. Not all of these control sequences always need to be present in a recombinant vector as long as the desired gene is capable of being transcribed and translated. "Operably linked" refers to an arrangement of elements where the components described in this way are configured to perform their usual function. In this way, the control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements do not need to be contiguous with the coding sequence, as long as they function to direct the expression of the same. In this way, for example, the intervention of the transcribed sequences not yet translated may be presented between a promoter and the coding sequence and the promoter may still be considered "operably linked" to the coding sequence. A control element, such as a promoter, "directs the transcription" of a coding sequence in a cell when the RNA polymerase will bind to the promoter and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence. A "host cell" is a cell that has been transformed, or is capable of transformation, by an exogenous nucleic acid molecule. A cell has been "transformed" by exogenous DNA when the exogenous DNA has been introduced into the cell membrane. The exogenous DNA can be integrated or not (covalently linked) on the chromosomal side that makes up the genome of the cell. In prokaryotes and yeasts, for example, the DNA can be maintained in an episomal element, such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which exogenous DNA has become integrated into the chromosome so that it is inherited by fixed cells through chromosomal replication. This ability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of fixed cells containing the exogenous DNA. The term "derivative of", as used herein, denotes a real or theoretical source or origin of molecule or immunogen. For example, an immunogen that is "derived from" a particular hormone molecule will have a close sequence similarity to a relevant portion of the hormone. In this manner, an immunogen that is "derived from" a GnRH molecule can include the entire wild type GnRH sequence, or it can be altered by insertion, deletion or substitution of the amino acid residues, while the derived sequence provides an immunogen corresponding to the target hormone. Immunogens derived from a denoted molecule will contain at least one epitope specific to the denoted molecule. By "mammalian subject" is meant any member of the mammalian class, including, but not limited to, rodents, cattle, pigs, sheep, goats, horses and primates and companion animals such as dogs and cats. The term does not denote a particular idea. In this way, they are proposed to be covered adult, newborns and fetuses.

B General Methods Before describing the present invention in detail, it will be understood that this invention is not limited to particular formulations or process parameters as such may vary, of course. It is also to be understood that the terminology used in the present invention is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting. Although a number of compositions and methods similar or equivalent to those described herein may be used in the practice of the present invention, preferred materials and methods are described herein. The central point in the present invention is the discovery that the efficiency and particularly, the uniformity, of vaccination against an endogenous immunogen can be greatly implemented in mammalian subjects through the administration of vaccine compositions to the ear instead of the administration intramuscular in the neck. In commercially significant domestic animals, the ear provides a desirable site for these injections since the ear is not generally consumed by humans. This avoids the presence of residual immunogens and / or other vaccine components (e.g., oil) in the consumable tissue at the time of killing, particularly when these residues may have adverse effects on humans. In cattle, the ear is the ideal place for vaccination since it is not consumed. In pigs, the ear is also a preferred site for these vaccinations since it provides an easily sensitive location for subcutaneous or intradermal injection. Accordingly, one aspect of the invention relates to targeted targeting of vaccine compositions containing one or more endogenous immunogens. The distribution is carried out by administering the vaccine composition to an ear of the subject. The vaccine compositions are used to induce the production of antibodies capable of neutralizing the bioactivity of a target endogenous hormone, hormone receptor, agonist or antagonist; or are used to produce an immune response against an endogenous type of target cell (eg, a cancer or otherwise diseased cell). These "auto" molecules must become immunogenic in order to be reproduced by the immune system of the vaccinated subject. Vaccine compositions in this manner generally comprise one or more epitopes derived from an endogenous molecule, and are provided as base compositions in nucleic acid and / or peptides. The endogenous immunogen can be derived from peptide hormones, such as ACTH, CRF, GNRH, GnRH, cholecystokinin, dynorphins, endorphins, endothelin, fibronectin fragments, galanin, gastrin, insulin, proinsulin, growth hormone, EGF, Somatostatin, SNX-111, BNP, insulinotropin, glucagon, ANP, GTP-binding protein fragments, leukocytes, magainin, mastoparans, dermaseptine, sistemiha, neuromedins, neurotinsin, pancreastat ina, pancreatic polypeptide, vasoactive intestinal polypeptide (VIP), substance P, secretin, thymosin, and the like. The immunogen can also be derived from a glycoprotein hormone (e.g., thyroid stimulating hormone (TSH), follicle stimulating hormone (FSH), luteinizing hormone (LH), placental hormones, and chorionic chorionic gonadotropin (hCG), or a steroid hormone (e.g., gonadal steroid hormones such as estrogens, androgens, and progesterones) Other endogenous immunogens can be derived from peptide hormone receptors (e.g., insulin receptor, angiotensin receptor, growth hormone receptor, and similar), or any number of the superfamily of steroid hormone receptors Immunogens derived from hormone agonists (activin) and antagonists (eg, inhibin) also find use in the present vaccine compositions, as well as antigens from tumor, for example, any of the various MAGE (E antigen associated with melanoma), including MAGE 1, 2, 3, 4, etc. (Boon, T. (1993) Scíentific American p 82-89); any of several tyrosinases, MART 1 (melanoma antigen recognized by T cells), mutant ras; p53 mutant; p97 mlelanoma antigen; CEA (carcinoembryonic antigen); and the like; or embryonic proteins that have been re-expressed by transformed cells, or antigens that are not truly tumor-specific, but are 1 prevalent or overexpressed in mammalian tumor tissue. It will be generally understood that the immunogenicity of endogenous molecules can be significantly increased by producing immunogenic forms of these molecules comprising multiple copies of selected epitopes. Accordingly, in one aspect of the invention, vaccine compositions containing endogenous immunogenic multimers are provided in the form of nucleic acid or peptide for targeted delivery to an ear of the subject. The endogenous immunogens can also be conjugated to a suitable carrier in order to produce an immune response in a stimulated host. Suitable carriers are generally polypeptides or proteins that include antigenic regions of a protein derived from an infectious material such as a viral surface protein, or a carrier peptide sequence. These carriers serve: to non-specifically stimulate the activity of the T-helper cells and to assist in the targeting of an immunogen of interest to the antigen-presenting cells (APC) for the processing and presentation of the cell surface in association with the molecules of the histocompatibility main complex (MHC). Several carrier systems have been developed for this purpose. For example, peptide small haptens are often coupled to peptide carriers such as keyhole limpet hemocyanin (Bittle et al (1982) Nature 298: 30-33), bacterial toxins such as tetanus toxoid (Muller et al. (1982) Proc. Nati, Acad. Sci. USA 7: 9: 569-573) ovalbum and mycoglobin of whale sperm, to produce an immune response. These coupling reactions typically result in the incorporation of several moles of the peptide hapten per mole of the carrier protein. Other suitable carriers for use with the present invention include rotavirus VP6 polypeptides, or functional fragments thereof, as described in U.S. Patent No. 5,071,651. Also useful in a fusion product of a viral protein and one or more epitopes of a target molecule of interest, fusion products that are made by the methods described in U.S. Patent No. 4,722,840. Still other suitable carriers include cells, such as lymphocytes since the presentation in this form mimics the natural mode of presentation in the subject, which gives rise to the immunized state. Alternatively, the endogenous immunogens can be coupled to erythrocytes, preferably the erythrocytes of the subject. Methods of coupling the peptides to proteins or cells are known to those skilled in the art. Distribution systems useful in the practice of the present invention can also utilize carriers in the form of particles. For example, the pre-formed particles have been used as platforms on which the immunogens can be coupled and incorporated. Proteosome-based systems (Lowell et al (1988) Science 240: 800-802) and immune-mimicking complexes (Morein et al (1984) Nature 308: 457-460) are also known in the art. Carrier systems using recombinantly produced chimeric proteins that self-assemble into particles can also be used with the present invention. For example, the yeast retrotransposon, Ty, codes for a series of proteins that mount on virus-like particles (ty-VLPs).; Kingsman et al (1988) Vaccines 6_: 304-306). In this manner, a gene, or fragment thereof, which codes for the endogenous immunogen of interest can be inserted into the TyA gene and expressed in the yeast as a fusion protein. The fusion protein retains the ability to self-assemble into particles of uniform size. The useful, different virus-like carrier systems are based on HBsAg, (Valenzuela et al. (1985) Bio / Technol.3: 323-326; U.S. Patent No. 4,722,840; Depeyroux et al. (1986); Science 233: 472-475), the Hepatitis B core antigen (Clarke et al (1988) Vaccines 88 (Ed.H. Ginsberg, et al.) Pp. 127-131) L Poliovirus (Burke et al. 1988) Nature 322: 81-82), and the Tobacco Mosaic Virus (Haynes et al. (1986) Bio / Tecnol. 4_: 637-641). Especially preferred carriers include serum albumins, limpet hemocyanin, ovalbumin, whale sperm myoglobin, leukotoxin molecules, and other proteins well known to those skilled in the art. The protein carriers can be used in their native form or their content of functional groups can be modified for example, by succinylation of lysine residues or reaction with Cys-thiolactone. A sulfhydryl group can also be incorporated into the carrier (or antigen) for example, by reacting the amino functions with 2-iminothiolane or the N-hydroxysuccinimide ester of 3- (-dithiopyridyl propionate.) Suitable carriers can also be modified to incorporate the separating arms (such as hexamethylene diamine molecules or other bifunctional molecules of similar size) for the binding of the peptide immunogens.The carriers can be physically conjugated to the endogenous of interest, using normal coupling reactions. Alternatively, the chimeric molecules can be prepared recombinantly for use in the present invention, such as by fusion of a gene encoding a suitable polypeptide carrier to one or more copies of a gene, or fragment thereof, which codes for an endogenous immunogen, selected.

