CN1179181A - GnRH-leukotoxin chimeras - Google Patents

Abstract

The present invention discloses new immunological vector systems, DNA encoding them, and uses of these systems. The vector system comprises a chimeric protein comprising a leukotoxin polypeptide fused to a selected GnRH multimer consisting essentially of at least one repeating GnRH decapeptide sequence or at least one corresponding to the selected GnRH decapeptide sequence. The sequence of at least one epitope of the GnRH molecule consists of repeating units. In the present invention, the selected GnRH sequences may all be the same, or may correspond to different derivatives, analogs, variants or epitopes of GnRH, as long as the GnRH sequences can elicit an immune response. The function of the leukotoxin is to enhance the immunogenicity of the GnRH multimer fused therein.

Description

GnRH-leukotoxin chimera

The present invention generally relates to immunological vector systems. In particular, the invention relates to leukotoxin-GnRH chimeras comprising more than one copy of a GnRH polypeptide. It was shown to be more immunogenic than individual GnRH polypeptides.

In vertebrates, the synthesis and release of two gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are regulated by a polypeptide called gonadotropin-releasing hormone (GnRH) (formerly named LHRH). Thus, one way to control reproduction in animal populations is to reduce GnRH levels, for example by anti-GnRH immunization, resulting in a reduction in LH and FSH levels with concomitant disruption of the estrous cycle and spermatogenesis. See eg., Adams et al., J. Anim. Sci. (1990) 68:2793-2802.

In particular, early studies of the GnRH molecule had shown that repeated injections of synthetic GnRH peptides might raise antisera (Arimura et al., Endocrinology (1973) 93(5):1092-1103). Additionally, GnRH antibodies have been produced in many species by chemically conjugating GnRH to an appropriate carrier and administering the conjugate in an appropriate adjuvant (Carelli et al., Proc. Natl. Acad. Sci.( 1982) 79:5392-5395). The use of recombinant fusion proteins comprising GnRH or GnRH analogues in peptide vaccines to immunize various domesticated animals and farm animals or to suppress their reproductive function has been reported (Meloen et al., Vaccine (1994) 12 (8) : 741-746; Hoskinson et al., Aust. J. Biotechnol. (1 990) 4: 166-170; and International Publication No. WO92/19746, published November 12, 1992; published March 7, 1991 WO 91/02799; WO 90/11298 published October 4, 1990; WO 86/07383 published December 18, 1986).

However, due to the extremely low immunogenicity of GnRH peptides and due to the difficulty in controlling the chemical conjugation process, efforts to provide adequate immunosterilized products have failed, resulting in substantially heterogeneous GnRH conjugates with little certainty. In addition, GnRH-based peptide vaccines have a low success rate in achieving uniform effects even after repeated vaccinations in individual animals. In this regard, since GnRH is a small "self" molecule, which is usually not recognized by the immune system of the test animal, it is less immunogenic and cannot induce a significant immune response against endogenous GnRH. The GnRH construct cannot be a successful immunosterilized vaccine product.

It is generally believed that the immunogenicity of viral antigens, small proteins or endogenous materials can be enhanced by preparing immunogenic forms of these molecules containing multiple copies of selected epitopes. In this regard, it has been shown that 2 or 4 repeats of peptide 9-21 of herpes simplex virus type I glycoprotein D (Ploeg et al., J. Journal of Immunological Methods (1989) 124:211-217), the antigen of Plasmodium falciparum 2-6 repeats of the cyclosporosome tetrapeptide NPNA (Lowell et al., Science (1988) 240: 800-802), 2 or 4 copies of the major immunogenic site of VP1 of Oral Telluride virus (Broekhuijsen et al., J.gen.Virol.(1987) 68:3137-3143) and the construct (Meloen et al., Vaccine (1994) 12(8):741-746) on the basis of the tandem repeat of GnRH-like polypeptide can Effectively enhance the immunogenicity of these molecules.

Furthermore, small proteins or endogenous substances may also be combined with appropriate carriers in order to elicit a pronounced immune response in the stimulated animal. Suitable vectors generally comprise polypeptides derived from antigenic regions of proteins of the infectious agent, such as viral surface proteins, or vector peptide sequences. The role of these carriers is to non-specifically stimulate T helper cell activity and bind to molecules of the major histocompatibility complex (MHC) to direct antigen processing and presentation of the peptide on the cell surface of antigen-presenting cells.

Several vector systems have been developed for this purpose. For example, small peptide antigens are usually associated with keyhole limpet serum protein (Bittle et al., Nature (1982) 298:30-33), tetanus toxoid (Muller et al., Proc. Natl. Acad. Sci. USA (1982) ) 79:569-573), ovalbumin, and sperm whale myoglobin protein carrier conjugation to generate an immune response. These coupling reactions usually result in the incorporation of several moles of peptide antigen per mole of carrier protein. Although the presence of multiple copies of a peptide antigen generally enhances immunogenicity, the vector can elicit strong immunity independent of the peptide antigen, which may suppress immune responses to peptide vaccines in secondary immunizations (Schutze et al, J. Immunology (1985 ) 135:2319-2322).

Antigen release systems are also based on special carriers. For example, preformed particles have been used as platforms for antigen coupling and incorporation. Viruses based on proteosomes (Lowell et al., Science (1988) 240:800-802), immunostimulatory complexes (Morein et al., Nature (1984) 308:457-460), and HBsAg have been developed. Particle (Neurath et al., Mol. Immunol. (1989) 26:53-62) and rotavirus internal coat protein (Redmond et al., Mol. Immunol. (1991) 28:269-278) system.

Vector systems have also been designed using recombinantly produced chimeric proteins that self-assemble into particles. For example, the yeast retrotransposon, Ty, encodes a series of proteins that assemble into virus-like particles (Ty-VLPs; Kingsman, S.M., and A.J. Kingsman Vacc. (1988) 6:304-306). A foreign gene has been inserted into the TyA gene and expressed in yeast as a fusion protein. Fusion proteins retain the ability to self-assemble into particles of uniform size.

Other chimeric protein particles have been tested such as HBsAg (Valenzuela et al., Bio/Technol. (1985) 3:323-326; U.S. Pat. No. 4,722,840; Delpeyroux et al., Science (1986) 233:472-475) , hepatitis B virus core antigen (Clarke et al., Vaccine 88 (Ed.H.Ginsberg, et al., 1988) pp.127-131), poliovirus (Burke et al., Nature (1988) 332 :81-82), and tobacco mosaic virus (Haynes et al., Bio/Technol. (1986) 4:637-641). However, the utility of these vectors is limited by the limited size of active agents that can be inserted into structural proteins without interfering with particle assembly.

Finally, a chimeric system was designed using the Pasteurella hemolytica leukotoxin (LKT) polypeptide fused to the selected antigen. See, e.g., International Publication Nos. WO 93/08290, published April 29, 1993, and WO 92/03558, published March 5, 1992, and U.S. Patent Nos. 5,238,823 and 5,273,889. Peptide antigen chimeras comprising LKT carrier moieties provide enhanced immunogenicity of the chimeras by providing T-cell epitopes with broad-spectrum class reactivity, thereby eliciting a T-cell-dependent immune response in immunized subjects . In this regard, induction of appropriate T-cell help is critical for generating an immune response to the antigenic portion of the chimeric peptide, especially when the antigen is an endogenous molecule. However, until now, there has been no description of the use of leukotoxic polypeptide vectors in combination with multiple epitopes of GnRH peptides.

The present invention is based on the construction of a new fusion gene between the Pasteurella hemolytica leukotoxin gene, its variant and the nucleotide sequence encoding multiple GnRH polypeptides. These constructs produced chimeric proteins that exhibited surprisingly enhanced immunogenicity when compared to the immune response elicited by GnRH administration alone.

Thus, in one embodiment, the invention relates to a chimeric protein comprising a leukotoxin polypeptide fused to a multimer consisting essentially of one or more selected GnRH polypeptides, and the role of the chimeric leukotoxin portion is to enhance Immunogenicity of GnRH polypeptides. In particular, a GnRH multimer may correspond to more than one copy of a selected GnRH polypeptide or epitope, or multiple tandem repeats of a selected GnRH polypeptide or epitope. Alternatively, the GnRH multimer can be located at the carboxy- or amino-terminal or internal sites of the leukotoxin polypeptide. GnRH multimers may also correspond to molecules of the general formula GnRH-X-GnRH, wherein X is selected from the group consisting of peptide bonds, amino acid spacers, and (GnRH) _n , wherein n is greater than or equal to 1, and wherein " A "GnRH" can include any GnRH polypeptide.

Also disclosed is a vaccine composition comprising the chimeric protein and a pharmaceutically acceptable carrier, and a method of providing a selected GnRH multimer in a host, comprising administering an effective amount of the vaccine composition.

In another embodiment, the invention relates to a DNA construct encoding the chimeric protein. The DNA construct comprises a first nucleotide encoding a leukotoxin polypeptide operably linked to a second nucleotide sequence encoding more than one copy of the GnRH epitope.

In yet another embodiment, the present invention relates to an expression cassette comprising the DNA construct of (a) above and (b) control sequences directing transcription of the construct, thereby enabling transcription and translation of the construct in a host cell.

In another embodiment, the invention relates to host cells transformed with these expression cassettes.

Another embodiment of the invention provides a method of producing a recombinant polypeptide. The method comprises (a) providing a population of host cells as described above and (b) culturing the population of cells under conditions such that the polypeptide encoded by the expression cassette is expressed.

