Modified GnRH peptide for immune castration of mammals, conjugate thereof, preparation method, composition and kit

Abstract

The invention belongs to the fields of molecular biology, medical science and immunology, and in particular relates to a preparation for medical purposes. In particular, the present invention relates to modified peptides of gonadotropin releasing hormone (GnRH) and conjugates thereof for immune castration of mammals, in particular pigs, to methods of preparing modified GnRH antigens, compositions and kits comprising said vaccine compositions, it aims to improve the quality of mammalian carcasses, animal welfare, food use (feed conversion), produce less waste and use less water, thereby promoting efficient sustainable production (ESG).

Description

TECHNICAL FIELD

[0001] The present invention belongs to the field of molecular biology, medical science and immunology, and in particular relates to formulations for medical purposes. In particular, the present invention relates to modified Gonadotropin Releasing Hormone (GnRH) peptides and conjugates thereof for immunocastration of mammals, in particular pigs, to methods of preparing modified and conjugated GnRH antigens, compositions and kits comprising said vaccine compositions. BACKGROUND

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

[0003] GnRH vaccines stimulate the production of antibodies to inactivate endogenous GnRH. The anti-GnRH immune response suppresses the release of LH and FSH, which are essential for gonadal development and maintenance, the testes in males and the ovaries in females. Thus, the reduction in LH and FSH levels leads to the loss of reproductive function.

[0004] Since GnRH is a short peptide (10 amino acids long) and naturally circulates in the body, the development of immunocastration techniques based on the generation of an immune response to GnRH is challenging. Thus, the 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 known as carrier proteins.

[0005] The available vaccines with such constructs / molecules have achieved varying degrees of success in different species, but low immunogenicity remains a technical problem to be overcome.

[0006] One of the advantages of immunocastration of pigs is the reduction of the sexual odor of male pigs. Odor is associated with the sexual maturation and hormone production of male pigs, making the meat of uncastrated animals unsuitable for consumption (BABOL et al., 1998). Thus, the consumption of pork from uncastrated animals depends on its processing. More specifically, it is known that sexual odor is produced by the accumulation or combination of the following compounds: androstenone (5a-androst-16-en-3-one) and skatole (3-methylindole). Therefore, in current pig production, most of the animals destined for slaughter are surgically or immunocastrated males (HENNESSY et al., 1997).

[0007] Surgical castration (gonadectomy) of young male pigs is a common procedure in pig production worldwide (THUN et al., 2006). In addition, article 104 of the Brazilian Regulation for Inspection of Animal Origin and Industry (RISPOA) prohibits the slaughter of pigs that have not been castrated. In Brazil, the last current legislation regarding surgical castration of pigs is the Regulatory Instruction No. 113 of the Ministry of Agriculture, Livestock and Supply (MAPA) of June 1, 2018. This regulatory instruction establishes the conditions for surgical castration of pigs, aiming to promote animal welfare and ensure the quality of animal-origin products. However, surgically castrated animals have negative impacts on animal technical performance, carcass quality, and animal welfare. These factors play a decisive role in the search for more sustainable alternatives to pig castration and have driven the development of the present invention.

[0008] The principle of immunocastration is based on the application of a vaccine containing a modified form of GnRH conjugated to a protein, which induces the formation of antibodies against GnRH (ZAMARATSKAIA et al., 2008). The use of the pig's own immune system to suppress GnRH disrupts the transmission of GnRH from the release site in the hypothalamus to the action site in the pituitary, thus disrupting the hypothalamus-pituitary-gonad axis, by establishing an immunological barrier. GnRH suppression prevents it from stimulating the pituitary to secrete LH and FSH, thus reducing testicular development and the synthesis of steroid hormones (THUN et al., 2006), including androstenone (ZAMARATSKAIA et al., 2008), the main hormone responsible for the odor of carcasses. Immunocastrated animals exhibit improvements in carcass quality, food use (feed conversion), produce less waste, and use less water, contributing to efficient and sustainable production (ESG).

[0009] The main advantages of immunocastration are:

[0010] (1) Improved meat quality: immunocastration can improve meat quality, making it more tender and juicy, as immunized pigs have less intramuscular fat and higher content of unsaturated fatty acids;

[0011] (2) Reduced costs: immunocastration eliminates the need for surgical castration of pigs, reducing handling and transportation costs, in addition to avoiding behavioral problems associated with castration;

[0012] (3) Reduced environmental impact: immunocastration can reduce the environmental impact of pig production, as it avoids the generation of waste associated with surgical castration, such as the disposal of testicular tissue and the use of anesthetics;

[0013] (4) Higher feed efficiency: immunized pigs better utilize feed nutrients, which can result in better performance and higher profitability for producers (according to Santos et al. (2012), immunized castrated animals have a daily weight gain approximately 8.3% higher than castrated animals); and

[0014] (5) Improved animal welfare: immunization to castration helps to improve animal welfare as it avoids the pain and stress associated with surgical castration. Unregistered pigs have high levels of testosterone, androstenone and skatole, showing more aggressive behavior and less meat tenderness.