Nucleic acids In general, nucleic acid-based vaccines for use with the present invention will include pertinent reactions coding for an endogenous immunogen, appropriate control sequencing, and optionally, therapeutic, auxiliary nucleotide sequences. The nucleic acid molecules are prepared in the form of vectors that include the elements necessary to direct transcription and translation in a recipient cell. In order to increase an immune response in an immunized subject, the nucleic acid molecules can be administered in conjunction with auxiliary substances, such as pharmacological agents, adjuvants, cytokines, or in conjunction with the distribution of vectors encoding the response modifiers. biological, such as cytokines and the like. The nucleotide sequences selected for use in the present invention can be derived from known sources, for example, isolating them and cells or tissue containing desired gel or nucleotide sequence using standard techniques, or by using recombinant or synthetic techniques . Once the coding sequence for the endogenous immunogen has been prepared or isolated, these sequences can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those skilled in the art, and the choice of an appropriate cloning vector is a matter of choice. Ligations to other sequences, for example, auxiliary molecules or carrier molecules, are performed using standard procedures known in the art. One or more portions of endogenous immunogens of the chimera can be fused 5 'and / or 3' to a desired auxiliary sequence or carrier molecule. Alternatively, one or more portions of endogenous immunogen can be located at sites internal to the carrier molecule, or these portions can be placed at terminal and internal locations on the chimera. Alternatively, the DNA sequences encoding the endogenous immunogens of interest, optionally linked to carrier molecules, can be prepared synthetically instead of cloned. The DNA sequences can be designed with appropriate codons for the particular sequence. The complete sequence of the immunogen is then assembled for the overlap of oligonucleotides prepared by normal methods and assembled in a complete coding sequence. See, for example, Edge (1981) Nature 292: 756; Na bair et al. (1984) Science 223: 1299; and Jay et al (1984) J. Biol. Chem. 259: 6311. The coding sequence is then placed under the control of suitable control elements for expression in a suitable host tissue in vivo. The choice of control elements will depend on the subject and the type of preparation used. In this way, if the endogenous transcription and translation machinery of the subject will be used to express the immunogens, control elements compatible with the particular subject will be used. In this regard, several promoters are known in the art for use in mammalian systems. For example, typical promoters for expression of mammalian cells include the SV40 anterior promoter, a CMV promoter such as the CMV immediate forward promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter ( Ad MLP), and the promoter of simple herpes, among others. Other non-viral promoters, such as a promoter derived from the murine metallothionein gene, also found use for the expression of mammals. Typically, transcription termination and polyadenylation sequences will also be present, located 3 'to the translation stop codon. Preferably, a sequence for the optimization of translation initiation, located 5 'to the coding sequence, is also presented. Examples of the transcription / polyadenylation terminator signals include those derived from SV40, as described in Sambrook et al, supra, as well as the terminator sequence of bovine growth hormone. Introns, which contain splice donor and acceptor sites, can also be designed in constructions for use with the present invention. The enhancer elements can also be used herein to increase the expression levels of the constructions. Examples include the SV40 former gene enhancer (Dijkema et al. (1985) EMBO J. 4_: 761), the enhancer / promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma virus (gorman et al. (1982) Proc. Nati. Acá. Sci. USA 7_9: 6777) and the elements derived from human CMV (Boshart et al. (1985) Cell 4_1: 521), as well as the elements included in the sequence of intron A of CMV. Once prepared, the nucleic acid compositions can be delivered to the ear of a subject using known methods. In this regard, several techniques for immunization with DNA encoding the antigen have been described. U.S. Patent No. 5,589,466 to elgner et al.; Tangh et al. (1992) Nature 358: 152; Davis et al. (1993) Hum. Molec. Genet 2: 1847; Ulmer et al. (1993) Science 258: 1745; Wang et al. (1993) Proc.

Nati Acad. Sci. USA 90_: 4156; Eisengraun et al. (1993) DNA Cell Biol. 12_: 791; Fynan et al. (1993) Proc. Nati Acad. Sci. USA 90_: 12476; Fuller et al. (1994) AIDS Res. Human Retrovir. 1_0: 1433; and Raz et al (1994) Proc. Nati Acad. Sci. USA 9_1: 9519. General methods for the distribution of nucleic acid molecules to cells can also be used, such as liposome-mediated gene transfer. See, for example, Hazinski et al. (1991) A. J. Respir. Cell. Mol. Biol. 4_: 206-209; Brigham et al. (1989) Am. J. Med. Sci. 298: 278-281; Canonical et al. (1991) Clin. Res. 3_9: 219A; and Nabel et al. (1990) Science 249; 1285-1288. In this manner, the nucleic acid vaccine compositions can be delivered in either the liquid form or in the form of particles using a variety of known techniques.

