Modified gnrh peptide and its conjugate for mammalian immunocastration, manufacturing method, composition and kit

Abstract

The present invention falls within the fields of molecular biology, medical sciences and immunology, and especially relates to preparations for medical purposes. In particular, the present invention relates to a modified peptide of the gonadotropin-releasing hormone (GnRH) and its conjugate for immunocastration of mammals, especially pigs, to the method of manufacturing the modified GnRH antigen, to a composition and to a kit comprising said vaccine composition, for the purposes of improving mammal carcass quality, animal welfare and food utilisation (feed conversion), producing less waste and using less water, thus contributing to effectively sustainable production (ESG).

Description

Modified GnRH Peptide and its Conjugate for Mammalian Immunostaining, Manufacturing Method COMPOSITION AND KIT FIELD OF INVENTION

[0001] The present invention is within the fields of Molecular Biology, Medical Sciences and Immunology, and in particular relates to preparations for medical purposes. In particular, the present invention relates to a modified gonadotropin-releasing hormone (GnRH) peptide and its conjugate for immunocastration of mammals, especially swine, to a method for manufacturing the modified GnRH antigen and conjugate, to a composition and to a kit comprising said vaccine composition. BACKGROUND OF THE INVENTION

[0002] GnRH is secreted by the pituitary gland and acts as the primary reproductive hormone by regulating the release of two major gonadotropic hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In turn, LH and FSH induce the development of the gonads and the release of sex steroids.

[0003] GnRH vaccines stimulate the production of antibodies to inactivate endogenous GnRH. The anti-GnRH immune response inhibits the release of LH and FSH, which are necessary for the development and maintenance of the gonads—testes in males and ovaries in females. Thus, reduced LH and FSH levels lead to loss of reproductive function.

[0004] The development of immunocastration techniques based on the generation of the immune response to GnRH is challenging due to the fact that GnRH is a short peptide ( 10 amino acids long) and is naturally found circulating in the body. Therefore, this peptide is recognized by the immune system as a "self-protein." To overcome these problems, GnRH has been conjugated to various antigenic molecules, such as transport proteins, also called carrier proteins.

[0005] Available vaccines with such constructs / molecules have resulted in different levels of success in different species, but low immunogenicity remains a technical difficulty to be overcome.

[0006] Among the advantages provided by swine immunocastration is the reduction of boar taint in male pigs. The odor is related to sexual maturity and hormone production in male pigs, making meat from non-castrated animals unfit for consumption (BABOL et al., 1998). Thus, the consumption of pork from non-castrated animals is conditioned by its processing. More specifically, it is known that boar taint occurs due to the accumulation or association of the following compounds: androstenone (5a-androst-16-en-3-one) and skatole (3-methylindole). Thus, in current swine production, a large proportion of animals destined for slaughter are surgically or immunologically castrated males ((HENNESSY et al., 1997)).

[0007] Surgical castration (gonadectomy) of young male pigs is a common procedure in swine production worldwide (THUN et al., 2006). Furthermore, the slaughter of uncastrated pigs was prohibited in Brazil by article 104 of the Regulation for Sanitary and Industrial Inspection of Products of Animal Origin (RIISPOA). In Brazil, the latest current legislation on surgical castration in pigs is Normative Instruction No. 113, of I oJune 2018, from the Ministry of Agriculture, Livestock, and Supply (MAPA). This normative instruction establishes the conditions for surgical castration of pigs, with the aim of promoting animal welfare and ensuring the quality of animal products. However, surgically castrated animals negatively impact zootechnical performance, carcass quality, and animal welfare. These factors were decisive in the search for a more sustainable alternative for pig castration and motivated the development of the present invention.

[0008] The principle of immunocastration is based on the application of vaccines containing a modified form of GnRH conjugated to a protein that induces the formation of antibodies directed against GnRH (ZAMARATSKAIA et al., 2008). The use of the pig's own immune system to suppress GnRH interrupts the hypothalamic-pituitary-gonadal axis by establishing an immunological barrier that interrupts the passage of GnRH from the site of release in the hypothalamus to the site of action in the pituitary gland. Suppression of GnRH prevents it from stimulating the secretion of LH and FSH by the pituitary gland, consequently reducing testicular development and the synthesis of steroid hormones (THUN et al., 2006), including androstenone ZAMARATSKAIA et al. , 2008 ) , the main hormone responsible for carcass odor .Immunocastrated animals show improvements in carcass quality, food utilization (feed conversion), produce less waste and use less water, contributing to a. effectively sustainable production (ESG).