These and other embodiments of the present invention will be readily apparent to those of ordinary skill in the art from the disclosure herein. Brief description of the drawings

Figures 1A and 1B show the nucleotide and amino acid sequences of the GnRH constructs used in the chimeric leukotoxin-GnRH polypeptide fusion gene. Figure 1A depicts GnRH-1 comprising a single copy of the GnRH decapeptide; Figure 1B depicts GnRH-2 comprising four copies of the GnRH decapeptide when n=1, and 8 copies of GnRH when n=2.

Figure 2 depicts the structure of plasmid pAA352, where tac is the hybrid trp::lac promoter from Escherichia coli; bla represents the β-lactamase gene (ampicillin resistance); ori is the origin of replication of the plasmid based on ColE1; lktA is Pasteurella haemolytica leukotoxin structural gene; and lac1 is the E. coli lac operon repressor. Arrows indicate the direction of transcription/translation of the leukotoxin gene. The size of each component is not drawn to scale.

Figures 3-1 to 3-9 show the nucleotide sequence and deduced amino acid sequence of leukotoxin 352 (LKT352). Sequences of the structural gene and flanking vector regions of LKT352 are shown.

Figure 4 shows the structure of plasmid pCB113 carrying leukotoxin-GnRH (LKT-GnRH) fusion gene.

Figures 5-1 to 5-8 show the nucleotide sequence and deduced amino acid sequence of the LKT-GnRH chimeric protein derived from pCB113. The nucleotide and deduced amino acid sequences of the LKT-GnRH chimeric protein from pCB112 were identical to those of the chimeric protein derived from pCB113, except for the two insertions of the sequence of multiple copies of GnRH, as described in Figure 4 above.

Figure 6 shows the structure of plasmid pCB111 carrying leukotoxin-GnRH (LKT-GnRH) fusion gene.

Figures 7-1 to 7-5 show the nucleotide sequence and deduced amino acid sequence of the LKT-GnRH chimeric protein derived from pCB111. The nucleotide and deduced amino acid sequences of the LKT-GnRH chimeric protein from pCB114 were identical to those of the chimeric protein derived from pCB111 described above in Figure 6 except for the sequence of two insertions of multiple copies of GnRH.

Figure 8 shows the nucleotide sequence and deduced amino acid sequence of the blunt-ended fusion point of the truncated leukotoxin gene of plasmid pCB111 (Figure 8-2), which was removed from LKT352 by digestion with restriction enzymes BstB1 and Nael. DNA fragment (about 1300bp in length) (Figure 8-1).

Unless otherwise indicated, the methods of the present invention will employ conventional techniques of molecular biology, microbiology, virology, recombinant DNA techniques, and immunology, which are within the skill of the art. A full description of such techniques is found in the literature. See, eg, Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual; DNA Cloning , Vol. I and II (DNGlover ed.); Oligonucleotide Synthesis (MJ Gait ed.); Nucleic Acid Hybridization (BD Hames and SJ Higgins eds. ); Animal Cell Culture (RKFreshney ed.); Fixed Cells and Enzymes (IRL Publishing); B.Perbal, Molecular Cloning Experiment Guide ; Series, Methods in Enzyme (S.Colowick and N.Kaplan eds., Academic Press, Inc.) and Handbook of Experimental Immunology , Vols. I-IV (DM Weir and CC Blackwell eds., Blackwell Scientific Publishing). A. Definition

In describing the present invention, the following terminology will be used and defined as set forth below.

The term "gonadotropin-releasing hormone" or "GnRH" refers to a decapeptide secreted by the hypothalamus that controls the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in vertebrates (Fink, G., British Medical Bulletin ( 1979) 35:155-160). The amino acid sequence of GnRH is highly conserved among vertebrates, especially among mammals. In this regard, GnRH (formerly named LHRH) derived from most mammals including human, bovine, porcine and ovine GnRH has the amino acid sequence pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly - _NH2 (Murad et al., Hormones and Hormone Antagonists, Pharmacological Principles of Therapeutics , Sixth Edition (1980) and Seeburg et al., Nature (1984) 311:666-668).

As used herein, "GnRH polypeptide" includes molecules derived from native GnRH sequences, as well as recombinantly produced or chemically synthesized GnRH polypeptides having amino acid sequences substantially homologous to native GnRH while retaining immunogenicity, as described below. Thus, the term includes derivatives and analogs of GnRH with single or multiple amino acid additions, substitutions and/or deletions within the peptide or at the amino or carboxyl termini. Thus, in the present invention, "GnRH polypeptide" includes molecules with native sequence, such as the molecule depicted in Figure 1A (with an N-terminal Gin residue instead of pyro Glu (pyro Glu) residue), and with other amino acid appended , a substituted and/or deleted molecule that retains the ability to elicit the formation of antibodies that cross-react with naturally occurring GnRH. Of particular concern herein are repeats of oligomeric GnRH polypeptides as depicted in Figure 1B (wherein each selected GnRH polypeptide comprises an N-terminal Gln substitution, and in which each other GnRH polypeptide comprises an Asp residue substitution at position 2 ). Epitopes of GnRH are also within the scope of this definition.

The term "epitope" refers to the site on an antigen or hapten to which a specific antibody molecule binds. Since GnRH is a very small molecule, the identification of epitopes within which an antibody response can be elicited is readily accomplished using techniques known in the art. See, e.g., Geysen et al.Proc.Natl.Acad.Sci.USA (1984) 81:3998-4002 (General Method for Rapid Synthesis of Peptides to Position Immunogenic Epitopes in a Given Antigen); U.S. Pat. No. 4,708,871 ( methods for identifying and chemically synthesizing epitopes of antigens); and Geysen et al., Molecular Immunology (1986) 23:709-715 (a technique for identifying peptides with high affinity for a given antibody).

As used herein, the term "T-cell epitope" refers to a characteristic structure of a peptide capable of inducing T-cell immunity to a peptide construct or related hapten. In this regard, it has been accepted by those of ordinary skill that T-cell epitopes comprise linear peptide determinants with an extended configuration within the peptide-binding cleft of the MHC molecule (Unanue et al., Science (1987) 236:551-557) . The conversion of polypeptides into MHC class II-associated linear peptide determinants (typically 5-14 amino acids in length) defines "antigen processing" and is accomplished by antigen-presenting cells (APCs). Specifically, T-cell epitopes are determined by local features of the short peptide structure, such as primary amino acid sequence properties, including charge and hydrophobicity, and certain types of secondary structure, such as helicity, that do not depend on the folding of the entire polypeptide. . In addition, it is generally believed that short peptides that can be recognized by helper T-cells are generally amphiphilic structures, including a hydrophobic side chain (reactive with MHC molecules) and a hydrophilic side chain (reactive with T-cell receptors), (Margalit et al., Computer deduced T-cell epitopes, newly produced vaccines Marcel-Dekker, Inc, ed.G.C. Woodrow et al., (1990) pp.109-116), the other amphipathic structure has an α-helical configuration (See eg, Spouge et al., J. Immunol. (1987) 138:204-212; Berkower et al., J. Immunol. (1986) 136:2498-2503).

Therefore, protein fragments comprising T-cell epitopes can be easily predicted using a number of computer programs. (See e.g., Margalit et al., In silico prediction of T-cell epitopes, Novel Vaccines Marcel-Dekker, Inc, ed. G.C. Woodrow et al., (1990) pp. 109-116). Typically these programs compare the amino acid sequence of the peptide to sequences known to induce T-cell responses, looking for amino acid patterns believed to be required for T-cell epitopes.

An "immunogenic protein" or "immunogenic amino acid sequence" is a protein or amino acid, respectively, that elicits an immune response in a subject to which it is administered. In the present invention, "GnRH immunogen" refers to a GnRH molecule that stimulates an immune response when entering a host. In this regard, GnRH immunogens include multimers corresponding to more than one selected GnRH polypeptide sequence; in particular, multimers, multiple or tandem repeats of selected GnRH polypeptide sequences having multiple or tandem repeats A multimer of GnRH epitopes, or a conceivable multimer of their combinations.

An "immune response" to an antigen or vaccine is the generation of a cellular and/or antibody-mediated immune response in the host to the composition or vaccine used. Typically, such a response includes, but is not limited to, one or more of the following results; antibodies, B cells, helper T cells, suppressor T cells, and/or Generation of cytotoxic T cells and/or γδ T cells. The immune response can be detected using any of a number of immunoassays known in the art.

The term "leukotoxin polypeptide" or "LKT polypeptide" means a protein comprising at least one T-cell epitope and originating from a family of molecules characterized by the carboxy-terminal consensus amino acid sequence Gly-Gly-X-Gly-X-Asp (Highlander et al., DNA (1989) 8:15-28), wherein X is Lys, Asp, Val or Asn. These proteins include leukotoxins originating from Pasteurella haemolytica and Actinobacillus pleuropneumoniae, and E. coli alpha-hemolysin (Strathdee et al., Infect. Immun. (1987) 55:3233-3236; Lo, Can. J. Vet. Res. (1990) 54:S33-S35; Welch, Mol. Microbiol. (1991) 5:521-528) and others. This family of toxins is known as the "RTX" family of toxins (Lo, Can. J. Vet. Res. (1990) 54:S33-S35). Additionally, the term "leukotoxin polypeptide" refers to a leukotoxin polypeptide that is chemically synthesized, isolated from an organism expressing it, or recombinantly produced. Furthermore, the term means an immunogenic protein having an amino acid sequence substantially homologous to contiguous sequences present in a particular native leukotoxin molecule. Accordingly, the term includes full-length and partial sequences and analogs thereof. Although natural full-length leukotoxins have leukotoxin activity, the term "leukotoxin" also means molecules that retain immunogenicity but lack the cytotoxic properties of natural leukotoxins. The nucleotide sequences and corresponding amino acid sequences of several leukotoxins are known. See, e.g., U.S. Patent Nos. 4,957,739 and 5,055,400; Lo et al., Infect. Immun. (1985) 50:667-67; Lo et al., Infect. Immun. (1987) 55:1987-1996; Strathdee et al. (1987) 55:3233-3236; Highlander et al., DNA (1989) 8:15-28; Welch, Mol. Microbiol. (1991) 5:521-528. In chimeras produced according to the invention, selected leukotoxin polypeptide sequences enhance the immunogenicity of fusion GnRH multimers by providing T-cell epitopes containing small peptide fragments 5-14 amino acids in length Among other things, the small peptide fragments can be complexed with MHC class II molecules for presentation to and activation of T-helper cells. As discussed further below, these T-cell epitopes are present throughout the leukotoxin molecule and are believed to be localized in the N-terminal portion of the leukotoxin, ie between amino acid residues 1-199.