Peptides Peptide-based vaccine compositions can also be produced using a variety of methods known to those skilled in the art. In particular, endogenous immunogens can be isolated directly from native sources, using normal purification techniques. Alternatively, immunogens can be produced recombinantly using the nucleic acid expression systems described above, and purified using known techniques. Peptide immunogens can also be synthesized, based on the described amino acid sequences or amino acid sequences derived from the DNA sequence of a molecule of interest, using chemical polymer synthesis such as solid phase peptide synthesis. These methods are known to those skilled in the art. See, for example, J. M. Stewart and J. D. Uyoung Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co. , Rockford, IL (1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, V01. 2, Academic Press, New York, (1980), pp. 3-254, and for solid phase peptide synthesis techniques; and M. Bodansky, Principies of Peptide Synthesis, Springer-Verlag, Berlin (1984) and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, supra, Vol. 1, for classical synthesis of solutions . Peptide immunogens can also be produced by cloning the coding sequence thereto into any suitable expression or replicon vector. Numerous cloning vectors are known to those skilled in the art, and selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning, and of host cells that can be transformed, include the bacteriophage lambda (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria) , pGVHOd (gram-negative bacteria), pLAFRl (gram-negative bacteria), pME290 (gram-negative bacteria not E. coli), pHV14 (E. coli and Bacillus subtilis), pBd9 (Bacillus), pIJ161 (Streptomyces), pUCß (Streptomyces), YIp5 (Saccharomyces), YCpl9, (Saccharomyces) and the bovine papilloma virus (mammalian cells). See in general, DNA Cloning: Vols. I & II, supra; T. Maniati et al., Supra; B. Perbal, supra. The gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and optionally, an operator, so that the DNA sequence of interest in the RNA is transcribed by a suitable transformant. The coding sequence may or may not contain a signal peptide or leader sequence. Peptide immunogens can be expressed using, for example, the tac promoter of E. coli or the promoter of the protein A gene (spa) and the signal sequence. Guiding sequences can be removed by the bacterial host in post-translational processing. See, for example, U.S. Patent Nos. 4,431,739; 4,425,437; 4,338,397. In addition to the control sequences, it may be desirable to add regulatory sequences that allow the regulation of the expression of the immunogen sequences relative to the growth of the host cell. Regulatory sequences are known to those skilled in the art, and examples include those that elicit the expression of a gene to be active or inactive in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, integrating sequences. An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequence which is such that the coding sequence is transcribes under the "control" of the control sequences (ie, RNA polymerase that binds to the DNA molecule in the control sequences that transcribe the coding sequences.) Modification of the sequences encoding the immunogen with endogenous For example, in some cases, it may be necessary to modify the sequence so that the control sequences can be joined in the appropriate orientation.; that is, to maintain the reading frame. The control sequences and other regulatory sequences can be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site. In some cases, it may be desirable to add sequences that cause secretion of the immunogen from the host organism, with subsequent cleavage of the secretory signal. It may also be desirable to produce mutants or analogues of the endogenous immunogen. Mutants or analogs can be prepared by deleting a portion of the sequence encoding the immunogen, or if present, a portion of the sequence encoding the desired carrier molecule, by inserting a sequence, and / or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art. See, for example, Sambrook et al., Supra; DAN Cloning, Vols. I. and II, supra; Nucleic Acid Hybridizat ion, supra. Endogenous immunogens can be expressed in a wide variety of systems, including insect, mammalian, bacterial, viral and yeast expression systems, all well known in the art. For example, insect cell expression systems, such as vaculovirus systems, are known to those skilled in the art and described in for example Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for insect cell / vaculovirus expression systems are commercially available in the form of equipment from, inter alia, Invitrogen, San Diego CA ("MaxBac" team). Similarly, mammalian bacterial cell expression systems are well known in the art and described in for example, Sambrook et al., Supra. Yeast expression systems are also known in the art and are described in for example, Yeast Genetic Engineering (Barr et al., Eds., 1989) Butterworths, London. A number of host cells suitable for use with the above systems are also known. For example, mammalian cell lines are known in the art and include immortalized cell lines available from the American Species Culture Collection (ATCC), such as, but not limited to, Chinese Hamster Ovary (CHO) cells, HeLa cells, baby hamster kidney cells (BHK), monkey kidney cells (COS), human hepatocellular carcinoma cells (for example, Hep G2), bovine kidney cells from Madin-Darby ("MDBK"), as well as others. Similarly, bacterial hosts such as E. coli., Bacillus subtilis and Streptococcus spp., Will find use with the present expression constructs. Yeast hosts useful in the present invention include inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltose, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schi zosaccharomyces pombe and Yarrowia lipoluytica. Insect cells for use with the vaculovirus expression vectors include, inter alia, Aedes aegypti, autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni. Depending on the expression system and the selected host, the endogenous immunogens are produced by culturing host cells transformed by an expression vector described above under conditions whereby the immunogen is expressed. The expressed immunogen is then isolated from the host cells and purified. If the expression system secretes the immunogen in growth media, the product can be purified directly from the media. If it is not segregated, it can be isolated from the cellular phones. The selection of appropriate growth conditions and methods of recovery are within the skill of the experience of the technique. Subjects can be immunized against endogenous immunogens by the administration of vaccine compositions including the peptides described above. Prior to immunization, it may be desirable to further increase the immunogenicity of a particular immunogen. This can be achieved in any of the several ways known to those skilled in the art. For example, the immunogen can be administered bound to a secondary carrier. These carriers are described in detail above. Immunogens can also be administered via a carrier virus that expresses the same. Carrier viruses that found use in the present include, but are not limited to, vaccinia and other pox viruses, adenoviruses, and herpes viruses. By way of example, the vaccinia virus recombinants expressing the proteins can be constructed as follows. The DNA encoding a particular protein is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and the flanking vaccinia DNA sequences such as the thymidine kinase (TK) coding sequence. This vector is then used to transport cells that are simultaneously infected with vaccinia. The homologous recombination serves to insert the vaccinia promoter plus the gene encoding the desired immunogen in the viral genome. The resulting recombinant TK can be selected by culturing the cells in the presence of 5'-bromodeoxyuridy and by collecting the viral plaques resistant thereto. Typically, the mammalian subject is immunized in the ear with the endogenous immunogen, either administered alone, or in admixture with a pharmaceutically acceptable carrier or excipient. Suitable carriers are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or buffering agents. Vaccines are usually prepared as injectable products, either as solutions or liquid suspensions, or as solid forms that are suitable for solution or suspension in liquid vehicles before injection. The preparation can also be emulsified or the active ingredient encapsulated in liposome vehicles. The active immunogenic ingredient is often mixed with carriers containing excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable carriers are for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.

In addition, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants that improve the effectiveness of the vaccine. Suitable adjuvants include, for example, mura lyl dipeptides, avridine, aluminum hydroxide, oils, saponins and other substances known in the art. Current methods for preparing these dosage forms are known, or will be apparent, to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 18a edition, 1990. The composition or formion to be administered will contain an amount of the appropriate endogenous immunogen to achieve the immunized state desired in the subject being treated. . Formed controlled vibration formions are made by incorporating the endogenous immunogens into carriers or vehicles such as liposomes, non-resorbable waterproof polymers such as ethylene vinyl acetate copolymers and Hytrel® copolymers, swellable polymers such as hyogels, or resorbable polymers such as collagen and certain polyacids or polyesters such as those used to make resorbable sutures. Vaccine compositions can also be prepared in a solid form for distribution to the ear of a subject. For example, partice, solid formions can be prepared for distribution from commercially available needleless injection devices. Alternatively, solid dose implants may be provided for implantation in the ear of a subject, for example, using a trocar. See, for example, Spitzer et al. (1978) Theriogenology l_0: 181-200; and Bretzlaff et al. (1991) Am. J. Vet. Kes. 52: 1423-1426. Additionally, immunogens can be formed into vaccine compositions in either the neutral form or the salt form. The pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acids such as for example hydrochloric or phosphoric acid, or organic acids such as acetic acid, oxalic, tartaric, mandélico, and similars. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ylamino -et anol, histidine, procaine, and the like. Other vaccine compositions may include adjuvants to further increase the immunogenicity of the endogenous immunogen. Adjuvants may include, for example, emulsifiers, muramyl dipeptides, avridine, aluminum hydroxide, oils, saponins and other substances known in the art. More particly, the emulsifiers can be used as adjuvants. Compounds which may serve as emulsifiers herein include natural and synthetic emulsifying agents, as well as .anionic, cationic and nonionic compounds. Among the synthetic compounds, the anionic emulsifying agents include, for example, the potassium, sodium and ammonium salts of lauric and oleic acid, the calcium, magnesium and aluminum salts of fatty acids (i.e., metallic soaps), and organic sulfonates such as sodium lauryl sulfate. Synthetic cationic agents include, for example, cetyltri ethylammonium bromide, while synthetic nonionic agents are exemplified by glyceryl esters (e.g., glyceryl oleate), polyoxyethylene glycol esters and ethers, and fatty acid esters of sorbitan (eg, sorbitan monopalmitate) and its polyoxyethylene derivatives (eg, polyoxyethylene sorbitan monopalmitate). Natural emulsifying agents include gum arabic, gelatin, lecithin and cholesterol. Other suitable adjuvants can be formed with a cytose component, such as an individual oil, a mixture of oils, a water-in-oil emulsion, or an oil-in-water emulsion. The oil can be a mineral oil, a vegetable oil, or an oil of animal origin. Mineral oil, or oil-in-water emulsions in which the oily component is mineral oil are preferred. In this regard, a "mineral oil" is defined herein as a mixture of liquid hydrocarbons obtained from petroleum via a distillation technique; the term is synonymous with "liquid paraffin", "liquid petrolatum", and "white mineral oil". The term is also proposed to include "light mineral oil," that is, an oil that is obtained in a similar manner by the distillation of petrolatum, but which has a slightly lower specific gravity than white mineral oil. See, for example, Remington's Pharmaceutical Sciences, supra, at pages 788 and 1323. A particularly preferred oily component is the oil-in-water emulsion sold under the trade name EMULSIGEN PLUS ™ comprising a light mineral oil as well as 0.05% formalin, and 30 mcg / mL of gentamicin as preservatives), available from MVP Laboratories, Ralston, Nebraska, or the adjuvant VSA-3 which is a modified form of the EMULSIGEN PLUSTM adjuvant. Suitable oils of animal origin include, for example, cod liver oil, halibut oil, shad oil, rough orange oil and shark liver oil, all of which are commercially available. Suitable vegetable oils include, without limitation, canola oil, almond oil, cottonseed oil, corn oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil, and oils. Alternatively, a number of aliphatic nitrogen bases can be used as adjuvants with the vaccine formulations. For example, known immunological adjuvants include amines, quaternary ammonium compounds, guanidines, benzamidines and tiouroniums (Gall, D. (1966) Immunology _11_: 369-386). Specific compounds include dimethyldioctadecylammonium bromide (DDA) (available from Kodak) and N, N-dioctadecyl-N, N-bis (2-hydroxyethyl) propanediamine ("avridine"). The use of DDA as an immunological adjuvant has been described, for example, Kodak Laboratory Chemicals Bulletin 5_6 (1): 1- 5 (1986); Adv. Drug Deliv. Rev. 5_ (3): 163-187 (1990); J. Controlled Relase 7: 123-132 (1988); Clin. Exp. Immuno 1. 7_8 (2) -: 256-262 (1989); J. I unol. Methods: 97 (2): 159-164 (1987); Immunology 58_ (2): 245-250 (1986); and Int. Arch. Allergy Appl. Immunol. 6_8_ (3): 201-208 (1982). The ridine is also well known as an adjuvant. See, for example, U.S. Patent No. 4,310,550 to Wolff, III et al., Which discloses the use of N, N-higher alkyl-N ', N' -bis (2-hydroxyethyl) propanediamines in general , and especially avridine, as vaccine adjuvants. U.S. Patent No. 5,151-267 to Babiuk, and Babiuk et al. (1986) Virology 159: 57-66, also relate to the use of avridine as a vaccine adjuvant. The vaccine composition is formulated to contain an effective amount of the endogenous immunogen, the exact amount that is readily determined by one skilled in the art, wherein the amount depends on the animal being treated, the ability of the animal's immune system to synthesize antibodies. , and the degree of protection desired. For peptide-based vaccine formulations, approximately 1 μg to 1 mg, more generally 5 μg to 200 μg of immunogen per mL of injected solution, should be adequate to promote an immune response when administered. If a chimera is used per peptide-carrier, the ratio of immunogen to carrier in the vaccine formulation will vary based on the particular immunogen carrier selected to construct those molecules. Effective doses can be easily established by one skilled in the art by routine testing establishing dose response curves. The subject is immunized by administration of one of the vaccine compositions described above in the ear in at least one dose, and preferably two doses. In addition, the animal can be administered with as many doses as required to maintain a state of immunity. Any pharmaceutical dispensing medium suitable for distributing the vaccine composition to the subject's ear can be employed. For example, conventional needle syringes, compressed gas (air) or spring injectors (US Patent Nos. 1,605,763 to Smoot; 3,788,315 to Laurens; 3,853,125 by Clark et al .; 4,596,556 to Morrow et al., And 5,056,830 to Dunlap), liquid jet injectors (US Patent Nos. 2,754,818 to Scherer, 3,330,276 to Gordon, and 4,518,385 to Lindmayer et al.), And particle injectors (Patents of the United States Nos. 5,149,655 to McCabe et al. ,204,253 to Sanford et al.) Are all suitable for the distribution of vaccine compositions. Preferably, the vaccine composition is administered subcutaneously, subdermally, or intradermally, to the ear of the subject, eg, the pinna of the outer ear. If a jet injector is used, an individual jet of the liquid vaccine composition is injected under high pressure and velocity, for example 1200-1400 PSI, thereby creating an opening in the skin and penetrating to suitable depths for the immunization. When particularly small volumes of the vaccine are to be delivered by jet injection, for example, amounts of less than about 0.1 mL, it may be more effective to distribute the vaccine to the dorsal surface without hair of the ear to avoid adverse effects of the body hair. The following are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