[0015] In summary, unregistered pigs have high levels of testosterone, androstenone and skatole, showing more aggressive behavior and less tenderness in meat. Surgically castrated pigs, i.e. pigs with testicles removed, are less aggressive than uncastrated pigs, but are more weak. In turn, immunized castrated pigs are less aggressive (CRONIN et al., 2003) and have better feed conversion, daily weight gain (Daily Weight Gain / DWG), final carcass weight, and therefore higher meat yield (PAULY et al., 2009) than surgically castrated pigs.

[0016] In this context, the main object of the present invention is to develop a modified GnRH peptide and conjugates thereof for the preparation of a composition with high immunogenicity for immunocastration of mammals, particularly pigs, to develop a method for manufacturing conjugated modified GnRH antigens, to develop a vaccine composition comprising said conjugates and a kit comprising said vaccine composition and instructions for its use.

[0017] In particular, the modified GnRH peptide of the present invention is linked / conjugated to a carrier through a covalent bond and can be represented by one of the following structures:

[0018] Formula 1

[0019] BACKGROUND

[0021] Unless otherwise indicated, the terms used in this specification in the context of the present disclosure and in the specific context of each term's use in the present disclosure have their ordinary meanings. Certain terms used to describe the present disclosure will be discussed in the following detailed description or elsewhere in the specification to provide further guidance to the practitioner in relation to what is described by the present disclosure.

[0022] It should be understood that the same thing can be said in different ways. Accordingly, alternative language and synonyms can be used for any term discussed herein. No special significance is to be given to the use of any particular term herein over another. Synonyms for certain terms are provided. Some synonyms can be more or less preferred depending on the context.

[0023] In view of the foregoing, the expression "modified GnRH" and "modified GnRH peptide" in the present application refers to the amino acid polypeptide sequence of the GnRH hormone and the modification purposes described herein, and further refers to the active principle of the claimed vaccine composition. Thus, "modified GnRH" and "modified GnRH peptide" can be understood as referring to the expression of a vaccine antigen conjugated to a carrier protein, which is capable of being recognized and triggering an immune system response.

[0024] Some studies in the literature propose vaccines for the immunocastration of pigs. One of these vaccines, developed by the company Zoetis, under the trade name Vivax®, inhibits the activity of GnRF and the production of testosterone, reducing the production and accumulation of undesirable substances. The patent application corresponding to the vaccine composition is BR 1020190027932, which includes the following objects: vaccine, method of preparation of the 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 comprising CpG, sterol, phospholipid and oil, wherein the vaccine is an oily formulation or a water-in-oil emulsion, there is no mention of the type of GnRH used. However, in the description of the application, it is envisaged that GnRH encompasses the naturally occurring mammalian GnRH (SEQ ID NO: 11 (pyro-EHWSYGLRPG-NH2)) and includes GnRH analogs capable of inhibiting the release of gonadotropins by the pituitary, thus inhibiting the release of steroid hormones, estradiol, progesterone and testosterone.

[0025] However, in BR 1020190027932, other examples of GnRH analogs are provided, including but not limited to leuprolide (pyrGlu-His-Trp-Ser-Tyr-DLeu-Arg-Pro-NHEt; SEQ ID NO: 12), buserelin (pyrGlu-His-TrpSer-Tyr-D-Ser(TBU)-Leu-Arg-Pro-NHEt; SEQ ID NO: 13), goserelin (pyrGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-NHEt; SEQ ID NO: 14) and histrelin (pyrGlu-His-Trp-Ser-Tyrp-Gly-Leu-Arg-Pro-NHEt; SEQ ID NO: 15). In addition, the document discloses the use of immunostimulating carriers, namely: diphtheria toxin, ricin, exotoxin pseudomonas, alpha-ethyl carbamate, rat grape antiviral protein (PAP, pokeweed), proteins that inhibit ribosomes, especially those that inhibit barley, wheat, corn, rye, gelatin and abrin ribosomes, and certain cytotoxic chemicals, such as melphalan and daunomycin. However, the document BR 1020190027932 does not mention GnRH analogs, in particular, it does not mention the 9-amino acid GnRH containing a chloroacetyl-like modification at the amino terminus, nor does it disclose the modified 9-amino acid peptide conjugated to albumin (BSA) or diphtheria toxoid (DT) as disclosed in the present invention.