As used herein, a leukotoxin polypeptide "lacking leukotoxin activity" refers to a leukotoxin polypeptide as described above lacking in comparison to natural full-length leukotoxins (such as the full-length Pasteurella hemolytica leukotoxins described in U.S. Pat. Nos. 5,055,400 and 4,957,739). Leukotoxin polypeptides that are markedly cytotoxic but still retain immunogenicity and at least one T-cell epitope. The leukotoxic activity of leukotoxin polypeptides can be detected using known assays such as the lactate dehydrogenase release assay described by Korzeniewski et al., Journal of Immunological Methods 64: 313-320, wherein the leukotoxic activity of the leukotoxin polypeptide is detected by lactate dehydrogenation from bovine neutrophils. Hydrogenase release was used to measure cytotoxicity. A molecule was identified as leukotoxic if it caused a statistically significant release of lactate dehydrogenase compared to a control non-leukotoxic molecule.

In the present invention, constructs comprising LKT-GnRH chimeras of leukotoxin polypeptides lacking leukotoxin activity provide several important benefits. First, since injection of active toxins into individuals can lead to local cell death (PMNs and macrophages) and in turn severe inflammatory responses and abscesses at the site of injection, there is a need for leukotoxin polypeptides that lack leukotoxin activity. In this regard, leukotoxin activity that results in the killing of macrophages can result in reduced antigen presentation, thus eliciting a suboptimal immune response. As found in the non-leukotoxic LKT polypeptides used to produce the fusion proteins of the invention, removal of the cytotoxic portion produces a truncated LKT gene that is expressed at levels much higher than that of full-length LKT. In addition, the use of a non-leukotoxic LKT polypeptide in a fusion constructed according to the invention that retains sufficient T-cell antigenicity reduces the overall amount of leukotoxic-GnRH antigen that is sufficient for the host to produce sufficient response to the selected GnRH. Necessary for the B-cell response of the peptide. Specific examples of immunogenic leukotoxin polypeptides that lack leukotoxin activity include LKT352 and LKT111 described in greater detail below.

"LKT352" means the protein derived from the lktA gene in plasmid pAA352 (Figure 2, ATCC Accession No. 68283). The nucleotide sequence and corresponding amino acid sequence of this gene are described in International Publication No. WO91/15237 and are shown in FIG. 3 . This gene encodes a 931 amino acid, approximately 99 kDa truncated leukotoxin that lacks the cytotoxic portion of the molecule. The truncated genes thus produced are expressed at much higher levels than the full-length molecule (over 40% of total cellular protein compared to 1% for the full-length form) and are easier to purify. In the present invention, the derived LKT352 does not necessarily have to physically originate from sequences in plasmid pAA352. Rather, it may be produced by any means including, for example, chemical synthesis or recombinant production. In addition, the amino acid sequence of the protein need only be substantially homologous to the described sequence. Therefore, as long as the LKT polypeptide enhances the immunogenicity of the antigen it binds and also lacks leukotoxin activity, sequence variations can exist.

"LKT111" means a leukotoxic polypeptide derived from a gene in plasmid pCB111 (Figure 6, ATCC Accession No. 69748). The nucleotide sequence and corresponding amino acid sequence of this gene are shown in FIG. 7 . This gene encodes a truncated form of leukotoxin that was developed from the recombinant leukotoxin gene present in plasmid pAA352 (Figure 2, ATCC Accession No. 68283) by removing an internal DNA fragment approximately 1300 bp in length. The LKT111 polypeptide has a molecular weight of approximately 52 kDa (compared to the 99 kDa LKT 352 polypeptide), but retains the N-terminal portion from LKT352 containing the T-cell epitopes necessary for adequate T-cell immunogenicity and the N-terminal portion from LKT352 containing C-terminal portion of appropriate restriction sites for production of fusion proteins of the invention. In the present invention, the LKT111 leukotoxic peptide does not necessarily originate from the sequence present in the plasmid pCB111. Rather, it can be produced in any manner including, for example, chemical synthesis or recombinant production. In addition, the amino acid sequence of the protein need only be substantially homologous to the described sequence. Therefore, as long as the protein enhances the immunogenicity of the antigen it binds and also lacks leukotoxin activity, sequence variations can exist.

A leukotoxin-GnRH polypeptide chimera exhibits "enhanced immunogenicity" when it has a greater ability to elicit an immune response than the corresponding GnRH multimer alone. These enhanced immunogenicity can be determined by administering to animals specific leukotoxin-GnRH polypeptides and GnRH multimeric controls and comparing antibody titers against both using standard assays such as radioimmunoassays and ELISAs known in the art.

A "recombinant" protein or polypeptide refers to a polypeptide produced by recombinant DNA techniques, ie, from cells transformed with an exogenous DNA construct encoding the desired polypeptide. "Synthetic" proteins or polypeptides are those prepared by chemical synthesis.

A DNA "coding sequence" or a "nucleotide coding sequence" for a particular protein is a DNA sequence that is transcribed and translated into a polypeptide in vivo or in vitro under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (eg, mammalian) DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the coding sequence.

DNA "control sequences" are collective terms for promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory regions, enhancers, etc., which together regulate the transcription and translate.

One coding sequence is "operably linked" to another coding sequence when RNA polymerase transcribes the two coding sequences into mRNA, which is subsequently translated into the chimeric polypeptide encoded by the two coding sequences. The coding sequences need not be contiguous one after the other, so long as the transcribed sequences are eventually processed to produce the desired chimeric protein. A control sequence is "operably linked" to a coding sequence when it controls the transcription of the coding sequence.

A control sequence "directs the transcription of a coding sequence" in a cell when RNA polymerase binds the promoter sequence and transcribes the coding sequence into mRNA, which is subsequently translated into the polypeptide encoded by the coding sequence.

A "host cell" is a cell that has been or is capable of being transformed by an exogenous DNA sequence.

A cell has been "transformed" by exogenous DNA when the exogenous DNA has been introduced into the cell membrane. The exogenous DNA may or may not be integrated (covalently linked) into the chromosomal DNA making up the genome of the cell. In prokaryotes and yeast, for example, foreign DNA can exist episomally, such as plasmids. With respect to eukaryotic cells, a stably transformed cell is one in which the foreign DNA has become integrated into a chromosome so that it is heritable to daughter cells by chromosomal replication. This stability is demonstrated by the ability of eukaryotic cells to form cell lines or clones of subpopulations containing the exogenous DNA.

Two DNA or polypeptide sequences are "substantially homologous" when at least about 80%, preferably at least about 90%, most preferably at least about 95%) of the nucleotides or amino acids match over a defined length of the molecule. In Southern hybridization experiments, eg, under the severe conditions established for a particular system, substantially homologous DNA sequences can be identified. Determining appropriate hybridization conditions is known to those skilled in the art. See, eg, Sambrook et a., supra; DNA Cloning , Vol I and II, supra; Nucleic Acid Hybridization , supra.

A "heterologous" region of a DNA construct is an identifiable segment of DNA attached to or present within another DNA molecule that is not naturally associated with other molecules. Therefore, when the heterologous region encodes a bacterial gene, the gene is usually flanked by DNA from the bacterial gene that is not flanked in the genome of the source bacteria. Another example of a heterologous coding sequence is a construct of a coding sequence that itself does not occur in nature (eg, a synthetic sequence having codons that differ from the native gene). As used herein, allelic variation or naturally occurring mutational events do not produce heterologous regions of DNA.

"Vertebrate subject" means any member of the subphylum Chordate, including, but not limited to, mammals such as rodents, cattle, pigs, sheep, goats, horses and humans; domesticated animals such as dogs and cats; including Examples include chickens, roosters and hens of turkeys and other chicken-like birds, domestic, wild and game birds. The term does not denote a particular age. Therefore, both adult and newborn animals are included. B. Overall Approach

Central to the present invention is the discovery that leukotoxin polypeptides, when coupled to selected GnRH polypeptide repeat regions (or multimers), confer higher immunogenicity on the associated GnRH components. In this regard, the leukotoxin polypeptide functions as a carrier protein to deliver the selected GnRH multimer to the subject's immune system in a highly immunogenic form. Therefore, chimeric proteins constructed in accordance with the invention can be formulated into vaccine compositions that provide enhanced immunogenicity to the presence of GnRH polypeptides. Fusion of the leukotoxin gene to a selected GnRH polypeptide also facilitates purification of the chimeric protein from cells expressing it.