[0009] The main advantages of immunocastration are: (1) Improvement in meat quality: immunocastration can improve meat quality, making it more tender and juicy, as immunized pigs have less intramuscular fat and a higher content of unsaturated fatty acids; (2) Cost reduction: immunocastration eliminates the need to surgically castrate pigs, which reduces handling and transportation costs, in addition to avoiding behavioral problems associated with castration; (3) Lower environmental impact: immunocastration can reduce the environmental impact of swine production, as it avoids the production of waste associated with surgical castration, such as the disposal of testicular tissue and the use of anesthetics; (4) Greater feed efficiency: immunized pigs make better use of the nutrients in their feed, which can result in better productive performance and greater profitability for producers (According to studies by Santos et al. (2012), immunocastrated animals have a daily weight gain approximately 8.3% higher than castrated animals); and (5) Improved animal welfare: Immunocastration can contribute to improved animal welfare by avoiding the pain and stress associated with surgical castration. Unregistered pigs have high levels of testosterone, androstenone, and skatole, more aggressive behavior, and less tender meat.

[0010] In summary, unregistered pigs have high levels of testosterone, androstenone and skatole, and have more aggressive behavior and less tender meat. Surgically castrated pigs, that is, pigs that have had their testicles removed, are less aggressive than non-castrated pigs, but are more weakened. In turn, immunocastrated pigs are less aggressive (CRONIN et al., 2003) and have better feed conversion, average daily weight gain (ADG), final carcass weight, and consequently higher meat yield than surgically castrated pigs (Pauly et al. (2009).

[0011] In this scenario, the main objectives of the present invention were the development of a modified GnRH peptide and its conjugate for the preparation of compositions with high immunogenicity for the immunocastration of mammals, especially swine, the development of a method for manufacturing a conjugated modified GnRH antigen, the development of a vaccine composition comprising said conjugate and a kit comprising said vaccine composition and its instructions for use.

[0012] Particularly, the modified GnRH peptide of the present invention is linked / conjugated to the carrier by a covalent bond and can be represented by one of the following structures: Formula 1 CHLOROACETYL-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2ou CHLOROACETYL-HWSYGLRPG-NH2 STATE OF THE TECHNIQUE

[0013] Unless otherwise specified, terms used throughout this specification have their usual meanings in the art within the context of the disclosure and the specific context in which each term is used. Certain terms used to describe the disclosure are discussed below, or elsewhere in the descriptive report, to provide additional guidance to the practitioner regarding the description of the disclosure.

[0014] It should be appreciated that the same thing can be said in different ways. Consequently, alternative language and synonyms may be used for any of the terms discussed here. No special significance should be attached to whether a term is elaborated or discussed here. Synonyms for certain terms are provided, but the example of some synonyms does not exclude the potential use of others not listed here.

[0015] Considering the above, the expressions "modified GnRH" and "modified GnRH peptide" in the context of the present application refer to a 9-amino acid polypeptide sequence of the hormone GnRH and to the modifications described herein for its ends, and also refer to the active ingredient of the claimed vaccine composition. Thus, "modified GnRH" and "modified GnRH peptide" are expressions that can be understood as referring to the vaccine antigen that is conjugated to a carrier protein, capable of being recognized and triggering a response from the immune system.

[0016] Some studies in the literature propose vaccines for immunocastration in pigs. One such vaccine, commercially known as Vivax®, developed by Zoetis, inhibits GnRF activity and testosterone production, thereby reducing both the production and accumulation of undesirable substances. The request for patent corresponding to the aforementioned vaccine composition is BR 1020190027932, which comprises the following objects: a vaccine, a method of preparing said vaccine and use thereof. Although it is described that the vaccine composition comprises a) an antigen comprising GnRH, b) an adjuvant comprising an immunostimulatory oligonucleotide containing CpG, a sterol, a phospholipid and an oil, wherein said vaccine is an oily formulation or an A / O emulsion, no references are made to the type of GnRH used. However, in the descriptive report of said application, it is provided that GnRH encompasses naturally occurring mammalian GnRH (SEQ ID NO: 11 (pyro-EHWSYGLRPG-NH2)) and includes GnRH analogues that are capable of inhibiting the release of gonadotropins by the pituitary gland and, thus, inhibiting the release of steroid hormones, estradiol, progesterone and testosterone.

[0017] Furthermore, in BR 1020190027932, additional examples of GnRH analogs are presented which include, without limitation, leuprolide (pyrGlu-His-Trp-Ser-Tyr-DLeu-Leu-Arg-Pro-NHEt; SEQ ID NO: 12), buserelin (pyrGlu-His-TrpSer-Tyr-D-Ser(Tbu)-Leu-Arg-Pro-NHEt; SEQ ID NO: 13), deslorelin (pyrGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-NHEt; SEQ ID NO: 14) and fertirelin (pyrGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-NHEt; SEQ ID NO: 15). Furthermore, the document reveals the use of immunostimulant carriers, namely: diphtheria toxin, ricin toxin, pseudomonas exotoxin, alpha-amanitin, rat grape antiviral protein (PAP, pokeweed), ribosome-inhibiting proteins, especially ribosome-inhibiting proteins from barley, wheat, corn, rye, gelonin, and abrin, as well as certain cytotoxic chemicals, such as, for example, melphalan and daunomycin. However, document BR 1020190027932 does not refer to GnRH analogues, specifically to a 9-amino acid GnRH, comprising chloroacetyl-type modifications at the amino-terminal end, nor does it disclose a modified 9-amino acid peptide conjugated to albumin (BSA) or diphtheria toxoid (DT), as disclosed by the present invention.