[0026] The document representing Linder I, W., & Robey, F. A. is entitled "Automated synthesis and use of N-chloroacetyl-modified peptides for the preparation of synthetic peptide polymers and peptide-protein immunogens" and relates to a method for incorporating an N-chloroacetyl moiety at the amino terminus of a synthetic peptide using standard procedures with an automated peptide synthesizer. The N-chloroacetyl-modified peptide reacts with a thiol-containing protein such as 4-mercapto butyramide-modified bovine serum albumin (BSA) to form a stable protein-peptide conjugate.

[0027] In addition, the document representing Linder I, W., & Robey, F. A. describes the self-polymerization or cyclization of synthetic peptides by the incorporation of cysteine into the synthetic peptide, by the reaction of the free thiol group with the chloroacetyl group. However, contrary to what is proposed in the present invention, this document does not describe the modification of the C-terminal of the carboxyl GnRH peptide to an amide C-terminal (with a carbamoyl group), which modification results in the efficacy of the vaccine described herein (as shown in the efficacy study results of Tables 6, 7 and 8 below).

[0028] The document representing Chua, B. Y., Al Kobaisi, M., Zeng, W., Mainwaring, D., & Jackson, D. C. entitled "Chitosan Microparticles and Nanoparticles as Biocompatible Delivery Vehicles for Peptide and Protein-Based Immunocontraceptive Vaccines" describes the production of immunological contraceptive vaccines based on chitosan nanoparticles or microparticles as LHRH-based vehicles. Such particles have been modified to contain chloroacetyl groups, which are used as covalent bonding sites for peptides containing cysteine residues. However, in addition to the fact that the document does not describe GnRH peptides containing a C-terminal modified with a carbamoyl group, the document does not describe conjugation through SATA, but rather through the cysteine residue.

[0029] Similarly to the above-mentioned document, the document owned by Mezö, Gábor et al. entitled "Synthesis and Structural Characterization of Bioactive Peptide Conjugates using Thioether Linkage Approaches" describes the elongation and conjugation of GnRH peptides to carriers derived from chloroacetylated tetrahydrofuran with cysteine residues. However, in the document owned by Mezö, Gábor et al., the conjugation of the peptide to the carrier is not carried out through SATA, but rather through the cysteine residue. In addition, although the peptide to be conjugated has a C-terminal modified as a carbamoyl, the modification is determined based on the conjugation strategy, and not for the stabilization of the peptide and its efficacy, which is a factor that implies immunocastration of animals. In addition, the document refers to GnRH-III, which is a different peptide from the one described in the present invention.

[0030] The document entitled "New GnRH-like peptide construct to optimize efficient immunocastration of male pigs by immunoneutralization of GnRH" by Oonk, H. B., et al. discloses the development of dimeric and modified GnRH at amino acid positions to allow conjugation of the modified GnRH to ovalbumin. The conjugates are applied to the production of vaccines for immunocastration of pigs. However, this document discloses modifications of amino acid residues of GnRH, not the C-terminal modification proposed by the present invention.

[0031] The document entitled "The Occurrence of O-Acylation during Biotinylation of Gonadotropin-releasing Hormone and Analogs" by Miller, Brian T., et al. relates to the chemical reaction of GnRH and analogs thereof with a sulfoester of biotin N-hydroxysuccinimide, either directly through a thioether bond or through the insertion of a spacer. However, different types of conjugation are discussed in the document, wherein the conjugated peptides lack the C-terminal modification as disclosed by the present invention.

[0032] In this sense, and unlike the solutions found in the state of the art, the present invention proposes a modified GnRH vaccine antigen with high immunogenicity, which has efficacy for the immunocastration of mammals, particularly pigs, the C-terminal end of the peptide being an amidated C-terminal end (with a carbamoyl) and not a carboxyl. The results presented herein show that the amidated C-terminal peptide (with a carbamoyl) has superior efficacy to the carboxyl C-terminal peptide.

[0033] Modified antibodies that induce a more effective immune response in an individual. Thus, the present application explicitly demonstrates that, for example, the modified GnRH peptide with an amidated C-terminal end (with a carbamoyl) has superior efficacy in terms of efficacy compared to the unmodified C-terminal carboxyl end peptide of the art. The modification of the GnRH peptide combined with the use of a solubilizing agent (urea) further leads to a surprising effect on the claimed composition compared to the state of the art documents, since the results presented herein show that the modified GnRH peptide is more exposed to the immune system, resulting in a greater amount of neutralizing antibodies, i.e. an even more effective immune response is induced in an individual. Thus, the present application explicitly demonstrates that, for example, the modified GnRH peptide with an amidated C-terminal end (with a carbamoyl) has superior efficacy in terms of efficacy compared to the unmodified C-terminal carboxyl end peptide of the art.