Thus, exemplified herein are leukotoxin chimeras comprising leukotoxin fused to more than one GnRH peptide sequence. Particularly contemplated embodiments of the invention include chimeras comprising a leukotoxin polypeptide fused to a GnRH multimer, wherein said multimer consists essentially of at least one repeating GnRH decapeptide sequence, or consists of at least one decapeptide sequence corresponding to at least one selected The sequence of the epitope of the GnRH molecule consists of repeating units. In addition, the selected GnRH peptide sequences may all be the same, or may correspond to different derivatives, analogs, mutants or epitopes of GnRH as long as they retain the ability to elicit an immune response. Figure 1A depicts the nucleotide sequence of a representative GnRH decapeptide. The GnRH sequence of the present invention is modified by replacing the pyroglutamic acid present in the native sequence with an N-terminal glutamine. This particular substitution allows the molecule to retain the native glutamic acid structure but also the uncharged structure of pyroglutamic acid. Thus, the resulting peptide does not require cyclization of glutamic acid residues and can be produced in the absence of the conditions necessary for cyclization to occur.

Since the GnRH sequence is relatively short, it can be readily produced using the synthetic techniques described in detail below. In the present invention, to help elicit an appropriate immune response to endogenous GnRH in vertebrates, leukotoxin polypeptide sequences are used to confer immunogenicity to bound GnRH polypeptides (as carrier proteins). In this way, immunization with GnRH can modulate fertility in vaccinated animals by disrupting the estrous cycle or sperm production. A detailed discussion of GnRH can be found in US Patent No. 4,975,420.

Additionally, this paper is of particular note for providing reliable and effective alternatives to invasive sterilization methods currently used in domestic and farm animal husbandry such as surgical castration, surgical hystero-oophorectomy and the like. The use of leukotoxin-GnRH chimeras in vertebrates according to the present invention provides an effective alternative to immunosuppressive regenerative activity, wherein the construct achieves uniform inactivation of regenerative activity in immunized animals. In this regard, in order to provide a successful alternative to surgical methods, suitable sterilization vaccine products must uniformly inactivate regenerative capacity when individual animals respond to small vaccinations. This property is of particular importance for the immunosterilization of herd animals, and in particular, it desirably immunocastrates male piglets to prevent "wealth contamination" produced by androgenic steroid synthesis in the male piglets' normally functioning testes . See, eg, Meloen et al., Vaccines (1994) 12(8):741-746. Due to the insufficient immunogenicity of GnRH peptides and/or related carrier systems, and the resulting inability of various existing GnRH-based vaccines to induce adequate immune responses to endogenous GnRH, past efforts to develop this product Efforts have not yielded constant results.

Therefore, in order to obtain a GnRH peptide antigen with higher immunogenicity, the leukotoxin-GnRH polypeptide chimera designed by the present invention contains a GnRH portion corresponding to more than one selected GnRH polypeptide sequence. This property is based on the recognition that endogenous proteins can generally effectively confer self-antigenicity by multimerization of their epitopes, as described above. Specifically, the GnRH portion of the novel chimera designed in accordance with the present invention may comprise multiple or tandem repeats of selected GnRH sequences, multiple or tandem repeats of selected GnRH epitopes, or any suitable combination thereof. In this regard, GnRH epitopes can be identified using the techniques detailed above, or fragments of the GnRH protein can be tested for immunogenicity and active fragments that can replace the entire polypeptide for use in compositions. Figure 1B depicts the sequence of a particular GnRH moiety of the invention, wherein four GnRH sequences, represented by (1), (2), (3) and (4), respectively, are separated by triplets of amino acid spacers, The spacer sequence contains various combinations of serine and glycine residues. In this oligomer, every other GnRH sequence (represented by (2) and (4) respectively) contains a non-conservative amino acid substitution at the second position of the GnRH decapeptide, replacing the Asp residue present in the native GnRH sequence The His residue. The resulting interphase GnRH multimeric sequence provides a highly immunogenic GnRH antigenic peptide useful in the fusion proteins of the invention. The inventors also specifically contemplate other GnRH analogs corresponding to any single or multiple amino acid additions, substitutions and/or deletions that may be used in repeat or interspaced multimeric sequences.

In addition, the particular GnRH moieties depicted in Figure IB contain spacer sequences between the GnRH moieties. In order to confer enhanced immunogenicity on this construct, the inventors specifically contemplated the use of various spacer sequences between selected GnRH polypeptides. Thus, in the present invention, selected spacer sequences may encode various types of portions that are one or more amino acids in length. The selected spacer group may preferably provide an enzymatic cleavage site so that the expressed chimera can be processed in vivo by proteolytic enzymes (APC's or the like), resulting in a plurality of peptides - each containing at least one carrier-derived moiety (leukotoxin Part of) the T-cell epitope - and preferably fused to the substantially complete GnRH polypeptide sequence. Additionally, spacers can be constructed so that the junction region between selected GnRH components contains foreign sequences that are apparent to the immunized animal, thereby conferring enhanced immunogenicity to the combined GnRH peptide. Additionally, spacer sequences may be constructed to provide T-cell antigenicity, such as sequences encoding amphipathic and/or alpha-helical peptide sequences generally recognized in the art as providing immunogenic helper T-cell epitopes. In this regard, the choice of particular T-cell epitopes provided by such spacers may vary depending on the particular vertebrate species inoculated. Although specific GnRH moieties containing spacer sequences are exemplified, the inventors also contemplate GnRH multimers comprising immediately adjacent GnRH sequences without intervening spacer sequences.

The leukotoxin-GnRH polypeptide complex can be readily produced recombinantly as a chimeric protein. The GnRH portion of the chimera can be fused to the 5' or 3' end of the leukotoxin portion of the molecule, or the GnRH portion can be located at an internal site on the leukotoxin molecule. The nucleotide sequence encoding the full-length P. haemolytica Al leukotoxin has been determined, see eg, Lo, Immunity to Infection (1987) 55: 1987-1996; US Patent No. 5,055,400. In addition, several variant leukotoxin gene sequences are disclosed herein.

Likewise, the coding sequences of porcine, bovine, and ovine GnRH have been determined, (Murad et al., Hormones and Hormone Antagonists, Pharmacological Principles of Therapeutics , Sixth Edition (1980)), and the cDNA of human GnRH has been cloned, So its sequence is well established (Seeburg et al., Nature (1984) 311:666-668). Other GnRH polypeptides of known sequence have been disclosed, such as the GnRH molecules present in salmon and chicken (International Patent Application Publication No. WO 86/07383 published December 18, 1986). In this regard, it is worth noting that GnRH is highly conserved in vertebrates, especially mammals, and that porcine, bovine, ovine and human GnRH sequences are identical. The desired leukotoxin and GnRH gene clones can be isolated and linked together using recombinant techniques known to those of ordinary skill in the art. See, eg, Sambrook et al., supra.

Alternatively, DNA sequences encoding chimeric proteins can be prepared synthetically rather than cloned. The DNA sequence can be designed with appropriate codons encoding a specific amino acid sequence. In general, one will select preferred codons for the intended host if the sequence is to be used for expression. Complete sequences were assembled from overlapping oligonucleotides prepared by standard methods and assembled into complete coding sequences. See, eg, Edge, Nature (1981) 292:756; Nambair et al. Science (1984) 223:1299; Jay et al. J. Biol. Chem. (1984) 259:6311.

Once the coding sequences for the chimeric proteins are prepared or isolated, they can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those skilled in the art and which vector to use is simply a matter of choice. Examples of recombinant DNA vectors used for cloning and their transformed host cells include lambda phage (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (Gram-negative bacteria), pGV1106 (Gram-negative coli), pKAFR1 (Gram-negative bacteria), pME290 (non-Escherichia coli Gram-negative bacteria), pHV14 (Escherichia coli and Bacillus ), YIp5 (yeast), YCp19 (yeast) and bovine papillomavirus (mammalian cells). See generally DNA Cloning : Vols. I and II, supra; T. Maniatis et al., supra; B. Perbal, supra.

The fusion gene is placed under the control of a promoter, a ribosome binding site (for bacterial expression), and an optional operator (collectively referred to herein as "control" elements) so that the DNA sequence encoding the chimeric protein is activated by the This expression construct is transcribed into RNA in vector-transformed host cells. The coding sequence may or may not contain a signal peptide or leader sequence. Chimeric proteins of the invention can be expressed using, for example, the native Pasteurella hemolytica promoter, the E. coli tac promoter or the protein A gene (spa) promoter and signal sequence. Leader sequences can be removed by the bacterial host in post-translational processing. See, eg, US Patent Nos. 4,431,739; 4,425,437; 4,338,397.

In addition to control sequences, it may also be desirable to include regulatory sequences to regulate the expression of the protein sequence relative to the growth of the host. Regulatory sequences are known to those skilled in the art, and examples include those sequences that cause the expression of a gene to be turned on or off in response to chemical or physical stimuli, including the presence or absence of a regulatory compound. Other types of regulatory elements may also be present in the vector, e.g. enhancer sequence.

Expression vectors are constructed such that the particular fused coding sequence is positioned in the vector with appropriate regulatory sequences, the coding sequence relative to the control sequences, positioned and oriented such that the coding sequence is transcribed under the "control" of the control sequences (i.e., within the control sequence The coding sequence is transcribed by RNA polymerase bound to the DNA molecule). To achieve this, it may be necessary to modify the sequence encoding the particular chimeric protein of interest. For example, in some cases it may be necessary to modify the sequence so that it can be ligated to the control sequences in the proper orientation, ie, to maintain the reading frame. Control and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector (cloning vector as described above). Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and appropriate restriction sites.