[0018] The document in the name of Linder I, W., & Robey, FA, entitled Automated synthesis and use of N-chloroacetyl-modified peptides for the preparation of synthetic peptide polymers and peptide-protein immunogens rrefers to a method for incorporating N-chloroacetyl moieties into the amino termini of synthetic peptides using a standard program with an automated peptide synthesizer. The N-chloroacetyl-modified peptides react with sulfhydryl-containing proteins, such as 4-mercaptobutyrimide-modified bovine serum albumin, to form stable protein-peptide conjugates.

[0019] Furthermore, the document in the name of Linder I, W., & Robey, FA describes that, upon incorporation of cysteine ​​into the synthetic peptide, self-polymerization or cyclization of the synthetic peptide occurs by the reaction of the free sulfhydryl with the chloroacetyl group. However, contrary to what is proposed by the present invention, the document does not describe the modification at the C-terminal end of the GnRH peptide from carboxyl to carbamide, a modification that results in the efficacy of the vaccine described herein (as demonstrated in the results of the efficacy study in Tables 6, 7, and 8 below).

[0020] The document by Chua, BY, Al Kobaisi, M., Zeng, W., Mainwaring, D., & Jackson, DC, entitled "Chitosan Microparticles and Nanoparticles as Biocompatible Delivery Vehicles for Peptide and Protein-Based Immunocontraceptive Vaccines," describes the production of immunocontraceptive vaccines based on chitosan nanoparticles or microparticles as LHRH-based vehicles. These particles were modified to contain chloroacetyl groups, which serve as covalent binding sites for peptides containing cysteine ​​residues. However, in addition to not describing a GnRH peptide comprising the C-terminal end modified with carbamide, the document does not describe conjugation via SATA, but rather via cysteine ​​residues.

[0021] Similar to the document described above, the proprietary document by Mezõ, Gábor, et al., et al., entitled Synthesis and Structural Characterization of Bioactive Peptide Conjugates using Thioether Linkage Approaches, describes GnRH peptides elongated with cysteine ​​residues and conjugated to a carrier derived from chloroacetylated tetratufitsine. However, in the proprietary document by Mezõ, Gábor, et al., et al., the conjugation of the peptide to the carrier is not carried out via SATA but rather via cysteine ​​residues. Furthermore, although the peptide to be conjugated has a C-terminal modification to carbamide, this modification was determined based on a conjugation strategy, but not for peptide stabilization and its efficacy, a factor that implies in the immunocastration of animals. Furthermore, the aforementioned document refers to GnRH - ITT, which is a different peptide than that addressed in the present invention.

[0022] The patent document of Oonk, HB, et al., entitled "New GnRH-like peptide construct to optimize efficient immunocastration of male pigs by immunoneutralization of GnRH," discloses the development of a dimerized GnRH modified at an amino acid position to allow conjugation of the modified GnRH to ovalbumin. This conjugate was applied in the production of vaccines for swine immunocastration. However, this document discloses a modification in an amino acid residue of GnRH but not the modification at the C-terminal, as proposed in the present invention.

[0023] The paper by Miller, Brian T., et al., entitled "The Occurrence of O-Acylation during Biotinylation of Gonadotropin-releasing Hormone and Analogs," deals with the chemical reaction between GnRH and its analogs with sulfonated biotin N-hydroxysuccinimide esters through a thioether bond directly, or by the insertion of a spacer. However, that paper discusses a different type of conjugation, in which the conjugated peptide does not have the C-terminal modification, as disclosed in the present invention.

[0024] In this sense, unlike the solutions found in the prior art, the present invention proposes a modified GnRH vaccine antigen of high immunogenicity with efficacy for immunocastration of mammals, especially swine, with the C-terminal end of said peptide being carbamide instead of carboxyl. The results presented here show that the peptide with a carbamide C-terminal end had superior efficacy to the peptide with C-terminal carboxyl end.