[0034] Thus, the above prior art, even in combination with each other, does not lead to the teachings described in a novel way before the present application. In particular, the above prior art neither describes nor enables a modified GnRH peptide having an amidated C-terminus (with a carbamoyl group) conjugated to a carrier via a SATA and having the N-terminus of GnrH conjugated with a chloroacetyl functionalization. Furthermore, it should be mentioned that the above prior art similarly does not describe or even suggest the use of urea as a solubilizer and GnRH exposure in the immunocastration of mammals, in particular in pigs. SUMMARY

[0036] Firstly, the present application aims at proposing a modified GnRH peptide defined in SEQ ID NO.: 1.

[0037] In a second case, the present application aims at proposing a conjugate comprising a modified GnRH peptide and a carrier protein.

[0038] In a third case, the present application aims at proposing a vaccine composition with high immunogenicity comprising said GnRH conjugate modified in a pharmaceutically acceptable formulation.

[0039] Furthermore, in a fourth case, the present application aims at proposing a kit comprising the vaccine composition of the present application and instructions for its use.

[0040] Nevertheless, in a fifth case, the present application aims at proposing the use of a conjugated modified GnRH peptide in the manufacture of a vaccine composition for the immunocastration of mammals, in particular in the immunocastration of pigs, wherein the modified GnRH peptide consists of the following structure: ClAc-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 or chloroacetyl-HWSYGLRPG-NH2.

[0041] In a sixth case, the present application aims at proposing a method for the preparation of a conjugated modified GnRH peptide antigen, which method refers to the active principle of the composition claimed in the present application. BRIEF DESCRIPTION OF DRAWINGS

[0042] In order to obtain a complete and integral visualization of the object of the present application, the following drawings are described below.

[0043] Figure 1 shows a reaction scheme of the synthesis antigen production process, wherein 1A represents step 1 of the carrier protein functionalization and 1B represents step 2 of the SATA group deprotection reaction.

[0044] Figure 2 Figure 2 shows a reaction scheme of the conjugation reaction of the carrier protein-SATA with the chloroacetyl functionalized GnRH peptide of step d of the method for the preparation of a conjugated modified GnRH synthetic antigen.

[0045] Figure 3 A schedule of the vaccine regimen for in vivo preclinical studies is shown.

[0046] Figure 4 shows SDS-Page and Western Blotting of anti-GnRH antibody labeling against different GnrH peptide conjugated carrier proteins. 4A). Lanes: 1. Molecular weight marker (4 μL); 2. Wild type diphtheria toxoid carrier conjugated with GnRH peptide (Supplier 1), C-terminal not modified with carbamoyl, without urea (10 μL); 3. Wild type diphtheria toxoid carrier conjugated with GnRH peptide (Supplier 1), C-terminal modified with carbamoyl, urea (10 μL); 4. Recombinant diphtheria toxoid carrier conjugated with GnRH peptide (CRM product 197 - purchased from FinaBio Biosolutions LLC), C-terminal not modified with carbamoyl, without urea (lot 001) (10 μL); 5. Recombinant diphtheria toxoid carrier conjugated with GnRH peptide, C-terminal not modified with carbamoyl, without urea (lot 002) (10 μL); 6. Wild type diphtheria toxoid carrier before conjugation with GnRH peptide (Supplier 1) (pre-conjugation control) (10 μL); 7. Recombinant diphtheria toxoid carrier before conjugation with GnRH peptide (pre-conjugation control) (10 μL). 4B). Lanes: 1. Molecular weight marker (4 μL); 2. Bovine albumin (BSA) carrier conjugated with GnRH peptide, without modification of the C-terminal with carbamoyl and urea; 3. Wild diphtheria toxoid carrier conjugated with GnRH peptide (Supplier 2), without modification of the C-terminal with carbamoyl and urea; 4. Bovine albumin (BSA) carrier conjugated with GnRH peptide, with modification of the C-terminal with carbamoyl and urea (10 μL); 5. Wild diphtheria toxoid carrier conjugated with GnRH peptide and modified with carbamoyl and urea (10 μL) of the C-terminal; 6. BSA carrier before conjugation with GnRH peptide (pre-conjugation control) (10 μL); 7. Wild diphtheria toxoid carrier before conjugation with GnRH peptide (Supplier 2) (pre-conjugation control) (10 μL). Antibodies used: Primary antibody: GnRH I (HU11B) monoclonal mouse IgG - Santa Cruz Biotechnology (sc32292 lot: # B0619), dilution: 1 / 125; Secondary antibody: Anti-mouse IgG-peroxidase - SIGMA ALDRICH (A8924 code, lot: SLBZ3812), dilution: 1 / 10,000. DETAILED DESCRIPTION

[0048] The present invention aims to propose a modified GnRH peptide with chloroacetyl and carbamoyl end consisting of the following sequence: ClAc-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 and having the chemical structure as shown below.