In some cases, it will be desirable to add sequences that cause secretion of the polypeptide from the host organism, followed by cleavage of the secretion signal. It may also be desirable to produce mutants or analogs of the desired chimeric protein. Mutants or analogs can be prepared by deletion of a portion of a sequence encoding a protein, insertion of a sequence, and/or substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-specific mutagenesis, are known to those skilled in the art. See, eg, T. Maniatis et al., supra; DNA Cloning , Vols. I and II, supra; Nucleic Acid Hybridization , supra.

Many prokaryotic expression vectors are known in the art.参见例如，美国专利号4,440,859；4,436,815；4,431,740；4,431,739；4,428,941；4,425,437；4,418,149；4,411,994；4,366,246；4,342,832；同时参见英围专利申请GB2,121,054；GB2,008,123；GB2,007,675；和欧洲专利申请103,395。 Yeast expression vectors are also known in the art. See, eg, US Patent Nos. 4,446,235; 4,443,539; 4,430,428; see also European Patent Applications 103,409; 100,561; 96,491.

Depending on the expression system and host chosen, the protein of the present invention is produced by growing host cells transformed with the expression vectors described above under conditions that express the desired protein. The chimeric protein is then isolated from the host cells and purified. If the expression system secretes the protein into the growth medium, the protein can be purified directly from the medium. If protein is not secreted, isolate from cell lysate. Selection of appropriate growth conditions and recovery methods is well known in the art.

Based on the determined amino acid sequence, the chimeric protein of the present invention can also be produced by chemical synthesis methods such as solid phase peptide synthesis. These methods are known to those skilled in the art. See, e.g., J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, Second Edition, Pierce Chemical Co., Rockford, IL (1984) and G. Barany and R.B. Merrifield, Peptides: Analysis, Synthesis, Biology, ed. E. Gross and J.Meienhofer, Vol.2, Academic Press, New York (1980), pp.3-254, for solid-phase peptide synthesis techniques; and M.Bodansky, Principles of peptide synthesis, Springer-Verlag, Berlin (1984) and E. Gross and J. Meienhofer, Eds., Peptides: Analysis, Synthesis, Biology, supra, Vol. 1, for typical solution synthesis.

Animal subjects can be immunized with a chimeric protein constructed according to the invention by administering a vaccine composition comprising said protein. It may be desirable to further enhance the immunogenicity of a particular chimeric protein prior to immunization. This can be accomplished by any of several methods known to those skilled in the art. For example, leukotoxin-GnRH polypeptide fusion protein can be administered in combination with a secondary carrier. For example, a fragment can be bound to a macromolecular carrier. Suitable carriers are typically large, slowly metabolized macromolecules such as: proteins; polysaccharides such as agarose, agar, cellulose, cellulose beads and the like; polymeric amino acids such as polyglutamic acid, polylysine, and the like; amino acid copolymers; and inactivated virus particles. Particularly useful protein substrates are serum albumin, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art.

These protein substrates can be used in their native form or their functional groups can be modified eg by succinylation of lysine residues or reaction with Cys-thiolactones. Sulfhydryl groups can also be incorporated into the carrier (or selected GnRH polypeptides) by, for example, reaction of functional amino groups with 2-iminothiolane or N-hydroxysuccinimide esters of 3-(4-dithiopyridyl)propionate . Suitable carriers may also be modified to incorporate spacer arms (such as hexylenediamine or other similarly sized bifunctional molecules) for binding the peptide.

Other suitable vectors for chimeric proteins of the invention include the VP6 polypeptide of rotavirus as disclosed in US Pat. No. 5,071,651, or functional fragments thereof. Also useful are fusion products of viral proteins and leukotoxin-GnRH immunogens, wherein the fusion products are made by the method disclosed in US Patent No. 4,722,840. Still other suitable vectors include cells such as lymphocytes, since the presentation in this form mimics the natural presentation found in the individual producing the immunized state. Alternatively, fusion proteins of the invention may be coupled to red blood cells, preferably the individual's own red blood cells. Methods of coupling peptides to proteins or cells are known to those skilled in the art.

The novel chimeric proteins of the invention can also be administered via vector viruses expressing them. Vector viruses that can be used in the present invention include, but are not limited to, vaccinia and other poxviruses, adenoviruses, herpesviruses. For example, a vaccinia virus recombinant expressing the novel chimeric protein can be constructed as follows. The DNA encoding the specific leukocyte GnRH chimeric protein is first inserted into an appropriate vector so that it is contiguous to the vaccinia promoter and flanked, for example, by a DNA sequence encoding thymidine kinase. Cells simultaneously infected with vaccinia were then transfected with this vector. The vaccinia promoter plus the gene encoding the chimeric protein of the invention was inserted into the viral genome by homologous recombination. The resulting TK-recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking for resistant viral plaques.

It is also possible to immunize an individual with a chimeric protein produced according to the invention, alone or in admixture with a pharmaceutically acceptable carrier or excipient. Typically, vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The formulation may also be emulsified or the active ingredient entrapped in liposome vehicles. The active immunogenic ingredient is often admixed with a carrier containing excipients which 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 carrier can, if desired, contain minimal amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the efficacy of the vaccine. Adjuvants can include, for example, muramyl dipeptides, avridine, aluminum hydroxide, oils, saponins and other substances known in the art. Particular methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, eg, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 18th ed., 1990. In any event, the composition or formulation to be administered will contain an amount of the protein suitable to achieve the desired immunization status in the individual being treated.

Other vaccine formulations suitable for other modes of administration include suppositories and, in some cases, aerosols, intranasal, oral formulations, and sustained release formulations. For suppositories, the carrier composition will include traditional combinations and carriers such as polyethylene glycols, or triacylglycerides. These suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w), preferably about 1% to about 2%. Oral carriers include those commonly used excipients, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium, stearates, sodium cellulose saccharine, magnesium carbonate, and the like. These oral vaccine compositions may be administered in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and contain from about 1% to about 30% active ingredient, preferably from about 2% to about 20%. .

Formulations for intranasal administration generally include carriers that neither cause irritation of the nasal mucosa nor significantly disrupt ciliary function. Diluents such as water, saline or other known substances can be used in the present invention. Nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol, algicide. Surfactants may appear to enhance the absorption of bulk proteins by the nasal mucosa.

By incorporating chimeric proteins into carriers such as liposomes, nonabsorbable encapsulating polymers such as ethylenevinylacetic acid copolymers and Hytrel® copolymers, imbibable polymers such as hydrogels, or absorbable polymers such as Collagen and certain polyacids or polyesters such as those used to make absorbable sutures make controlled or sustained release formulations. Chimeric proteins can also be provided using implanted pumplets known in the art.

In addition, chimeric proteins (or complexes thereof) can be formulated into vaccine compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the active polypeptide) and those formed with inorganic acids such as hydrochloric or phosphoric, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, sodium hydroxide, potassium, ammonium, calcium, or iron, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, promethazine, Caine, and whatnot.

To immunize an individual, the selected GnRH-leukotoxin chimera is administered parenterally, usually intramuscularly, in a suitable vehicle. However, other modes of administration such as subcutaneous, intravenous injection and intranasal delivery are also acceptable. Injectable vaccine formulations will contain an effective amount of the active ingredient in a carrier, the precise amount being readily ascertainable by one skilled in the art. Typical active ingredient ranges are from about 1% to about 95% (w/w) of the composition, or even higher or lower if appropriate. The amount administered will depend on the animal being treated, the ability of the animal's immune system to synthesize antibodies, and the degree of protection desired.

For such vaccine formulations, about 1 μg to 1 mg, more usually 5 μg to 200 μg of GnRH polypeptide per ml of injection solution is suitable for generating an immune response when administered. In this regard, the ratio of GnRH to leukotoxin in the leukocyte-GnRH antigen of the vaccine formulation will vary depending on the particular leukotoxin and GnRH polypeptide components chosen to construct these molecules. Specifically, in the leukotoxin-GnRH polypeptides used in the production of vaccine formulations according to the present invention, each fusion molecule has about 1-25% GnRH, preferably about 3-20% and most preferably about 7-17% GnRH polypeptide. Increasing the percentage of GnRH present in the LKT-GnRH antigen reduces the total amount of antigen that must be administered to an individual in order to elicit an effective B-cell response to GnRH. Effective dosages can be readily established by one of ordinary skill in the art by routine experimentation establishing dose response curves. An individual can be immunized by administering at least one dose, and preferably two doses, of a leukocyte-GnRH polypeptide. In addition, multiple doses can be administered to the animal to maintain the immune status.

The following are examples of specific embodiments for implementing the invention. These examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

C. Experimental materials and methods

Enzymes were purchased from commercial sources and used according to the manufacturer's instructions. Also purchase radioactive nucleotides and nitrocellulose filter paper from commercial sources.

In cloning of DNA fragments, all DNA manipulations were performed according to standard methods, except where indicated. 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 manufacturer's instructions. Double-stranded DNA fragments are separated on an agarose gel.

cDNA and genomic libraries were prepared using standard techniques in pUC13 and lambda gt11 phage, respectively. See DNA Cloning: Vol I and II, supra.

Pasteurella hemolytica biotype A, serotype 1 ("Al") strain B122 was isolated from the lungs of cattle succumbed to Pasteurella pneumoniae and stored in defibrinated blood at -70°C. Routine propagation was performed on blood agar plates or in brain heart infusion broth (Difco Laboratories, Detroit, MI) supplemented with 5% horse serum (Gibco Canada Ltd., Burlington, Canada). All cultures were incubated at 37°C.