[0025] The modification of the GnRH peptide coupled with the use of a solubilizing agent (urea) also results in a surprising effect in the claimed composition when compared to the prior art documents, since the results presented here show that the modified GnRH peptide was more exposed to the immune system, thus resulting in a greater quantity of neutralizing antibodies, that is, inducing an even more effective immune response in the individual. Thus, the present application demonstrates, unequivocally, that a modified GnRH peptide with a carbamide C-terminal end has superior effects, in relation to efficacy, for example, in comparison with the unmodified peptide with a carboxyl C-terminal end of the art.

[0026] Therefore, the aforementioned prior art, even when combined, would not result in the teachings described in a novel manner in the present invention. In particular, the aforementioned prior art does not describe, nor is it even capable of suggesting, a modified GnRH peptide with a carbamide C-terminal end conjugated to a carrier via SATA and with the GnrH N-terminus functionalized with chloroacetyl. Furthermore, it should be noted that the aforementioned prior art, similarly, does not describe, nor even suggest, the use of urea as a solubilizing and exposure agent for GnRH in the immunocastration of mammals, especially swine. SUMMARY OF THE INVENTION

[0027] In a first embodiment, the present invention aims to propose a modified GnRH peptide, as defined in SEQ ID NO: 1.

[0028] In a second embodiment, the present invention aims to propose a conjugate comprising a modified GnRH peptide and a carrier protein.

[0029] In a third embodiment, the present invention aims to propose a vaccine composition, with high immunogenicity, comprising said modified GnRH conjugate in a pharmaceutically acceptable formulation.

[0030] Furthermore, in a fourth embodiment, the present invention aims to propose a kit comprising the vaccine composition of the invention as well as its instructions for use.

[0031] Furthermore, in a fifth embodiment, the present invention aims to propose the use of the conjugated modified GnRH peptide in the manufacture of a vaccine composition for immunocastration of mammals, in particular in the immunocastration of swine, in which the modified GnRH peptide consists of the following structure: ClAc-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 or chloroacetyl-HWSYGLRPG-NH2.

[0032] In a sixth embodiment, the present invention aims to propose a method for the manufacture of conjugated modified GnRH peptide antigen, which refers to the active ingredient of the composition claimed in the present invention. BRIEF DESCRIPTION OF THE FIGURES

[0033] To obtain a complete and total view of the objective of this invention, the following Figures are presented, described below.

[0034] Figure 1 shows a diagram of the reactions in the synthetic antigen production process, in which IA represents step 1 of carrier protein functionalization and 1B represents step 2 of the SATA group deprotection reaction.

[0035] Figure 2 shows the scheme of the conjugation reaction of the carrier protein-SATA to the chloroacetyl-functionalized GnRH peptide in step d of the method for manufacturing the synthetic antigen of conjugated modified GnRH.

[0036] Figure 3 shows the scheme of the vaccine protocol used for the in vivo preclinical studies.

[0037] Figure 4 shows the SDS-Page and Western Blotting rrespectively, labeled against Anti-GnRH antibody of the different carrier proteins conjugated with GnrH peptide.4A) Columns: 1 - Molecular weight marker (4 uL); 2 - Wild-type diphtheria toxoid carrier (supplier 1) conjugated to GnRH peptide without modification of the C-terminal end to carbamide and without urea (10 uL); 3- Wild-type diphtheria toxoid carrier (supplier 1) conjugated to GnRH peptide with modification of the C-terminal end to carbamide and with urea (10 uL); 4 - Recombinant diphtheria toxoid carrier (product CRM 197 - purchased from FinaBio Biosolutions LLC) conjugated to GnRH peptide without modification of the C-terminal end to carbamide and without urea (Lot 001) (10 uL); 5 - Recombinant diphtheria toxoid carrier conjugated to GnRH peptide without modification of the C-terminal end to carbamide and without urea (Lot 002) (10 uL); 6 - Wild-type diphtheria toxoid carrier (supplier 1) before conjugation to GnRH peptide (control before conjugation) (10 uL); 7 - Recombinant diphtheria toxoid carrier before conjugation to. GnRH peptide (Control before conjugation) (10 uL); 4B) Columns: 1 - Molecular weight marker (4 uL); 2 - Carrier Bovine albumin (BSA) conjugated to the GnRH peptide without modification of the C-terminal end to carbamide and without urea (10 uL); 3- Carrier wild-type diphtheria toxoid (supplier 2) conjugated to the GnRH peptide without modification of the C-terminal end to carbamide and without urea (10 uL); 4 - Carrier Bovine albumin (BSA) conjugated to the GnRH peptide with modification of the C-terminal end to carbamide and with urea (10 uL); 5 - Wild-type diphtheria toxoid carrier (supplier 2) conjugated to GnRH peptide with C-terminal modification to carbamide and with urea (10 uL); 6 - BSA carrier before conjugation to GnRH peptide (control before conjugation) (10 uL); 7 - Wild-type diphtheria toxoid carrier (supplier 2) before conjugation to GnRH peptide (Control before conjugation) (10 uL).The antibodies used were: Primary antibody: GnRH I (HU11B) mouse monoclonal IgG - Santa Cruz Biotechnology (sc32292 Lot: # B0619), dilution: 1 / 125; Secondary Antibody: Anti-mouse IgG-Peroxidase - SIGMA ALDRICH (Code A8924 Lot: SLBZ3812), dilution: 1 / 10,000. DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention aims to propose a modified GnRH peptide with chloroacetyl and carbamyl ends, consisting of the following sequence: ClAc-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 and with the chemical structure as shown below. Formula