[0049] Formula 2

[0050]

[0051] Moreover, the aim of the present invention is to propose a conjugate comprising a modified GnRH peptide and a carrier protein, wherein the carrier protein is Bovine Serum Albumin (BSA) or wild type or recombinant diphtheria toxoid or other carriers with the feature of a primary amine of a surface exposed lysine residue.

[0052] Nevertheless, the aim of the present invention is a highly immunogenic vaccine composition comprising a conjugate of said GnRH with albumin or diphtheria toxoid (wild type or recombinant) or other carriers with the feature of a primary amine of a surface exposed lysine residue in a pharmaceutically acceptable formulation comprising DEAE-dextran or its conjugated salts or cationic polymers comprising PEI, chitosan, polypropylene imine, polylysine, polyamidoamine, polyallylamine, polydimethylammonium chloride, poly(isopropylacrylamide-co-acrylamide), co-acrylic acid, poly N-ethylvinylpiperidine bromide, polydimethylamino, polyethylene glycol, polytrimethylamino methacrylate ethyl ester hydrochloride.

[0053] Nevertheless, the aim of the present invention is to propose a kit comprising a vaccine composition for immunocastration of mammals, in particular pigs, and instructions for its use.

[0054] Moreover, another aim of the present invention is to describe the use of a conjugate comprising a modified GnRH peptide and a carrier for the preparation of a vaccine composition for the immunocastration of mammals, in particular for the immunocastration of pigs.

[0055] Moreover, the aim of the present invention is to propose a method for the preparation of a modified GnRH peptide antigen conjugated to a carrier protein, as described in the following steps.

[0056] GnRH modification

[0057] First, the structure of the GnRH peptide was modified. Initially, the GnRH molecule consists of a decapeptide containing a C-terminal (carboxyl terminal) and N-terminal (amino terminal) as follows: pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2. In the present invention, the decapeptide is converted into a 9 amino acid peptide containing a modified end, where the N-terminal is chloroacetyl and the C-terminal is carbamoyl instead of carboxamide, conjugated with albumin (BSA) or diphtheria toxoid in a pharmaceutically acceptable formulation containing DEAE-dextran or its conjugated salt or cationic polymers containing PEI, chitosan, polypropylene imine, polylysine, polyamidoamine, polyallylamine, polydialdehyde-based dimethylammonium chloride, poly(isopropylacrylamide-co-acrylamide), co-acrylic acid, poly-N-ethylvinylpyridine bromide, polydimethylaminoethyl methacrylate, polyethylene glycol and polytrimethylaminoethyl methacrylate hydrochloride. Thus, the modified GnRH is prepared by a chemical process (chemical acetylation) without the need to establish industrial production facilities for the production of recombinant vaccines, thus reducing the biosafety level requirements.

[0058] The method of preparation of the synthetic modified GnRH antigen conjugated by chemical acetylation comprises the following steps (Figure 1 and Figure 2 ):

[0059] a) functionalization of the carrier protein, which can be selected from bovine albumin, wild type or recombinant diphtheria toxoid or other carriers with primary amine features from surface-exposed lysine residues, using the SATA reagent (N-succinimidyl S-acetylthioacetate) in a reaction solution and EDTA Figure 1A );

[0060] b) incubation of the reaction for at least 2 hours under stirring at room temperature (20-30 °C), in which the SATA reagent binds to the primary amines of the carrier protein. SATA adds a thiol group to the primary amines of the protein by nucleophilic attack, abandoning the succinimide group Figure 1A and 1B );

[0061] c) performing a diafiltration process to eliminate free SATA reagent that did not functionalize the carrier;

[0062] d) conjugation of the modified GnRH peptide with the SATA functionalized carrier in the presence of a deacetylation solution, in which the conjugation reaction of the chloroacetyl of the SATA with the lysines of the carrier protein occurs. The conjugation occurs due to the nucleophilic attack of the thiol group released by the peptide to the hydroxylamine present in the deacetylation solution. In the chemical reaction, the peptide is modified, in which the final antigen obtained consists of several peptides bound to the carrier protein;

[0063] e) Incubate the reaction for at least 12 hours at a temperature of 20 to 30 °C under stirring;

[0064] f) After conjugation, add a reaction stop solution;

[0065] g) Diafiltration to eliminate GnRH peptides not conjugated to the carrier protein;

[0066] h) Sterilize the conjugated antigen by end filtration with a 0.22 pm filter to collect the sterile antigen solution in a sterile disposable bag; and

[0067] i) Collect the sample for quality analysis.