C. Experimental part Although the invention is broadly applicable to vaccination against any endogenous immunogen in a mammalian subject, the invention is exemplified herein with particular reference to active immunization against GnRH. Immunization against GnRH can be used to produce the spot of boars in commercial pigs, or surgical castration in cattle can be used as an alternative. Bonneau et al. (1995) Livestock Production 42. A number of GnRH immunogens, vaccine compositions containing these immunogens, and immunoneutralization methods against endogenous GnRH in subjects vaccinated using the vaccine compositions are described in the commonly owned US patent application. , serial number 08 / 69,865, filed August 9, 1996, and in International Publication No. WO 96/24675, published August 15, 1996. Thus, one embodiment of the invention relates to the distribution of a GnRH immunogen to the ear of a mammalian subject to provide an immune response directed against endogenous GnRH. The particular immunogen used may comprise one or more GnRH polypeptides, and / or one or more GnRH multimers. The selected GnRH immunogens can be used in their native form, or modified to provide a more immunogenic form, for example, by succinylation of lysine residues or reaction with cys-thiolactone. Additionally, the GnRH immunogen can be administered to the ear alone, or in combination with a suitable carrier molecule. Alternatively, the GnRH immunogen is conjugated to a macromolecular carrier, or a chimeric molecule can be used which includes leukotoxin fused to a GnRH polypeptide. More particularly, GnRH leukotoxin chimeras are formed which include a leukotoxin polypeptide fused to one or more GnRH multimers having at least one repeating GnRH decapeptide sequence, or at least one repeat unit and a corresponding sequence. to at least one epitope of a molecule selected from GnRH. The GnRH peptide sequences selected in the chimeras can all be the same, or they can correspond to different GnRH derivatives, analogs, variants or epitopes while retaining the ability to produce an immune response. A detailed discussion of GnRH can be found in U.S. Patent No. 4,975,420. Additionally, a nucleotide sequence representative of a GnRH decapeptide is depicted in Figure 1A. This GnRH sequence is modified by the substitution of a glutamine residue in the N-terminus in place of pyroglutatic acid which is found in the native sequence. This particular substitution provides a molecule that retains the native structure of glutamic acid but also retains the structure without change of pyroglutamate. Therefore, the resulting peptide does not require cyclization of the glutamic acid residue and can be reduced in the absence of conditions necessary to effect cyclization. Because the GnRH sequence is relatively short, it can be easily generated using synthetic techniques. In leukotoxin-GnRH chimeras, a leukotoxin polypeptide sequence is used to confer immunogenicity on the associated GnRH polypeptides (as a carrier protein) to help produce an adequate immune response towards endogenous GnRH in an immunized subject. This immunization with GnRH can regulate fertility in a subject vaccinated by the breakdown of estrus or spermatogenesis cycles. The leukotoxin-pol-peptide chimeras GnRH, particular, used herein contain one or more portions of GnRH having a plurality of selected GnRH polypeptide sequences. The GnRH portion of the chimera may comprise either multiple or tandem repeats of the selected GnRH sequences, multiple or tandem repeats of selected GnRH epitopes, or any conceivable combination thereof. Suitable epitopes of GnRH can be identified using routine techniques known in the art, or fragments of GnRH proteins can be tested for immunogenicity, and the active fragments used in the compositions in view of the complete polypeptide. When more than one multimer of GnRH is included in the chimeric molecules, each portion of GnRH may be the same as or different from the other portions of GnRH included in the molecule. The sequence of a particular GnRH multimer is represented in Figure IB where four GnRH sequences, indicated in (1), (2) _, (3), and (4) respectively, are separated by the amino acid separating sequences. of triplet comprising several combinations of serine and glisina residues. In this multimer, each GnRH sequence (for example, those indicated in (2) and (4), respectively) contain a non-conservative amino acid substitution in the second position of the GnRH decapeptide comprising an Asp residue instead of the His residue found in the native GnRH sequence. The multimeric sequence of alternating GnRH produced in this manner produces a highly immunogenic GnRH antigen. Other GnRH analogs corresponding to any addition, substitution and / or deletion of amino acids, single or multiple can be used in any repetitive or alternating multimeric sequence. In a preferred function of leukotoxin-GnRH, four copies of the GnRH portion depicted in Figure IB are fused to a molecule of leukotoxin such that the leukotoxin molecule is flanked, in its N- and C-term, for two copies of this GnRH multimer. The leukotoxin-GnRH immunogens can be produced recombinantly as a chimeric protein using the methods described above. The nucleotide sequence encoding the full length leukotoxin Al from P. haemolytica has been determined. See, for example, Lo, Infect. Immun. (1987) 55_: 1987-1996 and U.S. Patent No. 5,055,400. Additionally, several sequences of variant leukotoxin genes have been described, in U.S. Patent No. 5,476,657, International Publication No. WO 96/24675, published August 15, 1996, and in the United States Patent Application. Commonly owned, serial No. 08 / 694,865, filed August 9, 1996. Similarly, the coding sequence for which porcine, bovine and ovine GnRH have been determined (Murad et al. (1980) Hormones and Hormone antagonists, in The Pharmacological Basis of Therapeutics Sixth Edition), and the cDNA for human GnRH has been cloned so that its sequence has been well established (Seeburg et al (1984) Nature 311: 666-668). Additional GnRH polypeptides of known sequences have been described, such as the GnRH molecule that occurs in salmon and chickens (International Publication No. WO 86/07383, published December 18, 1986). The GnRH coding sequence is highly conserved in vertebrates, particularly in mammals; and the porcine, bovine, ovine and human sequences of GnRH are identical to each other. The desired leukotoxin and the GnRH genes can be cloned, isolated and ligated together using recombinant techniques, generally known in the art. For example, Sambrook et al., Supra. Particular examples of these GnRH immunogens are provided hereinbelow.