Example 1

Isolation of the leukotoxin gene from Pasteurella hemolytica

To isolate the leukotoxin gene, a gene library of P. haemolytica Al (strain B122) was constructed using standard techniques. See, Lo et al., Infect. Immun., supra; DNA Cloning: Vol. I and II , supra; and Sambrook et al., supra. The genomic library was constructed in the plasmid vector pUC13 and the DNA library in the phage λgt11. The resulting clones were used to transform E. coli and collected and screened for reactivity with sera from cattle that had survived P. haemolytica infection and boosted with concentrated culture supernatant of P. haemolytica to increase anti-leukotoxin antibody levels single clone. Positive clones were tested 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 clones were identified and restriction endonuclease maps were made to analyze these recombinants. One clone appeared to be identical to a leukotoxin gene cloned in the past. See, Lo et al., Infect. Immun., supra. For demonstration, smaller fragments were recloned and restriction maps compared. It was confirmed that about 4 kb of DNA had been cloned. Larger clones were gradually isolated by performing chromosome walking (5'-3' direction) in order to isolate full-length recombinants approximately 8 kb in length. This final construct was named pAA114. This construct contains the entire leukotoxin gene sequence.

IktA was treated with the Klenow fragment of DNA polymerase I plus nucleotide triphosphates, a Mael restriction endonuclease fragment from pAA114 containing the entire leukotoxin gene, and ligated into the SmaI site of cloning vector pUC13. This plasmid was named pAA179. From this, two expression constructs were made in the ptac-based vector pGH432:lacI digested with SmaI. One is pAA342, containing the 5'-AhaIII fragment of the lktA gene, and the other is pAA345, containing the entire MaeI fragment described above. Clone pAA342 expressed the truncated leukotoxin peptide at high levels, whereas pAA345 expressed the full-length leukotoxin at low levels. Therefore, the 3' end of the lktA gene (from the StyI BamHI fragment of pAA345) was ligated with StyI BamHI digested pAA342, resulting in plasmid pAA352. Figure 2 shows the structure of pAA352, and Figure 3 shows the nucleotide sequence and deduced amino acid sequence of the P. haemolytica leukotoxin produced from the pAA352 construct (hereinafter referred to as LKT 352).

Example 2

Construction of LKT-GnRH fusion

A representative LKT-GnRH fusion was constructed as follows. Oligonucleotides containing sequences corresponding to a single copy of GnRH and four multiple repeats of GnRH were constructed on the Pharmacia Gene Assembler using standard phosphoramidite chemistry. Figures 1A and 1B show the sequences of these oligonucleotides. The oligonucleotides were annealed and ligated into vector pAA352 (ATCC No. 68283, as above) which had been digested with the restriction endonuclease BamH1. This vector contains the Pasteurella hemolytica leukotoxin gene. E. coli strain MH3000 was transformed with the ligated DNA. A restriction endonuclease map was made to identify transformants containing the oligonucleotide insert.

An eight-copy GnRH tandem repeat was prepared by annealing four copies of the GnRH oligonucleotide and ligating them to the vector that had been digested with the restriction endonuclease BamH1. The oligomer was designed to disable the upstream BamH1 site upon insertion and ensure that insertion of other copies of the oligomer would be oriented in the correct reading frame. Figure 1B shows the sequence of this oligonucleotide. Plasmid DNA from E. coli MH3000 strain was then isolated and used to transform strain JM105. The recombinant plasmids were named pCB113 (LKT352: 4 copies of GnRH, ATCC Accession No. 69749) and pCB112 (LKT352: 8 copies of GnRH). Figure 4 shows the recombinant plasmid pCB113, and the plasmid pCB112 is identical to pCB113 except that multiple copies of the GnRH sequence (corresponding to the oligomer of Figure 1B) were inserted twice as described above. Figure 5 shows the nucleotide sequence of the recombinant LKT-GnRH fusion of pCB113. The nucleotide sequence of the recombinant LKT-GnRH fusion of pCB112 is the same, except that multiple copies of the GnRH sequence are inserted twice.

Example 3

Construction of truncated LKT carrier peptide

A truncated form of the recombinant leukotoxin peptide was constructed from the recombinant gene present in plasmid pAA352 (described above). The truncated LKT gene was generated by excising an internal DNA fragment approximately 1300 bp in length from the recombinant LKT gene as described below.

Plasmid pCB113 (ATCC Accession No. 69749), which contains the LKT352 polypeptide fused to four copies of the GnRH polypeptide, was digested with the restriction enzyme BstB1 (New England Biolabs). The resulting linearized plasmid was then digested with mungbean nuclease (Pharmacia) to remove single-stranded overhangs resulting from BstB1 digestion. The blunted DNA was then digested with the restriction enzyme Nae1 (New England Biolabs). And the digested DNA was added to 1% agarose gel, and the DNA fragments were separated by electrophoresis. A large DNA fragment of about 6190 bp was isolated and purified from an agarose gel using Gene Clean Kit (Bio 101), and the purified fragment was self-ligated using bacteriophage _T4 DNA ligase (Pharmacia). Competent E. coli JM105 cells were transformed with the resulting ligation mixture, and positive clones were identified based on their ability to produce aggregated proteins with a molecular weight of approximately 57 KDa. The resulting recombinant plasmid, designated pCB111 (ATCC Accession No. 69748), produces a truncated leukotoxic polypeptide (hereinafter LKT111) fused to four copies of the GnRH polypeptide. Figure 6 shows the structure of pCB111. Plasmid pCB114 is identical to pCB111 except that multiple copies of the GnRH sequence (corresponding to the oligomer of Figure IB) are inserted twice. Figure 7 shows the nucleotide sequence of the recombinant LKT-GnRH fusion of pCB111, the nucleotide sequence of the recombinant LKT-GnRH fusion of pCB114 is the same, the difference is that the multiple copies of the GnRH sequence are inserted twice.

The nucleotide sequence of the ligated fusion point of this clone has been confirmed by sequencing with a phage T7 polymerase sequencing kit (Pharmacia). Figure 8 shows the nucleotide sequences of these fusion points.

Example 4

Purification of LKT-antigen fusion

The recombinant LKT-GnRH fusions of Examples 2 and 3 were purified by the following method. For each fusion, 5-10 colonies of the transformed E. coli strain were inoculated in 10 ml of TB broth supplemented with 100 mg/ml of ampicillin and incubated for 6 hours at 37C on a G10 shaker, 220 rpm. 4 ml of this culture was diluted into two protected (batfled) Fernbach flasks containing 400 ml TB broth + ampicillin and incubated overnight as described above. Cells were harvested by centrifugation at 4000 rpm for 10 min in a 500 ml volume polypropylene bottle using a Sorvall GS3 centrifuge. The pellet was resuspended in an equal volume of TB broth (ie, 2 x 400 ml) containing ampicillin that had been pre-warmed to 37°C and the cells were incubated for 2 hours as described above.

To induce the synthesis of recombinant fusion proteins, 3.2 ml of isopropyl-B,D-mercaptogalactopyranoside (IPTG, Gibco/BRL), 500 mM dissolved in water, was added to each culture (final concentration = 4 mM) . The culture was incubated for 2 hours. Cells were harvested by centrifugation as described above, resuspended in 30 ml 50 mM Tris-HCl, 25% (w/v) sucrose, pH 8.0, and frozen at -70C. After standing at -70°C for 60 minutes, the frozen cells were thawed at room temperature and 5 ml of lysozyme (Sigma, 20 mg/ml in 250 mM Tris-HCl, pH 8.0) was added. The mixture was vortexed at high speed for 10 seconds, then placed on ice for 15 minutes. The cells were then added to 500ml of lysis buffer in a 1000ml beaker and stirred with a 2ml pipette to mix. The beaker containing the lysed cell suspension was placed on ice and sonicated with a Braun sonicator, large probe set at 100 watts, for a total of 2.5 minutes (5-30 seconds treatment with 1 minute break). An equal volume of the solution was placed in a Teflon SS34 centrifuge tube and centrifuged at 10,000 rpm for 20 minutes in a Sorvall SS34 rotor. Resuspend the pellet by high speed vortexing in a total of 100 ml sterile double distilled water and repeat the centrifugation step. The supernatant was discarded, the pellet was combined into 20 ml of 10 mM Tris-HCl, 150 mM NaCl, pH 8.0 (Tris buffered saline), and the suspension was frozen overnight at -20°C.

The reconstituted suspension was thawed at room temperature and added to 100 ml of 8M guanidine hydrochloride (Sigma) in Tris-buffered saline and mixed vigorously. Place a magnetic stir bar in the bottle and mix the dissolved sample for 30 minutes at room temperature. Transfer the solution to a 2000ml Erlenmyer flask and quickly add 1200ml Tris-buffer. The mixture was stirred for another 2 hours at room temperature. Aliquots of 500ml were placed into dialysis bags (Spectrum, 63.7mm diameter, 6000-8000MW cutoff, #132670 from Fisher Scientific), these were placed in 3500ml Tris-buffered saline +0 .SM guanidine hydrochloride in a 4000ml beaker. The beaker was placed in a 4°C room overnight on a magnetic stirrer, after which the dialysis buffer was replaced with Tris-buffered saline + 0.1M guanidine hydrochloride and dialysis was continued for 12 hours. The buffer was then replaced with Tris-buffer + 0.05M guanidine hydrochloride and dialysis was continued overnight. The buffer was replaced with Tris-buffer (without guanidine) and dialysis was continued for 12 hours. Repeat this practice 3 more times. Pour the final solution into a 2000ml plastic roller bottle (Coming) and add 13ml 100mM PMSF (in ethanol) to inhibit protease activity. The solution was stored at -20°C in 100 ml aliquots.