[0068] After approval of the final antigen quality by biological, physicochemical and microbiological methods (sterility) (Figure 4 shows the results of the SDS-Page and Western Blotting methods). From Figure 4A and 4B it can be observed that the labeling of the anti-GnrH antibodies occurs in the antigens (GnrH peptides and their conjugates) which are modified in the C-terminal end with carbamides and the presence of urea ( Figure 4A - column 3) and Figure 4B - columns 4 and 5). These results indicate that both the modification of the peptide with carbamides and the use of urea contribute to the binding process, the recognition of the antigen by the antibody and the stability of the antigen. Surprisingly, the use of urea plays a key role in the conjugation reaction of the peptide to the carrier, the stability of the conjugate and the exposure of the conjugated peptide to the recognition by the antibody. The ability of urea to form weak bonds and to uncoil proteins contributes to increase the exposure of the primary amines of the carrier lysines functionalized with SATA to the conjugation of the peptide and to the exposure of the conjugated peptide to each carrier molecule. The final antigen is prepared with a pharmaceutically acceptable formulation comprising DEAE-dextran or its conjugate salt or a cationic polymer comprising PEI, chitosan, polypropylene imine, polylysine, polyamidoamine, polyallylamine, polydiallyldimethylammonium chloride, poly(isopropylacrylamide-co-acrylamide), co-acrylic acid, poly-N-ethylvinylpiperidine bromide, polydimethylamino, methacrylic acid ethyl ester, polyethylene glycol, polytrimethylamino methacrylic acid ethyl ester hydrochloride. Thus, a modified GnRH with a molecular weight of approximately 1145.9-1147.9 Da is obtained, commercially and chemically known as N-chloroacetyl-GnRF (2-10) conjugated to a carrier protein.

[0069] With regard to the steps of the preparation method of the above-mentioned synthetic modified GnRH antigen, further technical details are as follows:

[0070] Step 1, functionalization of the carrier using SATA (SATA carrier) (Figure 1A )

[0071] a) adding carrier protein (bovine albumin or wild type or recombinant diphtheria toxoid) at a solubilization concentration of 5-10 mg / ml in the reaction solution;

[0072] b) adding 10-100 mM EDTA solution, stirring at least 50 rpm;

[0073] c) waiting at least 10 minutes and checking the pH value. If necessary, adjust the pH value between 8.0 and 9.0 with NaOH solution (1 M);

[0074] d) adding SATA reagent solubilized with DMSO (0.1-2% w / v);

[0075] e) waiting at least 10 minutes and checking the pH value. If necessary, adjust the pH value between 8.0 and 9.0 with NaOH solution (1 M);

[0076] f) at the end of the addition of components and pH adjustment, complete the final volume with the reaction solution, as follows:

[0077] Formula 3

[0078]

[0079] g) incubate the reaction under stirring, at least 50 rpm, and at room temperature (20-30 °C) for at least 2 hours;

[0080] h) clarify the SATA carrier solution of step 1 in a 1.0 pm maximum porosity PVDF filter;

[0081] i) diafiltrate the concentrated SATA carrier solution from step 1 using a continuous feedback system with 5-10 volumes of diafiltration solution from step 1, i.e. keeping the diafiltration volume 1 constant in which the sample is washed;

[0082] j) complete the SATA carrier solution of step 1 to the final volume with the diafiltrate of step 1;

[0083] k) measure the concentration of the SATA carrier in the spectrophotometer at a wavelength of 280 nm;

[0084] l) continue the calculation to evaluate the concentration of the SATA carrier in millimoles (mM) and mg / mL and record the results;

[0085] m) Before starting the second step of the reaction, the rate of protected groups, i.e. the average number of SATA incorporated in the carrier, is calculated. This measurement is performed by the Ellman's reaction, which is a colorimetric reaction in which Ellman's reagent changes color in the presence of free sulfhydryl groups and is absorbed at a wavelength of 412 nm;

[0086] n) The absorbance is measured in a spectrophotometer at a wavelength of 412 nm;

[0087] o) The rate of each carrier molecule and thiol in the following formula is calculated:

[0088] Formula 4

[0089]

[0090] Step 2, conjugation of SATA-carrier with chloroacetyl peptide-GnRH (deprotection reaction of SATA groups: Figure 1B )

[0091] a) To the flask containing the solubilizing peptide, 35-40% of the diafiltration 1 is added;

[0092] b) The stirring system is driven at least 50 rpm;

[0093] c) To the vial containing the solubilizing peptide, the SATA carrier solution of step 1 (final product of step 1) is added;

[0094] d) To the vial, 5-20% of the deacetylation solution is added;

[0095] e) The vial is closed and the reaction solution is maintained from step 2 at room temperature (20-30 °C) under stirring at least 50 rpm for at least 12 hours;

[0096] f) After the incubation time, 0.1-1% of the reaction stop solution (iodoacetic acid - 30 mg / mL) is added to the reactor, incubated for at least 2 hours and stirred under agitation at least 50 rpm;

[0097] g) The solution is clarified on a 0.5-1.0 pm porosity filter;

[0098] h) The GnRH carrier solution in step 2 is diafiltered by a continuous feedback system using a total volume of 5-10 volumes of diafiltration from step 2, i.e. maintaining the initial volume of the GnRH carrier solution in step 2;

[0099] i) At the end of the process, the final volume of the GnRH carrier solution of step 2 with diafiltration is completed, step 2;

[0100] j) In a biological safety cabinet, filter the GnRH carrier solution of step 2 on a 0.22 μιη filter.