Materials and methods Enzymes were purchased from commercial sources, and used according to the manufacturers' instructions. Radionucleotides and nitrocellulose filters were also purchased from commercial sources. In the cloning of DNA fragments, except where noted, all DNA manipulations were done according to normal procedures. See, Sambrook et al., Supra. Restriction enzymes, T4-DNA ligase, E. coli, DNA polymerase I, Klenow fragment, and other biological reagents were purchased from commercial suppliers and used according to the manufacturers' instructions. The double-stranded DNA fragments were separated on agarose gels. CDNA and genomic libraries were prepared by standard techniques in pUC13 and the bacteriophage lambda gtll, respectively. See, DNA CLONIÑG: Vols I and II, supra. Strain B122 of serotype 1 ("Al"), from biotype A of P. haemolytic was used from the lung of a calf that died of pneumonia and was stored at minus 70 ° C in defibrinated blood. Routine propagation was carried out on blood agar plates or brain-heart infusion broth (Difco Laboratoeis, Detroit, MI) supplemented with 5% (v / v) horse serum (Gibco Canada Ltd., Burlington, Canada). All cultures were incubated at 37 ° C.

Example 1 Construction of Leukotoxin Chimeras-GnRH 1. Isolation of the Leukotoxin Gene from P. haemolí t ica To isolate the leukotoxin genes, the gene libraries of P. haemolytica Al (strain B122) were constructed using standard techniques. See, Lo et al., Infect. Immun. , supra; DNA CLONING: Vols. I and II, supra; and Sambrook et al., supra. A genomic library was constructed in the Pucl3 plasmid vector and a DNA library constructed in the bacteriophage da gtll. The resulting genes were used to transform E. coli and individual colonies were mixed and titrated for the reaction with serum from a calf that has survived a P. haemolytic infection and that has been boosted with a concentrated culture supernatant of P. haemolytic to increase the levels of anti-leukotoxin antibodies. Positive colonies were detected for their ability to produce leukotoxin by incubating cell lysates with bovine neutrophils and subsequently measuring the release of lactate dehydrogenase from the latter. Several positive colonies identified these recombinants were analyzed by correlation with restriction endonucleases. One clone appeared to be identical to a previously cloned leukotoxin gene. See, Lo et al., Infect. Immun., Supra. To confirm this, small fragments were re-cloned and restriction maps were compared. It was determined that approximately 4 kilobases of DNA bases have been cloned. The progressively larger clones were isolated by carrying out a shift on the chromosome (5 'or 3' direction in order to isolate the full-length recombinants that were approximately 8 kb in length) The final construct was called pAA114.

This construct contained the complete sequence of the leukotoxin gene. LktA, a restriction endonuclease fragment of Mael from pAA114 that contained the complete leukotoxin gene, was treated with the Klenow fragment of DNA polymerase I plus the nucleotide triphosphates was read at the Smal site of the cloning vector pUC13. This plasmid was named pAA179. From this, two expression constructs were made in the ptac-based vector pGH432: lacI digested with Smal. One, pAA342, consisted of the 5'-AhaIII fragment of the lktA gene while the other, pAA345, contained the complete Mael fragment described above. The clone pAA342 expressed a truncated leukotoxin peptide at high levels, while the pAA345 expressed the full-length leukotoxin at very low levels. Therefore, the 3 'end of the lktA gene (BamHl fragment of Styl from pAA345) was ligated to pAA342 digested with Styl BamHl, yielding plasmid pAA352. The leukotoxin of P. haemolytic produced from the construction of pAA352 is subsequently referred to herein as LKT 352. The various truncated versions of the leukotoxin gene were expressed from_pAA114. These truncated forms were fusions with the β-galactosidase (lacZ) gene. Two fragments, LTX1.1 and LTX3.2, from a double digestion of EcoRV PstI, were isolated from pAA114 as purified restriction fragments (1.0 kb and 2.1 kb, respectively). These fragments were cloned into the cloning vector pTZ18R which were digested with HincII and PstI. The resulting vector, called pLTX3P1, was used to transform E.coli strain JM105. Transformed cells were identified by plating in the medium containing ampicillin plus Xgal and IPTG. The blue colonies indicated the presence of a functional lacZ gene. The DNA of the transformed cells was analyzed by digestion with restriction endonucleases and found to contain the 5 'end of the leukotoxin gene (lktC and lktA). A 5 'fragment of EcoRV / Pstl leukotoxin (from pLTX3P1) was subcloned into the cloning vector pBR325 which was digested with EcoR1 and Pst1. Plasmid pBR325 also contained the native leukotoxin promoter (purchased from pLTX3P.l) and a full-length lacZ gene, without promoter. The resulting construct was used to transform JM105 from E. coli and blue colonies were isolated from Xgal agar. The new construction was called pAA1081 (ATCC No. 67883). The leukotoxin of P. haemolytica produced from the pAAlOl construct is hereinafter referred to as "LKT 101". 2. Fusion Constructions of LKT-GnRH Representative LKT-GnRH fusions were constructed as follows. Oligonucleotides containing the sequences corresponding to an individual copy of GnRH and GnRH as four multiple repeats were constructed in a Pharmacia gene assembler using normal phosphoramidite chemistry The sequences of these oligonucleotides are shown in Figures IA and IB The present oligonucleotides were ligated into the vector pAA352 (ATCC No. 68283, and described above), which have been digested with the restriction endonuclease Bamhl.This vector contains the leukotoxin gene of P. haemolytic. The ligand was used to transform E. coli strain MH3000 The transformants containing the oligonucleotide inserts were identified by the correlation with restriction endonucleases.

A eight-copy GnRH tandem repeat sequence was prepared by binding the four-copy GnRH oligonucleotides and ligating them into a vector that has been digested with the restriction endonuclease BamHl. The oligomers were designed to disable the Ba Hl site at address 50 when inserted and to ensure that the insertion of additional copies of the oligomer will be oriented in the appropriate reading frame. The sequence of the present oligonucleotide is shown in Figure IB. The plasmid DNA from E. coli strain MH3000 was then isolated and used to transform strain JM105. The recombinant plasmids were designated pCBH3 (LKT 352: GnRH of 4 copies, ATCC Accession No. 69749) and pCB112 (LKT 352: GnRH of 8 copies).