To demonstrate that the fusion protein has been isolated, an aliquot of each preparation was diluted 20-fold in double distilled water, mixed with an equal volume of SDS-PAGE sample buffer, placed in a boiling water bath for 5 minutes and incubated in 12% polyacrylamide Electrophoresis on the gel. A recombinant leukotoxin control was also run at the same time.

All fusion proteins were expressed at high levels in the form of inclusion bodies. The molecular weight deduced according to the DNA sequence of the fusion protein is 104869 (LKT 352::4 copies of GnRH from pCB113); 110392 (LKT352::8 copies of GnRH from pCB112); 57542 (LKT111::4 copies of GnRH from pCB111 ); 63241 (LKT111::8 copies of GnRH from pCB114). The predicted molecular weight of the recombinant LKT352 molecule was 99338 and the predicted molecular weight of the recombinant LKT111 molecule was 51843.

Example 5

In vivo immune activity of LKT-GnRH fusion

To test the ability of the LKT-GnRH fusion in vivo to induce an immune response against GnRH in vivo, and to compare its response to other GnRH carrier conjugates, the following immunization assay was performed. Three groups of eight male pigs approximately 8 weeks of age (35-50 kg), which were specific pathogen free, were utilized. The animals were bred in the least disease-prone facility and vaccinated on days 0 and 21 of the experiment with the following formulation:

Group 1 - placebo (2 ml) consisting of a salt formulated with Emulsigen Plus adjuvant containing 15 mg DAA;

Group 2 - LKT352-GnRH formulated with the same adjuvant (250 μg LKT, prepared as described in the previous example) (2 ml);

Group 3 - VP6-GnRH, 0.5 μg VP6 and 5 μg GnRH, formulated with the same adjuvant (2 ml). VP6 formulations were prepared using the binding peptides described in US Patent No. 5,071,651.

Blood samples were taken on days 0, 21 and 35, clotted, centrifuged at 1500 g, and serum removed. Anti-GnRH serum antibody titers were determined by the RIA method of Silversides et al., J. Reprod. Immunol. (1985) 7: 171-184.

The results of this test showed that only those animals immunized with the LKT 352-GnRH formulation developed significant anti-GnRH titers (titers > 1:70). Neither the placebo nor the VP6-GnRH groups produced anti-GnRH titers. In the past, repeated vaccinations with GnRH doses in excess of 100 μg in combination with other carrier proteins were required to induce antihormonal titers. These results indicate that the LKT-GnRH vector system provides immunogens that are substantially more advanced than past vector systems.

Example 6

In vivo immune effects of multiple tandem GnRH repeats linked to LKT

To test the ability of recombinant LKT-GnRH fusion proteins containing multiple GnRH polypeptide repeats to induce an anti-GnRH immune response in vivo, the following vaccination assay was performed. Cultures of E. coli containing plasmids pCB113 and pCB175 (with 4 and 8 copies of GnRH linked to LKT352, respectively), and plasmids with 1 copy of GnRH linked to LKT352 were prepared as described above. Vaccines of each of the above cultures were formulated to contain 5 µg of GnRH equivalent in 0.2 ml of Emulsigen Plus. Three groups of 10 female mice were given two subcutaneous injections 23 days apart and blood samples were collected on days 23, 35 and 44 after the initial injection. Serum antibody titers against GnRH were determined by standard radioimmunoassay at final dilutions of 1:100 and 1:1000. Antibodies were considered undetectable if less than 5% iodinated GnRH was bound. The antibody titers thus obtained are summarized in Table 1.

The results of this study showed that equal doses of GnRH present in multiple tandem repeats (4 or 8 copies of GnRH) produced significantly higher antibodies than a single copy of GnRH (as measured by binding to iodinated native GnRH). Further, the above results indicate that fusion proteins containing 4 copies of GnRH tandem repeats linked to LKT352 represent the best form of immunogenic GnRH antigen, although immunogenicity may be affected by dose or species of individual tested. sampling time group 1 group 2 group 3 LKT 352::1 copy GnRH LKT 352::4 copy GnRH LKT 352::8 copy GnRH Number of individuals who responded Average Response (%) ^* Number of individuals who responded Average Response (%) ^* Number of individuals who responded Average Response (%) ^* 1:100 1:1000 1:100 1:1000 1:100 1:1000 1:100 1:1000 1:100 1:1000 1:100 1:1000 twenty three 0 0 - - 3 1 16 9 2 0 33 - 35 2 2 45 20 9 9 75 30 7 5 48 41 44 2 2 60 39 10 10 55 43 8 7 57 46 ^* Average response is the average binding of those animals that bound ^I125 -GnRH at more than 5%.

Table 1

Example 7

In vivo immune activity and biological effects of LKT352::GnRH and LKT111::GnRH fusions

To test the ability of fusion proteins containing multiple tandem repeats of GnRH (linked to LKT352 or LKT111 ) to elicit an anti-GnRH immune response in vivo and to demonstrate biological effects in vivo, the following vaccination experiments were performed. Cultures of E. coli containing plasmids pCB113 and pCB111 (4 copies of GnRH linked to LKT352 or LKT111, respectively) were prepared as described above. Vaccines were formulated for each of the above cultures; 0.2 ml of VSA-3 adjuvant (modified Emulsigen Plus adjuvant) contained 5 μg of GnRH equivalent, and a control immunization vaccine containing 0.2 ml of adjuvant was also prepared. Three groups of 5 male Swiss mice were injected subcutaneously twice at 21 day intervals, the first injection was given at 5-6 weeks of age (day 0). The test animals were sacrificed on day 49.

The immunological activity of the GnRH-LKT fusion was determined by measuring anti-GnRH antibody titers at a 1:1000 serum dilution using standard radioimmunoassay methods. The biological effects of the GnRH-LKT fusion were quantified by radioimmunoassay of serum testosterone levels with a sensitivity of 25 pg/ml, and testicular tissue was weighed and histologically examined. The results of this test are summarized in Table 2.

In the assay, all individual animals injected with the GnRH:LKT antigen had readily detectable antibody levels; however, the LKT111::GnRH fusion (from plasmid pCB111) exhibited outstanding immunogenicity, as demonstrated by the uniformity of responses and titers . Serum testosterone (produced by testicular Leydig cells) is secreted in a fluctuating manner, therefore, low values and exceptionally variable serum levels can be expected in normal individual animals. In the test, the control group (received 0.2 ml adjuvanted vaccine injection) had normal serum testosterone levels, while the two groups of treated individuals had essentially undetectable serum testosterone.

In addition, in experiments, histological evaluation of testicular tissue showed varying degrees of decline of Leydig cells, reduced diameter of spermatogenic tubules and disruption of spermatogenesis in treated individuals; however, testicular weights in treated animals remained near normal - even in the presence of high anti-GnRH antibody titers - although 2 out of 5 individuals who received the LKT111::4 copy GnRH fusion had clear signs of testicular regression.

Thus, these results show that multiple copies of GnRH linked to LKT352 or LKT111 constitute a potent immunogen; additionally demonstrate that vaccination with fusion proteins of the invention can elicit the production of antibodies capable of neutralizing endogenous GnRH in vivo, and Individual animals receiving such vaccinations may be accompanied by identifiable in vivo biological effects. animal group 1 group 2 group 3 control 5 μg LKT 352::4 copy GnRH 5 μg LKT 111::4 copy GnRH Antibody Titer ^* Testicular weight (mg) Serum testosterone Antibody Titer ^* Testicular weight (mg) Serum testosterone Antibody Titer ^* Testicular weight (mg) Serum testosterone 1 7 0 252 .04 73.0 282 .13 75.0 163 .00 2 4.0 327 .18 14.0 334 .10 59.0 296 .07 3 0.0 276 2.73 18.0 254 .03 54.0 260 .twenty four 4 0.0 220 .36 55.0 222 .05 66.0 265 .03 5 1.0 232 1.44 61.0 226 .19 64.0 50 .00 average 2.4 261 .95 44 263 .10 64 206 .07 standard error 1.4 19 .51 12 twenty one .03 4 45 .04 ^* % binding of I ¹²⁵ -GnRH at 1:1000 serum dilution

Table 2

Example 8

  In vivo immune activity of LKT::GnRH fusion in pig individuals

To test the ability of fusion proteins containing multiple tandem repeats of GnRH (linked to LKT352 or LKT111 ) to elicit an in vivo anti-GnRH immune response in pig individuals, the following vaccination experiments were performed. Containing plasmids pCB113, pCB111, pCB175, and pCB114 (LKT352::4 copies of GnRH, LKT111::4 copies of GnRH, LKT352::8 copies of GnRH, and LKT111::8 copies of GnRH, respectively) were prepared as described above. Escherichia coli culture. Vaccines from each of the above cultures were formulated to contain 50 µg of GnRH equivalents and administered in a 2.0 ml volume in VSA-3 adjuvant. Four groups of 5 male and 5 female 35 day old (at day 0) weaned piglets were injected on day 0 of the experiment and on day 21. Blood samples were collected on days 0, 21 and 35 and titers of anti-GnRH antibodies at a final dilution of 1:1000 were determined by standard radioimmunoassay. The measurement results are summarized in Table 3.