[0039] Furthermore, it is an objective of the present invention to propose a conjugate comprising the modified GnRH peptide and a carrier protein, in which the carrier protein is bovine serum albumin (BSA) or wild or recombinant diphtheria toxoid or another carrier that has the characteristic of primary amines of plant residues exposed to the surface.

[0040] Furthermore, the present invention also provides a highly immunogenic vaccine composition comprising the conjugate of said modified GnRH with albumin or diphtheria toxoid (wild or recombinant) or other carriers that have characteristics of primary amines from plant residues exposed to the surface in a pharmaceutically acceptable formulation comprising DEAE-Dextran or its conjugated salts or cationic polymers comprising REI, chitosan, polypropylene imine, poly-lysine, polyamideamine, polyallylamine, polydiallyldimethylammonium chloride, poly (isopropyl acrylamide - co-acrylamide), co-acrylic acid, poly N-ethylvinylperidine bromide, poly dimethylamino, ethylmethacrylate, polyethylene glycol, polytrimethylaminoethylmethacrylate hydrochloride.

[0041] Furthermore, the objective of the present invention is to propose a kit comprising the vaccine composition for immunocastration of mammals, especially pigs, as well as their instructions for use.

[0042] Furthermore, it is an additional objective of the present invention to describe the use of the conjugate comprising the modified GnRH peptide and a carrier in the preparation of a vaccine composition for immunocastration of mammals, especially in the immunocastration of swine.

[0043] Additionally, it is an objective of the present invention to propose a method for the manufacture of a modified GnRH peptide antigen linked to a carrier protein, as described in the following steps. GnRH Modifications

[0044] First, modifications were made to the structure of the GnRH peptide. Originally, the GnRH molecule consisted of a decapeptide containing C-terminal (carboxy-terminal) and N-terminal (amino-terminal) ends, as follows: pyroGlu-His-Trp-Ser-Tyr-Gly-Leu~Arg~Pro~Gly-NPu.In the present invention, the decapeptide was transformed into a peptide containing 9 amino acids with modified ends, with chloroacetyl at the N-terminus and carbamyl instead of carboxamide at the C-terminus, conjugated to albumin (BSA) or diphtheria toxoid in a pharmaceutically acceptable formulation comprising DEAE-Dextran or its conjugated salts or cationic polymers comprising PEI, chitosan, polypropylene imine, poly-lysine, polyamideamine, polyallylamine, polydiallyldimethylammonium chloride, poly(isopropyl acrylamide - co-acrylamide), co-acrylic acid, poly N-ethylvinylperidine bromide, polydimethylamino, ethylmethacrylate, polyethylene glycol, polytrimethylaminoethylmethacrylate hydrochloride. Thus, the modified GnRH. It is produced by a chemical process (chemical acetylation), that is, without the need for an industrial plant to produce the recombinant vaccine, which requires a lower level of biosafety.

[0045] The aforementioned method of manufacturing the synthetic antigen of modified GnRH conjugated by chemical acetylation comprises the following steps (Figures 1 and 2): a) functionalization of the carrier protein, which can be chosen from bovine albumin, wild or recombinant diphtheria toxoid or other carriers that have characteristics of primary amines of lysine residues exposed on the surface, with the SATA reagent (N-succinimidyl S-acetylthioacetate) in reaction solution and EDTA (Figure 1A); b) incubation of the reaction for at least 2 hours under agitation at room temperature (20-30°C), in which the SATA reagent binds to the primary amines of the carrier protein.SATA adds sulfhydryl groups to the primary amines of proteins by nucleophilic attack, and the succimimide group is abandoned (Figures 1A and 1B); c) diafiltration is performed to eliminate the free SATA reagent that did not functionalize the carrier; d) conjugation of the modified GnRH peptide to the SATA-functionalized carrier in the presence of the deacetylation solution, in which the chloroacetyl conjugation of SATA with the lysine of the carrier protein occurs. The conjugation occurs due to the nucleophilic attack of the peptide on the thiol group released by the hydroxylamine present in the deacetylation solution. In this chemical reaction, a... modification in the peptide, in which the resulting final antigen is composed of several peptides linked to the carrier protein; e) incubation of the reaction for at least 12 hours under agitation at a temperature of 20 to 30 °C; f) addition of the reaction stop solution after conjugation; g) diafiltration to eliminate GnRH peptide not conjugated to the carrier protein; h) sterilization of the conjugated antigen by terminal filtration with a 0.22 µm filter, to collect the sterile antigen solution in sterile disposable bags; ei) withdrawal of samples for quality analysis.