[0101] The vaccine composition of the present application, whose active principle is the modified GnRH peptide, also comprises a pharmaceutically acceptable carrier or carrier selected from the group consisting of wild-type or recombinant diphtheria albumin or toxoid or other carriers characterized by a primary amine feature with surface-exposed lysine residues.

[0102] Preclinical studies

[0103] In vivo tests

[0104] Starting from the construction of the modified GnRH peptide, a vaccine for the immunocastration of pigs was developed, whose safety and effectiveness tests included evaluations such as pre-slaughter assembly behavior, analytical determinations (quantification of testosterone, androstenedione, skatole and anti-GnRH), sensory evaluation (cooking test of meat and fat). Such evaluations were carried out in a comparative manner to demonstrate the performance of wild-type, recombinant and BSA diphtheria toxoid (DT) based formulations as carriers.

[0105] Table 1 below lists the groups of animals tested by administering the vaccine composition of the present application, comprising a modified GnRH conjugate and a pharmaceutically acceptable carrier or carrier, and the composition Vivax® available on the domestic market, under the trade name.

[0106] Table 1: Preclinical study groups, where group 1 refers to the group inoculated with Vivax® and group 2 refers to the group inoculated with the vaccine composition of the present application.

[0107]

[0108] Vaccination protocol

[0109] For the in vivo preclinical study, healthy pigs of approximately 128 days were used, which were inoculated on day D0 (first inoculation) and on day D28 (second inoculation). The study was carried out with 8 repetitions, with 3 animals per column, totaling 9 groups (n = 24). On day D51, the animals were slaughtered according to the recommended ethical procedures (D0 = day 0 - first inoculation and D28 = second inoculation). Figure 3

[0110] Thus, based on the vaccination protocol carried out on groups 1 and 2, results were obtained regarding the technical performance of the animals, carcass type and safety, which are presented and described below.

[0111] Table 2: This table shows the quantification of 3 performance parameters: daily weight gain - DWG (kg), daily feed intake - DFI (kg) and feed conversion - FC, comparing the groups treated with the Vivax® vaccine and the vaccine composition of the present application.​

[0112]

[0113] Table 3: This table presents the quantification of 7 performance parameters: live weight at slaughter (kg) [at 179 days of age], carcass weight / dressed (kg), dressing yield (%), lard thickness (mm), loin depth (mm), lean meat (%), and lean meat (kg), comparing the groups treated with the Vivax® vaccine and the vaccine composition of the application.

[0114]

[0115] Table 4: This table shows the serological results of the quantification of hormones in fat androstenone (pg / g), skatole (pg / g) and testosterone in serum (ng / mL), comparing the groups treated with the Vivax® vaccine and the vaccine composition of the application.

[0116]

[0117] Table 5: Risk reference table related to the content of androstenone / fat (pg / g) and skatole (pg / g) in fat to interpret the data in Table 4.

[0118]

[0119] Results

[0120] The effectiveness and safety tests of the target species were carried out in groups as described below:

[0121] - G1 : Negative (placebo)

[0122] - G2: Bovine serum albumin carrier of GnRH peptide with carboxyl at the C-terminus

[0123] - G3: Wild type DT carrier 2, GnRH peptide with carboxyl at the C-terminus

[0124] - G4: Recombinant DT charger containing GnRH peptide (CRM product 197 - purchased from the company FinaBio Biosolutions LLC), with carboxyl at the C-terminus

[0125] - G5: BSA carrier of GnRH peptide with aminourea modification at the C-terminus

[0126] - G6: Wild type DT carrier 1, modified GnRH peptide with carbamoyl at the C-terminus

[0127] - G7: recombinant DT vector containing a modified GnRH peptide (CRM product 197 - purchased from FinaBio Biosolutions LLC), with a modified GnRH peptide and containing a carbamoyl group at the C-terminal end.

[0128] - G8: wild type DT vector 2 with a GnRH peptide with a carbamoyl modification at the C-terminal end.

[0129] - G9: Vivax®

[0130] Table 6 shows the comparison according to the treatment with respect to the daily feed intake (DFI), the feed conversion (FC) and the daily weight gain (DWG).