Anti-GnRH antibodies were not detectable in any individual in the trial prior to immunization and were readily detectable in most individuals on day 35 (one individual in treatment group 4 died from an infection unrelated to treatment). The results of this experiment demonstrate that fusion proteins containing multiple GnRH repeats linked to LKT352 or LKT111 carrier polypeptides form useful immunogens in pig individuals. According to the estimated molecular weight of the decapeptide GnRH (1,200), LKT111 polypeptide (52,000) and LKT352 polypeptide (100,000), the percentage of GnRH in the LKT-GnRH antigen fusion is as follows: 4.9% (LKT352::4 copies of GnRH); 8.5% (LKT111::4 copies of GnRH); 9.3% (LKT352::8 copies of GnRH); and 15.7% (LKT111::8 copies of GnRH). Therefore, the actual results obtained in this way show that with the LKT-GnRH fusion containing the LKT111 polypeptide carrier, the total amount of the antigen (LKT-GnRH) administered to the individual can be halved (compared with the vaccine composition with the LKT352 carrier polypeptide system) i.e. The same anti-GnRH response was obtained. Number of animals group 1 group 2 group 3 group 4 LKT 352::4 copy GnRH 50μg LKT 111::4 copy GnRH 50μg LKT 352::8 copy GnRH 50μg LKT 111::8 copy GnRH 50μg Day 35 1:1000 dilution Day 35 1:1000 dilution Day 35 1:1000 dilution Day 35 1:1000 dilution 1 ♂47.7 ♀46.0 ♂68.3 ♂51.0 2 ♀50.3 ♂71.6 ♂65.1 ♂31.7 3 ♀66.0 ♀21.4 ♀50.7 ♀35.7 4 ♀70.2 ♂46.2 ♂4.7 ♀65.9 5 ♂17.3 ♀48.9 ♀38.3 ♀ 6 ♂18.3 ♂69.4 ♀17.4 ♂11.3 7 ♀14.7 ♂47.9 ♀51.4 ♀28.3 8 ♂37.0 ♀44.4 ♂18.0 ♂43.0 9 ♂26.0 ♂70.8 ♂83.5 ♀78.7 10 ♀2.7 ♀37.8 ♀24.2 ♂55.9 average 35.0 50.4 42.2 44.6 standard deviation 7.3 5.1 8.1 6.9 percentage of respondents 9/10 10/10 9/10 9/9

table 3

Example 9

Prediction of T-cell epitopes in recombinant LKT352 and LKT111 molecules

In order to predict potential T-cell epitopes in the leukotoxin polypeptide sequence used in the LKT-GnRH chimera of the present invention, the method suggested by Margalit and coworkers was carried out on the amino acid sequence corresponding to LKT molecule 1-199 as described in Table 4 (Margalit et al., J. Immunol (1987) 138:2213). In this method, the amino acid sequence of the leukotoxin polypeptide sequence is compared to other sequences known to induce T-cell responses and to the amino acid profile believed to be required for T-cell epitopes. Table 4 describes the comparison results.

As predicted, several short sequences of leukotoxic polypeptides were identified as potential T-cell epitopes following the principles suggested by Margalit et al (supra). Specifically, 9 sequences were identified as having (charged/Gly-hydrophobic-hydrophobic-polar/Gly) sequences (as indicated by profile "1" in Table 4), and 3 sequences were identified as having (charged/ Gly-hydrophobic-hydrophobic-hydrophobic/Pro-polar/Gly) sequence (as indicated by profile "2" of Table 4). Combining these data with the in vivo anti-GnRH activity elicited by the LKT352 and LKT111 vector systems of Examples 7 and 8 above, it can be seen that key T-cell epitopes are retained in the truncated LKT111 molecule, and that those epitopes may be contained in N-terminal part of LKT352 and LKT111 molecules.

Table 4

LKT sequence map corresponding to potential T-cell epitopes showing LKT amino acid sequence of profile "1"

GTID (aa's 27-30)

GITG (aa's 66-69)

GVIS (aa's 69-72)

HVAN (aa's 85-88)

KIVE (aa′s 93-96)

DLAG (aa's 152-155)

KVLS (aa's 162-165)

DAFE (aa's 171-174)

KLVQ (aa′s 183-186)

GIID (aa′s 192-195) shows the LKT amino acid sequence of profile "2"

RYLAN (aa's 114-118)

KFLLN (aa's 124-128)

KAYVD (aa′s 167-171) D. Industrial application

When administered to a vertebrate host, the leukotoxin-GnRH chimera of the invention can be used as an immunogen to immunize against endogenous GnRH, thereby inhibiting the reproductive function or reproductive capacity of the host.

Notwithstanding the specific utility exemplified by this specification, the novel chimeric molecules disclosed herein suggest a means of providing fusion proteins comprising more than one GnRH peptide sequence present in multiple or tandem repeats with the leukotoxin polypeptide portion of the molecule ( In some cases, the immunogenic epitope fusion is provided by a spacer peptide sequence present between selected GnRH sequences. The chimeric protein constructed by the present invention provides enhanced immunogenicity to the fusion GnRH peptide sequence, so that the vaccinated vertebrate host has enhanced an effective immune response to endogenous GnRH; two gonadotropins, luteinizing hormone (LH ) and follicle-stimulating hormone (FSH) synthesis and release are terminated, thereby rendering the host temporarily sterile. In this way, the new leukotoxin-GnRH construct could be used in immunosterilization vaccines as an alternative to the invasive sterilization methods currently used in domesticated and farm animal husbandry.

Other uses of the fusion molecules of the invention include population control, eg, disrupting the reproductive capacity of wild rodent populations. In this regard, LKT-GnRH fusion molecules can be used to replace currently used population control measures such as poisoning and the like. The fusion products of the invention may also be administered as constructs with slow and fast release components. In this way, the need for multiple vaccinations can be avoided. In addition, since the amino acid sequence of GnRH is highly conserved among species, it is possible to produce a leukotoxin-GnRH fusion vaccine product with a wide range of cross-species effects.

Thus, disclosed herein are various chimeric proteins comprising a leukotoxin fused to a selected GnRH polypeptide. While the preferred embodiment of the invention has been described in detail, obvious changes can be made without departing from the scope of the appended claims. Deposits of strains useful in the practice of the invention

Biologically pure cultures of the following strains have been deposited at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maeyland. The indicated registration number is registered after a successful viability test and payment of the application fee. This deposit is made under the provisions of the internationally recognized Budapest Treaty on the Deposit of Microorganisms for the Purposes of Patent Procedure and its Regulations. This guarantees the maintenance of viable cultures for 30 years from the date of deposit and requires the depositary to maintain for at least 5 years after depositing samples. These microorganisms are available from the ATCC under the terms of the Budapest Treaty, which guarantees permanent and unrestricted Obtain these cultures.

These deposits are made solely for the convenience of those skilled in the art and are not an acknowledgment of a deposit made to satisfy the requirements of 35 USC §112. The nucleic acid sequences of these plasmids, as well as the amino acid sequences of the polypeptides they encode, are incorporated herein by reference and, to the extent there is a discrepancy with the description herein, they are controlled. A license is required to make, use, or sell deposited material, but no such license is granted.

The strain storage date ATCC NO. Hemorrhizal Basilica serum type 1B122 February 1, 1989 53863 PAA352, Paa 352, March 30, 1990 in PCB113, February 1, 1995, 68283, 69749. pCB111 in Escherichia coli JM105 1 February 1995 69748

Claims (14)

1. chimeric protein that contains the leukotoxin polypeptide that merges with many bodies, these many bodies are made up of more than one selected GnRH polypeptide basically, and wherein the effect of the leukotoxin of said chimeric protein part is the immunogenicity that strengthens the many bodies of said GnRH.

2. according to the chimeric protein of claim 1, wherein said leukotoxin polypeptide has lacked the leukotoxin activity.

3. according to the chimeric protein of claim 2, wherein said leukotoxin is LKT352.

4. according to the chimeric protein of claim 2, wherein said leukotoxin is LKT111.

5. according to the chimeric protein of claim 1, the many bodies of wherein said GnRH contain the molecule that general formula is GnRH-X-GnRH, and wherein X is selected from by peptide bond, amino acid spacer groups, leukotoxin polypeptide and (GnRH) _nThe group of forming, wherein n is more than or equal to 1, and wherein GnRH comprises any GnRH polypeptide in addition.

6. according to the chimeric protein of claim 5, wherein X comprises the amino acid spacer groups that contains at least one T-cell helper epitope.

7. according to the chimeric protein of claim 1, wherein said chimeric protein comprises the aminoacid sequence that Fig. 5 describes, or homology basic with it aminoacid sequence suitable with function.

8. according to the chimeric protein of claim 1, wherein said chimeric protein comprises the aminoacid sequence aminoacid sequence suitable with function with homology basic with it that Fig. 7 describes.

9. contain the chimeric protein of any one and the vaccine composition of pharmaceutical acceptable carrier among the with good grounds claim 1-8.

10. one kind provides the method for selected many bodies of GnRH to animal subject, comprises the vaccine composition according to claim 9 of using significant quantity to said animal.

11. the DNA construct of any one chimeric protein among the coding claim 1-8.

12. an expression cassette comprises: (a) according to the DNA construct of claim 11; (b) control sequence of transcribing of the said construct of guidance, wherein said construct can transcribe in host cell and translate.

13. the host cell that transforms with the expression cassette of claim 12.

14. produce the method for recombinant polypeptide, comprising: the host cell group that claim 13 (a) is provided; (b) under the condition that said expression cassette encoded polypeptides is expressed, cultivate said cell mass.

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