[0046] After approval of the quality of the final antigen by bioanalytical, physicochemical, and microbiological methods (sterility) (Figure 4 shows the results of the SDS-Page and Western Blotting methods). From Figures 4a and 4b, it is possible to observe that antibody labeling against GnrH occurs in antigens (GnrH peptide and its conjugate) that had the carbamide modification at the C-terminal and the presence of urea (Figure 4A - column 3) and Figure 4B - columns 4 and 5). These results demonstrate that both the carbamide modification of the peptide and the use of urea contribute to the conjugation process, antigen-antibody recognition, and antigen stability. It is surprising that the use of urea plays a fundamental role in the conjugation reaction of the peptide to the carrier, conjugate stability, and exposure of the conjugated peptide for antibody recognition. The ability of urea to form weak bonds and unfold the proteins helped in the greater exposure of the primary amines of the carrier lysines that were functionalized with SATA for conjugation with the peptide and acted in the exposure of the conjugated peptides per carrier molecule. The final antigen is prepared with a pharmaceutically acceptable formulation comprising DEAE-Dextran or its conjugated salts or cationic polymers comprising PEI, chitosan, polypropylene imine, poly-lysine, polyamideamine, polyallylamine, polydiallyldimethylammonium chloride, poly (isopropyl acrylamide - co-acrylamide), co-acrylic acid, poly N-ethylvinylperidine bromide, poly dimethylamino, ethyl methacrylate, polyethylene glycol, polytrimethylaminoethyl methacrylate hydrochloride. Thus, a modified GnRH with a molecular weight of approximately 1145.9-1147.9 Da is obtained, commercially and chemically called N-Chloroacetl-GnRF (2-10) conjugated to the carrier protein.

[0047] Regarding the steps of the manufacturing method of the synthetic modified GnRH antigen described above, technical details are also presented: Step 1 - Functionalization of the carrier with SATA (SATA-carrier) (Figure IA) a) add the carrier protein (bovine albumin or wild-type or recombinant diphtheria toxoid) solubilized at a concentration of 5-10 mg / ml in the reaction solution; b) add 10-100 mM EDTA solution and shake for at least 50 rpm; c) wait at least 10 minutes and check the pH. If necessary, adjust the pH between 8.0 and 9.0 with NaOH solution (1 M); d) add the SATA reagent solubilized with DMSO (0.1 - 2% w / v); e) wait at least 10 minutes and check the pH. If necessary, adjust the pH between 8.0 and 9.0 with NaOH solution (IM); f) after adding the components and adjusting the pH, make up to the final volume with the reaction solution as follows: Formula 3 g) incubate the reaction under stirring at at least 50 rpm for a minimum period of 2 hours at room temperature (20-30 °C); h) clarify the SATA-carrier solution from step 1 through a PVDF filter with a maximum porosity of 1.0 pm; i) diafilter the concentrated SATA-carrier solution from step 1 using 5-10 volumes of diafiltration solution from step 1, through a continuous feedback system, that is, keeping the diafiltration volume 1 in which the sample is being washed constant; j) top up the SATA-carrier solution from step 1 to the final volume with the diafiltration solution from step 1; k) measure the SATA-carrier concentration in a spectrophotometer at a wavelength of 280 nm; l) proceed with the calculation to evaluate the SATA-carrier concentration in millimolar (mM) and in mg / mL and record the results; m) before starting the second step of the reaction, calculate the rate of protected groups, that is, the average number of SATA incorporated into the carrier. This measurement is carried out by the Ellmans reaction, which is a colorimetric reaction in which the Ellmans reagent changes color in the presence of free sulfhydryl groups and is absorbed at a wavelength of 412 nm; n) measure the absorbance in a spectrophotometer at a wavelength of 412 nm; o) calculate the rate of thiols per carrier molecule and in the formula below: Formula 4 Step 2 - SATA-carrier conjugation to the chloroacetyl-GnRH peptide (SATA group deprotection reaction - Figure 1B) a) add 35-40% of the diafiltration solution 1 to a vial containing the peptide solubilized in urea solution; b) activate the stirring system for at least 50 rpm; c) add the SATA-carrier solution from step 1 (final product of step 1) to the vial containing the solubilized peptide; d) add 5-20% of the deacetylation solution to the vial; e) close the vial and keep the reaction solution from step 2 stirring for at least 50 rpm for at least 12 hours, at room temperature (20-30°C); f) after completion of the incubation time, add 0.1-1% reaction stop solution (iodoacetic acid - 30 mg / mL) to the reactor and incubate for at least 2 hours and under stirring for at least 50 rpm; g) clarify the solution in a 0.5-1.0 pm porosity filter; h) diafilter the GnRH-carrier solution from step 2 using a total volume of 5-10 volumes of diafiltration solution from step 2, through a continuous feedback system, that is, maintaining the initial volume of the GnRH-carrier solution from step 2; i) at the end of the process, top up the GnRH-carrier solution from step 2 to the final volume with the diafiltration solution - Step 2; j) in a biological safety cabinet, filter the GnRH-carrier solution from step 2 through a 0.22 pm filter.