Construction of the shortened LKT Carrier Peptide A shortened version of the recombinant leukotoxin peptide was constructed from the recombinant gene present in the plasmid pAA352 (as described above). The shortened LKT gene was produced by deleting an internal DNA fragment of approximately 1300 bp in length from the recombinant LKT gene as follows. Plasmid pCB113 (ATCC Accession No. 69749) which includes the LKT 352 polypeptide fused to four copies of the GnRH polypeptide, was digested with the restriction enzyme BstBl (New England Biolabs). The resulting linearized plasmid was then digested with the mung-bean endonuclease (Pharmacia) to remove the individual strand leaving term produced by digestion with BstBl. The blunt-ended DNA was then digested with the restriction enzyme Nael (New England Biolabs), and the digested DNA was loaded onto a 1% agarose gel of one of the DNA fragments separated by electrophoresis. A large DNA fragment of approximately 61909 bp was isolated and purified from the agarose gene using a Gene Clean kit (Bio 101), the purified fragment was allowed to bind itself using the bacteriophage T4-DNA ligase (Pharmacia). The resulting ligation mixture was used to transform the competent E. coli JM105 cells, and the positive clones were identified by their ability to produce a thin protein having a molecular weight of approximately 57 KDa. The recombinant plasmid tested in this manner was designated pCBIII (ATCC Accession No. 69748), and produces a cut leukotoxin polypeptide (hereinafter referred to as "LKT 111"). Fused to four copies of the GnRH polypeptide. Plasmid pCB114 is identical to pCBlll except that the multiple copy GnRH sequence (corresponding to the oligomer of Figure IB) was inserted twice. 4. Construction of a LKT-GnRH Fusion that has GnRH multimers with Carboxi Terminal Groups and Amino Terminal Groups, of Eight Copies A fusion molecule of recombinant LKT-GnRH that has two GnRH multimers of eight copies, one arranged in the N'-terminus of LKT 111, and the other arranged in the C-term of LKT 111, was constructed from the fusion sequence of LKT-GnRH obtained from the plasmid pCB114 by ligating the GnRH sequence of multiple copies (corresponding to the oligomer of Figure IB) twice at the 5 'end of the LKTIII coding sequence. A synthetic nucleic acid molecule having the following nucleotide sequence: 5 '-ATGGCTACTGTTATAGATCGATCT-3' (SEQ ID No. 5) was ligated at the 5 'end of the multiple copy GnRH gene sequence. The synthetic nucleic acid molecule encodes a sequence of eight amino acids, Met-Ala-Thr-Val-Ile-Asp-arg-Ser (SEQ ID No. 6). The resulting recombinant molecule contains this in the order given in the 5 'to 3' direction: the synthetic nucleic acid molecule; a nucleotide sequence encoding a first GnRH multimer of 8 copies; a nucleotide sequence encoding the shortened LKT peptide (LKT 111); and a nucleotide sequence encoding a second GnRH multimer of 8 copies. The recombinant molecule was circulated and the resulting molecule was used to transform E.coli JM105 cells. competent. Positive clones were identified by their ability to produce a hybridized protein having a molecular weight of about 74 kDa. The recombinant plasmid formed in this manner was designated pCB122 which produces the LKTIII polypeptide fused to 16 copies of the GnRH polypeptide. A series of recombinant fusion molecules of LKT-GnRH were then derived from pCB122 as follows. The GnRH multimer of 8 copies at the 5 'end of the pCB122 construct was amplified using PCR. The copied GnRH multimer sequence was then modified to provide a GnRH insert that could be located at the Nsil site of the leukotoxin carrier in pCB122 and maintain the reading frame. The synthetic readings, which code for the additional amino acids flanking the GnRH insert, were also ligated to the insert. Flanking amino acids were required to successively use PCR to copy the GnRH insert and to ligate or bind the insert to the leukotoxin molecule. The resulting construct, called pCB133, contained 8 additional copies of GnRH that were inserted into the Nsil site of the shortened LKT peptide (LKT 111) in the construction of pCB122. An additional construct, called pCB132, was constructed in the same manner as pCB133, however, the 8-copy GnRH insert was inserted into the Stul site of the LKT 111 carrier in the construction of pCB122. A set of synthetic flanking sequences (different from those used in the construction of the pCBl33 construct) were added to the GnRH insert in order to ligate it to LKT 111. In this manner, pCB134 contains 8 additional copies of GnRH that were inserted into the Stul site of the cut LKT peptide (LKT 111) in the construction of pCB122. The Nsil insert of pCB133, which contains the 8-copy GnRH insert described above, was excised and ligated into the Nsil site at the pCB134 site to provide an additional construct called pCB135. The pCB135 construct produced a chimeric molecule comprising the LKT 111 polypeptide fused to the GnRH multimers (8 copies each) in 4 different locations, for a total of 32 copies of GnRH in the molecule. An additional construct, called pCB136, was derived from pCB122 by inserting into the Stul site of the LKT 111 sequence a synthetic polynucleotide encoding a number of "universal T cell epitope" peptide sequences interspersed among GnRH sequences. The universal T cell epitopes seem to stimulate the immune responses of T cells in all tested species. See, for example Sinigaglia et al. '(1988) Nature 336: 778-780; Panina-Bordignon et al. (1989) Eur. J. Immunol. 1'9: 2237-2242; O'Sullivan et al. (1990) J. Immun. 145: 1799-1808; and O'Sullivan et al. (1991) J. Immun. 147: 2663-2669. In particular, the polypeptide insert included in the 5 'to 3' direction, a sequence encoding the universal T cell epitope of tetanus toxin, a GnRH sequence, a sequence encoding the diphtheria toxin T cell epitope, a GnRH sequence, a coding sequence for the T cell epitope of whale sperm myoglobin, and a final GnRH sequence . Each GnRH sequence separated epitopes from adjacent T cells by two lysine residues that serves as the site of action for the enzymatic cathepsin. Cathepsin is a protease that is involved in the degradation of antigens for the presentation of the immune system.

. Purification of LKT-antigen fusions Recombinant fusions of LKT-GnRH were purified using the following procedure. For each fusion, five to ten colonies of the E. coli strains were inoculated in 10 mL of the TB broth supplemented with 100 micrograms / mL of ampicillin and incubated at 37 ° C for 6 hours on a G10 shaker, 220 rpm. Four mL of this culture was diluted in each of the two flasks with Fernbach deviators containing 400 L of the Tb + ampicillin broth and incubated overnight as described above. The cells were harvested by centrifugation for 10 minutes at 4,000 rpm in polypropylene bottles, 500 mL in volume, using a Sorvall GS3 bull. The pellet was redispersed in an equal volume of TB broth containing ampicillin that has been preheated to 37 ° C (i.e., 2 x 400 ml), and the cells were incubated for 2 hours as described above. 3.2 mL of isopropyl-B, D-t iogalactopyranoside (IPTG, Gibco / BRL), 500 mM in water (final concentration = 4 mM), were added to each culture in order to induce synthesis of the recombinant fusion proteins. The cultures were incubated for two hours. Cells were harvested by centrifugation as described above, redispersed in 50 mL 50 mM Tris-hydrochloride, 25% sucrose (w / v), pH 8.0, and frozen at -70 ° C. The frozen cells were thawed at room temperature after 60 minutes at -70 ° C, and 5 mL of lysozyme were added. (sigma, 20 mg / mL in 250 M Tris-HCl, pH 8.0). The mixture was vortexed at high speed for 10 seconds and then placed on ice for 15 minutes. The cells were then added to 500 mL of lysis buffer in a 1000 mL beaker and mixed by shaking with a 12 mL pipette. The beaker containing the suspension of used cells was placed on ice and treated with sound for a total of 2.5 minutes (5-30 seconds of explosion with 1 minute of cooling between each) with a Braun sonicizer, large, adjusted probe at a power of 100 watts. Equal volumes of the solution were placed in Teflon SS34 centrifuge tubes and centrifuged for 20 minutes at 10,000 rpm in a Sorvall SS34 rotor. The elements were redispersed in total of 100 mL of double distilled water, sterile when subjected to vortex at high speed, and repeat the centrifugation step. The supernatants were discarded and the elements combined in 20 mL of 10 mm Tris-HCl, 150 mm NaCl, pH 8.0 (saline buffered with Tris) and the suspension was frozen overnight at -20 ° C. The recombinant suspension was thawed at room temperature and added to 100 L of Guanidine-HCl (Sigma 8 M in Tris-buffered saline and mixed vigorously.) A magnetic stir bar was placed in the bottle and the solubilized sample was mixed with water. ambient temperature for 30 minutes The solution was transferred to a 200 mL Erlenmeyer matrix and 1200 mL of Tris-buffered saline were added rapidly, this mixture was stirred at room temperature for an additional 2 hours, 500 n mL aliquots were placed in bags of dialysis (Spectrum, diameter 63.7 mm cut of PM 6,000-8,000, Fisher Scientific # 132670) and these were placed in 4,000 mL beakers containing 3,500 mL of saline buffered with Tris + guanidine-HCl 0.5 M. beakers were placed in a room at 4 ° C on a magnetic stirrer overnight after which the dialysis buffer was replaced with solute Saline was quenched with Tri + Guanidine-HCl 0.1 M and the dialysis was continued for 12 hours. Then, the buffer was replaced with buffered solution with 0.05 M Tris + Guanidine HCl and the dialysis was continued overnight. The buffer was replaced with saline solution buffered with Tris (no guanidine), and dialysis was continued for 12 hours. This was repeated three more times. The final solution was poured into a 200 L plastic roller bottle (Corning) and 13 mL of 100 mM PMSF (in ethanol) were added to inhibit the protease activity. The solution was stored at -20 ° C in 100 mL aliquots. To confirm that the fusion proteins have been isolated, aliquots of each preparation were diluted 20 times in double-distilled water, mixed with an equal volume of SDS-PAGE sample buffer, placed in a boiling water bath for five minutes and run through polyacrylamine gels at room temperature. 12% Controls of recombinant leukotoxin were also run. All fusion proteins were expressed at high levels as inclusion bodies.