[0131] Table 6: Average daily weight gain (DWG), daily feed intake (DFI) and feed conversion (FC) according to the treatment. Different letters in the column a、b、c indicate differences in the Tukey test (p < 0.05).

[0132]

[0133] Table 7 shows the carcass classification parameters related to the percentage of lean meat, the lard thickness (mm) and the loin depth (mm) compared to the treatment carried out.

[0134] Table 7: Average values of the carcass classification parameters according to the treatment.

[0135]

[0136] Table 8 below shows a compilation of data on testicular weight (TW), body weight at slaughter, daily weight gain (DWG), feed conversion (FC), percentage of lean meat, lard thickness measurement, loin thickness measurement, androstenone dose, skatole dose, number of animals with mating behavior before slaughter, testosterone dose and anti-GnRH titers (GMT) before slaughter.

[0137] Table 8: The average values are provided in the table. Different letters in the line a、b、cDifferences expressed as Tukey's test (p < 0.05). TW: testis weight (Kg); slaughter weight: average body weight at slaughter (Kg); DWG: daily weight gain (Kg), FC: feed conversion; % lean; lard thickness (mm); loin: loin thickness (mm); androstane: androstanedione dose in fat (pg / g); coprostanol: coprostanol dose in fat (pg / g), mating: number of animals with mating behavior before slaughter; testosterone: serum testosterone dose (ng / mL), title: anti-GnRH dose (GMT) before slaughter.

[0138]

[0139] The results show that the group with the GnRH peptide with a carbamoyl modification in the C-terminal is the one that works best, presenting results numerically similar to the G9 group of the Vivax® vaccine (Tables 6. Animal technical performance and 7. Carcass classification) or superior results (Table 8. Mounting behavior, testosterone and titer).

[0140] In summary, the results provided demonstrate that the vaccine formulation of the present application is safe, effective and has similar performance in animals when using BSA or recombinant DT or wild-type DT as a carrier, compared to the Vivax® vaccine available in the domestic market.

Claims

1. A method of preparing conjugated modified GnRH peptide antigen comprising the steps of: a) functionalizing a carrier protein with SATA (N-succinimidyl S-acetylthioacetate) in the presence of a reaction solution and EDTA; b) incubating the reaction for at least 2 hours at a temperature of 20 to 30 °C under stirring; c) diafiltrating the reaction; d) conjugating the modified GnRH peptide with the SATA functionalized carrier protein in the presence of a deacetylation solution and urea; e) incubating the reaction for at least 12 hours at a temperature of 20 to 30 °C under stirring; f) adding a reaction termination solution after conjugation; g) diafiltering the reaction; and h) sterilizing the modified GnRH antigen by end filtration with a 0.22 μm filter.

2. The method of claim 1, wherein the carrier protein is bovine serum albumin (BSA), natural or recombinant diphtheria toxoid, or other carriers with primary amine character on surface exposed lysine residues.

3. The method of claim 1, wherein in step d) urea is used as a solubilizing agent and a presentation / exposure aid of the conjugated peptide on the antigen.

4. A modified GnRH peptide consisting of a sequence of nine amino acid residues, and being chloroacetylated at the N-terminus and carbamylated at the C-terminus, such as chloroacetyl-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (SEQ ID NO.: 1).

5. The modified GnRH peptide of claim 4, which is prepared by the chemical acetylation process of claim 1.

6. A conjugate comprising the modified GnRH peptide of claim 4 or 5 and a carrier protein.

7. The conjugate of claim 6, wherein the carrier protein is bovine serum albumin (BSA), natural or recombinant diphtheria toxoid, or other carriers with primary amine character on surface exposed lysine residues.

8. A vaccine composition comprising the modified GnRH conjugate of claim 6 or 7, and being in a pharmaceutically acceptable formulation containing DEAE-dextran or its conjugated salt, or a component containing one of the following cationic polymers: polyethylenimine (PEI), chitosan, polypropylenimine, polylysine, polyamidoamine, polyallylamine, polydiallyldimethylammonium chloride, poly(isopropylacrylamide-co-acrylamide), acrylic acid copolymer, poly(N- ethylvinylpiperidine bromide), poly(2-dimethylaminoethyl methacrylate), polyethylene glycol, polytrimethylaminoethyl methacrylate hydrochloride.

9. The composition of claim 8, comprising DEAE-dextran hydrochloride present at a concentration of 5 - 20% (preferably about 15%).

10. A kit comprising the vaccine composition of claim 8 or 9 and instructions for its use. ​ 11. Conjugate comprising a modified GnRH peptide according to claim 6 or 7 for the manufacture of a composition according to claim 8 or 9 for use in immunocastration of a mammal.

12. Use of a modified GnRH peptide according to claim 11, wherein the mammal is preferably a pig.

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