[0048] The vaccine composition of the present invention, whose active ingredient is a modified GnRH peptide, additionally comprises a pharmaceutically acceptable carrier or carrier selected from the group consisting of albumin or wild-type or recombinant diphtheria toxoid or other carriers that have characteristics of primary amines of surface-exposed lysine residues. Preclinical study In vivo tests

[0049] From the construction of the modified GnRH peptide, a vaccine for immunocastration of pigs was developed, whose safety and efficacy tests, which include evaluations such as pre-slaughter mounting behavior, analytical tests (quantification of testosterone, androstenone, skatole and anti-GnRH), sensory evaluation (meat cooking test with fat). These evaluations were carried out comparatively to demonstrate the performance between formulations based on wild-type and recombinant diphtheria toxoid (DT) and BSA as carriers.

[0050] Table 1 below shows the groups of animals tested with the administration of the vaccine composition of the present invention, comprising a modified GnRH conjugate and a pharmaceutically acceptable carrier or carrier, and the composition with the trade name Vivax® available on the national market. Table 1: Preclinical study groups, in which group 1 refers to the group vaccinated with Vivax® and group 2 refers to the group vaccinated with the vaccine composition of the present invention. Vaccination Protocol

[0051] For in vivo preclinical studies, healthy pigs approximately 128 days old were used and vaccinated on days DO (day zero - first vaccination) and D28 (second vaccination). The study was carried out in 8 replicates with 3 animals / pen, totaling 9 groups (n = 24). On day D51, the animals were slaughtered following the recommended ethical procedures (Figure 3).

[0052] Thus, based on the vaccination protocol carried out with In both groups 1 and 2, results were obtained on zootechnical performance, carcass typification and safety, which are presented and described below. Table 2: The table presents the quantification of 3 performance parameters: Daily weight gain - GPD (kg), Daily feed consumption - CDR (kg) and Feed conversion - CA, comparing the groups treated with the Vivax® vaccine and the vaccine composition of the present invention. Table 3: The table presents the quantification of 7 performance parameters: Live weight at slaughter (kg) [179 days old], Dead weight / carcass (kg), Carcass yield (%), Backfat thickness (mm), Loin depth (mm), Lean meat (%) and Lean meat (kg), comparing the groups treated with the vaccine Vivax® and the vaccine composition of the present invention. Table 4: The table presents serological results of the hormonal quantification of androstenone (pg / g), skatole (pg / g) in fat and testosterone (ng / mL) in blood serum, comparing the groups treated with the Vivax® vaccine and the vaccine composition of the present invention. Table 5: Risk reference table in relation to androstenone / fat (pg / g) and skatole (pg / g) levels in fat for the interpretation of the data in Table 4. Results

[0053] Tests were carried out regarding efficacy and safety in the target species in the groups described below: - G1: Negative (placebo) - G2: BSA carrier with GnRH peptide with carboxyl at the C-terminus - G3: Wild-type DT Loader 2 with C-terminal carboxyl GnRH peptide - G4: Recombinant DT Charger (product CRN 197 - acquired from FinaBio Biosolutions LLC) with GnRH peptide with carboxyl at the C-terminal - G5 : BSA loader with carbamide-modified GnRH peptide at the C-terminus - G6 : Wild-type DT loader 1 with carbamide-modified GnRH peptide at the C-terminus - G7: Recombinant DT Loader (CRM 197 product - acquired from FinaBio Biosolutions LLC) with carbamide-modified GnRH peptide at the C-terminus - G8: Wild-type DT Loader 2 with carbamide-modified GnRH peptide at the C-terminus - G9 : Vivax®

[0054] Table 6 presents the comparison according to treatments in relation to daily feed intake (DFI), feed conversion (FCR) and daily weight gain (DWG). Table 6: Average daily weight gain (ADWG), daily feed intake (DFI) and feed conversion (FCR) according to treatments. a ' b ' c different letters in the columns indicate difference by Tukey's test (p<0.05).