Example 2 GnRH Immunogen Administration The following study was conducted to compare the efficiency and uniformity of vaccination with GnRH immunogens administered either in the ear or intramuscularly in the neck of porcine subjects. Five different GnRH immunogens were used in the assay, particularly the leukotoxin-GnRH chimeras obtained from constructs pCbl22, pCB133, pCB134, pCB135 and pCB136 described above in Example 1. All recombinant GnRH immunogens were produced in E. coli, and were combined with oil adjuvant in VSA-3 water (manufactured by MVP Laboratories, Ralston, Nebraska). The vaccines were formulated to deliver 40 μg of the GnRH immunogen in a volume of 1.0 L when given by conventional syringe and needle, or 0.5 L when administered with a jet injector. Needle injections were given intramuscularly, 8 to 10 cm behind the ear and 6 to 10 cm on either side of the midline, using a 2 mL syringe and a 20 gauge 1 inch needle. Needleless injections (jet injections) were administered with the Biojetc 200 injection system, (manufactured by Bioject, Portland, Oregon). The jet injector was equipped with a specialized 1 mL syringe that has an orifice size that allows the jet of fluid to pierce the skin, and is deposited in a subcutaneous location on the ear. These ear injections occurred on the outer surface of the pinna, and were achieved by grasping the tip of the ear to immobilize it and create a flat surface that was able to withstand the pressure of the injection device as it remained on the surface of the ear. ear. The vaccine penetrated the skin and moved laterally in a thin sheet along the surface of the inner cartilaginous structure. The animal tolerated this procedure with little or no evidence of pain or exertion. Each of the five vaccine formulations was administered intramuscularly to 10 animals by needle injection into the neck, subcutaneously into different animals by jet injection to the ear. More particularly, the animals were injected to the left ear or neck on days 21 of age, and in the right ear or neck 35 days later. The animals were observed twice weekly to evaluate the reactions of the injection site. Blood samples were collected at the time of the booster injection, and 14 and 28 days later. The serum for GnRH antibodies was assessed by a standard procedure. In particular, serum samples were taken, appropriate dilutions were made in buffer, aliquots were placed in test tubes. A standard amount of 125-iodinated GnRH was then added to each tube. The final serum dilution was 1: 5,000 and 1: 20,000. After incubation for 48 hours at 5 ° C, a 1 ml aliquot of 1% carbon suspension in buffer was added. The tubes were centrifuged to pellet the carbon, and the radioactivity of the tubes containing the pellets was counted. In this method, the carbon absorbs the GnRH labeled with 125I, and a calculation is made to determine the amount of 125I-GnRH bound to the antibody in the sample. The results are expressed below in Table 1 as the percent of added 125 I-GnRH that binds to the sample. Higher values indicate higher antibody titers.

The mean titers of antibodies with the two different methods of injection are shown below in Table 2. a against b, p < .05 The serological evidence shown in Tables 1 and 2 shows a better response to the five GnRH immunogens when they are distributed to the ear by jet injector. These results clearly demonstrate that the ear is a preferred site for immunization with GnRH immunogens, providing a higher titer of antibodies as compared to immunizations distributed via intramuscular injection in the neck. The ear also provides a preferred site for the distribution of the vaccine since the tissue of the pinna is generally uniform from animal to animal, allowing the vaccine to be presented in a consistent manner.

Example 3 Administration of Compositions of Vaccine to the Ear The following study was carried out to compare the effectiveness of GnRH vaccination carried out via subcutaneous injection in the neck, or intradermal distribution in the ear. More specifically, two groups of 20 pigs each (10 males and 10 females) were injected either subcutaneously in the neck with 0.2 mL of vaccine containing 40 μg of the leucotoxin GnRH chimera obtained from the construction of pCB122 (Example 1), or 0.2 L of the same vaccine intradermally in the ear. The vaccine compositions contained the adjuvant VSA-3 (Example 2), and were distributed to the neck or ear via needle and syringe. The main injection was given at 21 days of age and the booster dose was administered at 35 days later. Blood was collected 14 and 28 days after the boost and analyzed for the anti-GnRH antibodies as described above in Example 2. The antibody titers were then expressed as% binding of 125 I-GnRH in serum diluted to 1: 5000 . These results are reported below in the. Table 3 * Mean values ± standard errors As can be seen, the antibody titers were higher when the vaccine was given in the ear, and these titers remained high throughout the trial (28 days after the booster immunization). These data confirm the usefulness of the ear as a vaccination site. 4 Administration of Compositions of Vaccine to the Ear In order to have additional access to the efficacy of the vaccination methods targeting the ear, the following study was carried out. In this study, an experimental group consisting of 20 pigs received either 0.2 or 0.4 mL of a vaccine composition containing a water-in-oil adjuvant (Seppic ISA-70, available from seppic, Inc. Castres, France). The vaccine composition included 40 μg of the leucotoxin-GnRH chimera obtained from the construction of pCB122 (see Example 2) and was given subcutaneously in the ear as an individual dose to 60-day-old pigs. Table 4 reports the titers of anti-GnRH antibodies (% binding of 125 I-GnRH in a 1: 5000 dilution) obtained from these animals on days 14, 28, 42 and 56 after injection.

* Average values ± standard errors As can be seen from the results reported in Table 4, an individual injection of the ear using a water-in-oil adjuvanted vaccine composition elicited a strong antibody response that increased even 56 days after the individual injection. These data indicate that the ear is a site that corresponds well to the different classes of adjuvants. The selection of these ear vaccine compositions provides advantages with respect to tissue residue, ease of administration, and safety of animal technicians when administering potentially dangerous vaccines.

Example 5 Comparison of Adjuvant Systems, Booster Vaccinations The following study was carried out in order to assess the efficacy of the target selection of the mammalian ear for booster vaccinations. The leukotoxin-GnRH chimeras obtained from the pCB122 construct (Example 1) were administered to cattle using either an oil-in-water adjuvant, or a water-in-oil adjuvant. More particularly, all the cattle used in the study were primed by vaccination with 200 μg of the chimera immunogen pCbl22 combined with a suitable adjuvant (an emulsion W-water in oil formed with a metabolizable oil (Squalene)) to provide a final volume of 2. 0. mL. The bait was carried out using needle and syringe to distribute the vaccine composition in the neck. For the administration of reinforcement, three experimental groups of cattle were established when reinforcing with the following vaccines: (Group 1) received 200 μg of the chimera immunogen pCB122 in a volume of 2.0 mL of a water-in-oil adjuvant (VSA3), performed administrations via subcutaneous injection to the neck using a normal needle and syringe; (Group 2) received 200 μg of the chimera immunogen pCB122 in a volume of 0.5 mL of the adjuvant VSA3, administrations were carried out via subcutaneous injection to the ear carried out using a jet injection device; and (Group 3) received 300 μg of the chimera immunogen pCB122 in a water-in-oil adjuvant (Seppic ISA-70), administrations were carried out via subcutaneous injection to the ear via the jet injection device. Table 5, below, provides the anti-GnRH antibody titers of each group of animals (reported as the% binding of 125 I-GnRH at a dilution of 1: 100) on day 21 and day 105 after administration. booster vaccination.

* Group mean ± standard error of the mean As can be seen from the data reported in Table 5, a booster vaccination administered to the ear (with either adjuvant formulation) provided an antibody response equivalent to subcutaneous booster vaccination that was administered to the neck.

Example 6 Vaccination of Individual Dose to the Mammalian Ear In yet a further study, 29 steers were once injected subcutaneously via the jet injector device, using 200 μg of the leukotoxin-GnRH chimera obtained from the construction of pCB122. The vaccine was formulated using a water-in-oil adjuvant (Seppic ISA-70). The anti-GnRH antibody titers for these steers (% binding of 125 I-GnRH at a dilution of 1: 100) on days 0, 21 and 35 after vaccination are reported below in Table 6.

Group mean ± standard error of the mean These data demonstrate that an individual dose of a GnRH vaccine composition administered to the ear provides a substantial, primary vaccine response at day 35. Thus, the ear is an effective vaccination site for both primary and primary vaccinations. reinforcement in cattle. Thus, methods for immunizing a mammal against an endogenous immunogen via administration of a vaccine composition to the ear have been described. Although preferred embodiments of the present invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and scope of the invention as defined by the appended claims.

Deposit of Useful Strains in the Practice of the Invention A deposit of biologically pure cultures of the following strains was made with the American Species Crop Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland. The indicated access number was assigned after the successful feasibility test, and the required fees were paid. The deposits were made under the provisions of the Budapest treaty in the International Recognition of deposit of Microorganisms for the Purpose of Patent Procedure and Regulations itself (Budapest Treaty). This ensures viable crop maintenance for a period of thirty (30) years from the date of deposit and at least five (5) years after the most recent request for the submission of a deposit sample by the depositor. The organisms will be available by the ATCC under the terms of the Budapest Treaty, which ensures the permanent and unrestricted availability of crops not yet determined by the North American Patent and Trademark Commission to be titled to it in accordance with 35 U.S.C. 5122 and the rules of the Commissioner according to this (including 37 C.F.R. 51.12). In granting a Patent, all restrictions on the availability to the public of the deposited crops will be irrevocably removed. These deposits are provided only as a convenience to those skilled in the art, and are not an admission that a deposit is required under 35.0.S.C. 5112. The nucleic acid sequences of these plasmids, as well as the amino acid sequences of the polypeptides encoded by them, are incorporated herein by reference and are controlled in the case of any conflict with the description herein. A license may be required to make, use or sell the deposited materials, and this license will not be granted hereby.