[0055] Table 7 presents the carcass typification parameters in relation to the percentage of lean meat, backfat thickness (mm) and loin depth (mm) in comparison according to the treatments performed. Table 7: Averages of carcass typification parameters according to treatments.

[0056] Table 8 below presents the compilation of data on testicular weight (TW), body weight at slaughter, daily weight gain (DWG), feed conversion (FCR), % lean meat, backfat thickness measurement, loin thickness measurement, androstenone dosage, skatole dosage, number of animals with pre-slaughter mounting behavior, testosterone dosage and anti-GnRH titer (GMT) at pre-slaughter. Table 8: The table presents the average values. a ' b ' cdistinct letters in the lines indicate difference by Tukey's test (p<0.05). PT: testicular weight (kg); Slaughter weight: average body weight at slaughter (kg); GPD: daily weight gain (kg), CA: feed conversion; % lean meat; Backfat: backfat thickness (mm); Loin: loin thickness (mm); Androst: androstenone dosage (pg / g) in fat, Skatole: skatole dosage (ng / mL) in fat, Mount: number of animals with mounting behavior pre-slaughter; Test: serum testosterone dosage (ng / mL), Titer: anti-GnRH dosage (GMT) pre-slaughter.

[0057] The results show that the groups with the GnRH peptide modified with carbamide in the C-terminal portion presented similar results (Table 6 - Zootechnical performance and Table 7 - carcass typification) or numerically superior (Table 8 - breeding behavior, testosterone and titer) to the G9 group of the Vivax® vaccine.

[0058] In summary, the results presented prove that the vaccine formulations of the present invention are safe for use in animals, are effective and present similar performance, using BSA or recombinant DT or wild-type DT as a carrier, when compared to the Vivax® vaccine available on the national market.

Claims

CLAIMS 1. Method of manufacturing a conjugated modified GnRH peptide antigen, characterized by the fact that it comprises the following steps: a) functionalizing the carrier protein with SATA (N-succinimidyl S-acetylthioacetate) in reaction solution and EDTA; b) incubating the reaction for at least 2 hours under stirring at a temperature of 20 to 30°C; c) diafiltering the reaction; d) conjugating the modified GnRH peptide to the carrier protein functionalized with SATA in the presence of the deacetylation solution and urea; e) incubating the reaction for at least 12 hours under stirring at a temperature of 20 to 30°C; f) adding reaction stop solution after conjugation; g) diafiltering the reaction; eh) sterilizing the modified GnRH antigen by terminal filtration with a 0.22 µm filter.

2. Method according to claim 1, characterized in that the carrier protein is bovine serum albumin (BSA) or wild-type diphtheria toxoid or recombinant diphtheria toxoid or another carrier that has a characteristic of primary amines in surface-exposed lysine residues.

3. Method according to claim 1, characterized in that in step d) urea is used as a solubilizing agent and as an aid to the exposure of the conjugated peptide in the antigen.

4. Modified GnRH peptide characterized by the fact that it consists of a sequence of nine amino acids with the ends modified with chloroacetyl at the N-terminal end and carbamyl at the C-terminal end, such as Chloroacetyl-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 of SEQ ID NO. :

1.

5. Modified GnRH peptide, according to claim 4, characterized by the fact that it is produced by a chemical acetylation process, as defined in claim 1.

6. Conjugate characterized by the fact that it comprises the modified GnRH peptide, as defined in claim 4 or 5, and a carrier protein.

7. Conjugate, according to claim 6, characterized by the fact that the carrier protein is bovine serum albumin (BSA) or wild or recombinant diphtheria toxoid or other carrier that has a characteristic of primary amines of lysine residues exposed to the surface.

8. Vaccine composition characterized by the fact that it comprises the modified GnRH conjugate, as defined in claim 6 or 7, in a pharmaceutically acceptable formulation comprising DEAE-Dextran or its conjugated salts or cationic polymers comprising PEI, chitosan, polypropylene imine, poly-lysine, polyamideamine, polyallylamine, polydiallyldimethylammonium chloride, poly(isopropyl acrylamide - co-acrylamide), co-acrylic acid, poly N-ethylvinylperidine bromide, poly dimethylamino, ethylmethacrylate, polyethylene glycol, hydrochloride poly trimethylaminoethyl methacrylate.

9. Composition, according to claim 8, characterized by the fact that it comprises DEAE-Dextran hydrochloride in a concentration between 5-20%, preferably in 15%.

10. Kit characterized by comprising the vaccine composition, as defined in claim 8 or 9, and instructions for use.

11. Use of a conjugate comprising the modified GnRH peptide, as defined in claim 6 or 7, characterized in that it is for the preparation of a composition, as defined in claim 8 or 9, for the immunocastration of mammals.

12. Use of modified GnRH peptide, according to claim 11, characterized by the fact that the mammal is preferably a pig.

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