LIST OF SEQUENCES (1) GENERAL INFORMATION (i) APPLICANT: BIOSTAR INC (ii) TITLE OF THE INVENTION: IMMUNIZATION AGAINST ENDOGENOUS MOLECULES (iii) NUMBER OF SEQUENCES: 6 (iv): CORRESPONDENCE ADDRESS: (A) RECIPIENT: GOG, STRATHY & HENDERSON (B) STREET: SUITE 2600, 160 ELGIN STREET (C) CITY: OTTA A (D) STATE: ONTARIO (E) COUNTRY: CANADA (F) POSTAL CODE: K1P IC3 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: Flexible disk (B) COMPUTER: compatible with IBM PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM: Patentln Relay # 1.0, Version # 1.30 (vi) DATA FROM THE CURRENT APPLICATION: (A) APPLICATION NUMBER: PCT / CA98 / 00059 (B) DATE OF SUBMISSION: 04-FEB-1998 (C) CLASSIFICATION: (vii) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: US 60 / 036,838 (B) DATE OF SUBMISSION: 05-FEB-1997 (viii) INFORMATION OF AGENT / LAWYER (A) NAME: ERRAT, JUDY A. (B) REGISTRATION NUMBER: (C) ORDER NUMBER / REFERENCE: 08-878618WO (ix) TELECOMMUNICATION INFORMATION (A) TELEPHONE: (613) -233-1781 (B) TELEFAX: (613) 563-9869 ORMATION FOR SEQ ID N0: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: genomic DNA (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 1 ... 30 (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 1: CAG CAT TGG AGC TAC GGC CTG CGC CCT GGC 30 Gln His Trp Ser Tyr Gly Leu Arg Pro Gly 1 5 10 (2) INFORMATION FOR SEQ ID NO: 2; (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 10 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 2: Gln His Trp Ser Tyr Gly Leu Arg Pro Gly 1 5 10 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 147 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 1 ... 147 (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 3: CAG CAT TGG AGC TAC GGC CTG CGC CCT GGC AGC GGT TCT CAA GAT TGG 48 Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp 15 20 25 AGC TAC GGC CTG CGT CCG GGT GGC TCT AGC CAG CAT TGG AGC TAC GGC 96 Ser Tyr Gly Leu Arg Pro Gly Gly Ser Ser Gln His Trp Ser Tyr Gly 30 35 40 CTG CGC CCT GGC AGC GGT AGC CAA GAT TGG AGC TAC GGC CTG CGT CCG 144 Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Ser Tyr Gly Leu Arg Pro 45 50 55 GGT 147 Gly) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 49 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 4: Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp 1 5 10 -15 Ser Tyr Gly Leu Arg Pro GIy Gly Ser Ser Gln His Trp Ser Tyr Gly 20 25 30 Leu Arg Pro Gly Ser Gly Ser Gln Asp Trp Being Tyr Gly Leu Arg Pro 35 40 45 Gly ) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 5: ATGGCTACTG TTATAGATCG ATCT 24 ) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 amino acids (B) TYPE: amino acids (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (i) DESCRIPTION FOR THE SEQUENCE: SEQ. ID No: 6: Met Ala Thr Val He Asp Arg Ser 1 5

Claims (31)

RE IVINDICAC I ONES

1. A method for inducing an immune response against an endogenous molecule selected from the group consisting of an endogenous hormone and an endogenous hormone receptor in a mammal, characterized in that it comprises administering to the ear of the mammalian subject an effective amount of a vaccine composition. comprising an immunogen derived from the molecule, and a pharmaceutically acceptable carrier, wherein the vaccine composition is capable of inducing an immune response against the molecule.

2. JE1 method according to claim 1, characterized in that the hormone is GnRH.

3. The method according to claims 1 6 2, characterized in that the vaccine composition is administered to the subject via the subcutaneous distribution in the pinna of the ear of the subject's ear.

4. The method according to claim 1 or 2, characterized in that the vaccine composition is administered to the subject via the intradermal distribution in the pavilion of the subject's ear.

5. The method according to claim 1 or 2, characterized in that the vaccine composition is administered in more than one dose.

6. The method according to claim 1 or 2, characterized in that the endogenous molecule in the vaccine composition binds to a carrier molecule.

7. The method according to claim 1 or 2, characterized in that the vaccine composition further comprises an adjuvant.

8. The method according to claim 7, characterized in that the adjuvant is an oil-in-water formulation.

9. The method according to claim 1 or 2, characterized in that the vaccine composition is administered with a device without a needle or jet injector.

10. The method according to claim 1 or 2, characterized in that the vaccine composition is administered in the solid form.

11. The method according to claim 10, characterized in that the vaccine composition is in the form of solid particles.

12. The method according to claim 10, characterized in that the vaccine composition is administered as a solid dose implant.

13. The method according to claim 1 or 2, characterized in that the vaccine composition comprises a nucleic acid molecule that codes for the endogenous molecule.

14. The method according to claims 1 6 2, characterized in that the mammal is bovine.

15. The method according to claim 1 or 2, characterized in that the mammal is porcine.

16. A method for distributing an endogenous, selected molecule to a mammal wherein the endogenous molecule is selected from the group consisting of an endogenous hormone and an endogenous hormone receptor, the method is characterized in that it comprises administering to the ear of the mammal an amount effective of a vaccine composition comprising the endogenous molecule and a pharmaceutically acceptable carrier.

17. The use of an endogenous molecule selected from the group consisting of an endogenous hormone and an endogenous hormone receptor, in the manufacture of a vaccine composition for inducing an immune response against the endogenous molecule in a mammalian subject, wherein the composition of vaccine is for administration to the ear of the mammalian subject.

18. The use of claim 17, wherein the hormone is GnRH.

19. The use of claim 17 or 18, wherein the vaccine composition is for administration to the subject subcutaneously or intradermally in the pinna of the ear of the subject.

20. The use of claim 17 or 18, wherein the vaccine composition is for administration in more than one dose.

21. The use of claim 17 or 18, wherein the endogenous molecule the vaccine composition is linked to a carrier molecule.

22. The use of claim 17 or 18, wherein the vaccine composition further comprises an adjuvant.

23. The use of claim 22, wherein the adjuvant is an oil-in-water formulation.

24. The use of claim 17 or 18, wherein the vaccine composition is for administration with a device without a needle or chlorine injector.

25. The use of claim 17 or 18, wherein the vaccine composition is for administration in the solid form.

26. The use of claim 17 or 18, wherein the vaccine composition is in the solid, particulate form.

27. The use of claim 25, wherein the vaccine composition is administered as a solid dose implant.

28. The use of claim 17 or 18, wherein the vaccine composition comprises a nucleic acid molecule that encodes the endogenous molecule.

29. The use of claim 17 or 18, wherein the mammalian subject is bovine.

30. The use of claim 17 or 18, wherein the mammalian subject is porcine.

31. The so of an endogenous molecule selected from a group consisting of an endogenous hormone and an endogenous hormone receptor, in the manufacture of a vaccine composition for distribution to a mammalian subject, wherein the vaccine composition is for the administration to the ear of the mammalian subject. Gln Xis Tro Ser T? R Glv Leu Ara Pro Glv GnRH-l: ... CAG CAT TGG AGC TAC GGC CTG CGC CCT GGC. ... GTC GTA ACC TCG ATG CCG GAC GCG GGA CCG. FIG. 1A (1) (2) Gln His Tro Ser Tyr Gl v Leu Ara Pro Gly Ser Gly Ser Gln Aso Tro Ser GnRH-2: .CAG CAT TGG AGC TAC GGC CTG CGC CCT GGC AGC GGT TCT CAA GAT TGG AGC. GTC GTA ACC TCG ATG CCG GAC GCG GGA CCG TCG CCA AGA GTT CTA ACC TCG 1 5 10 15 (3) Tyr Gl? Leu Ar < ? Pro Gly Gly Ser Ser Gln His Tro Ser Tyr Gl? Read Ara TAC GGC CTG CGT CCG 'GGT GGC TCT AGC CAG CAT TGG AGC TAC GGC CTG CGC ATG CCG GAC GCA GGC CCA CCG AGA TCG GTC GTA ACC TCG ATG CCG GAC GCG 20 25 30 (4) Pro Glv Ser Gly Ser Gln ASD Trp Ser Tyr Gly Leu Ara Pro Gly] í CCT GGC AGC GGT AGC CAA GAT TGG AGC TAC GGC CTG CGT CCG GGT. ' GGA CCG TCG CCA TCG GTT CTA ACC TCG ATG CCG GAC GCA GGC CCA. 35 40 45 49 ' FIG. 1 B