A GnRH polypeptide antigen, a pharmaceutical composition containing the GnRH polypeptide antigen, and its application.

Abstract

This invention provides a GnRH polypeptide antigen, wherein the amino acid sequence structure of the GnRH polypeptide antigen is Th+(J+GnRH)n or Th+(J+Th)n+(J+GnRH)n, where n is 1, 2, or 3; and the amino acid sequence of J is selected from -AA-, -GG-, -AG-, -GK-, or -EE-. This invention also provides the application of the above-mentioned GnRH polypeptide antigen in the preparation of animal sexual characteristic control drugs, and a pharmaceutical composition containing the above-mentioned GnRH polypeptide antigen. Experimental results show that the GnRH antigen polypeptide pharmaceutical composition of this invention can effectively inhibit the secretion of sex hormones, induce a reduction in testicular volume, achieve the effect of controlling animal sexual characteristics and reproductive capacity, and thus effectively control sex hormone-related indecent behavior in animals, while possessing good safety. The vaccine of this invention provides an effective solution for sexual characteristic regulation in the veterinary field.

Description

Technical Field

[0001] This invention relates to GnRH polypeptide antigens, particularly to a novel GnRH polypeptide antigen, and also to pharmaceutical compositions containing said antigen, and the use of said pharmaceutical compositions in the preparation of medicaments for controlling sexual dysfunction. Background Technology

[0002] GnRH (gonadotropin-releasing hormone, also known as luteinizing hormone-releasing hormone, LHRH) is a short peptide composed of ten amino acids secreted by the hypothalamus. GnRH acts on the anterior pituitary gland, promoting the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In male animals, luteinizing hormone reaches the testes via the bloodstream, stimulating the interstitial cells of the testes to produce testosterone. The main physiological functions of testosterone include stimulating and maintaining the normal development and growth of male reproductive organs (such as testicular tissue), promoting the appearance of secondary sexual characteristics, and maintaining the spermatogenic capacity of the testes. Therefore, GnRH is a key signaling molecule regulating the hypothalamic-pituitary-gonadal axis (HPG axis).

[0003] After reaching sexual maturity, male dogs and cats exhibit increased sex hormone secretion and estrus, leading to inappropriate behaviors. Immunocastration is a common method for controlling male sexual characteristics in dogs and cats. Immunocastration or sterilization is based on vaccines developed using GnRH peptides. For example, GnRH-based vaccines have been tested to lower testosterone levels in animals, thereby suppressing testicular function (Talwar et al., 2009). However, due to the natural tolerance of the immune system to self-antigens such as GnRH, an effective and durable immune response is often difficult to achieve.

[0004] Among the numerous contraceptive or castration vaccines developed, one type is conjugate vaccines, in which GnRH peptides bind to carrier proteins to enhance the immune response, showing potential effects in regulating male reproductive processes. Another type is nanoparticle-based GnRH vaccines, which have shown significantly improved antigen efficacy and targeted delivery. Furthermore, studies have explored subunit vaccines composed of specific fragments of GnRH peptides, revealing new possibilities and mechanisms of action in controlling reproductive function. These different types of GnRH peptide vaccines stimulate the immune system to produce antibodies against GnRH, interfering with the normal function of the hypothalamus-pituitary-gonadal axis, ultimately achieving control over reproductive activities or related behaviors.

[0005] Use of broad-spectrum Th antigenic epitopes: To overcome the challenge of insufficient immunogenicity of GnRH as a self-antigen, specific broad-spectrum Th antigenic epitope peptides are used in GnRH-based peptide vaccines. These T helper peptides are engineered to be highly immunogenic to multiple major histocompatibility complex (MHC) class II alleles, thereby promoting a robust T-cell response. By including broad-spectrum T helper peptides in vaccine formulations, the immune system can be effectively guided to recognize and generate an immune response to GnRH peptides, even as a self-antigen. This strategy significantly improves the efficacy of peptide-based vaccines in overcoming immune tolerance, particularly for veterinary applications requiring a consistent immune response.

[0006] Many research teams have conducted preliminary research on antigen modification and adjuvant screening for sterilization or castration vaccines. For example, GnRH is modified and then linked to a large molecular weight carrier protein to enhance immunogenicity. For instance, Chinese invention patent application CN 108047328 A addresses the drawbacks of large molecular weight carrier proteins, such as large steric hindrance, difficulty in quality control of chemical coupling, and significant side effects. It proposes the total synthesis of a modified GnRH compound with a Th epitope to form a fully synthetic polypeptide antigen with moderate molecular weight, significant immunogenicity, and controllable quality. Based on this, a suitable vaccine preparation method was designed according to the antigen characteristics, and the prepared vaccine produced high immunogenicity in rats. However, the inventors of this invention have verified that this animal castration vaccine does not achieve the expected results when applied to other animals such as dogs and cats.

[0007] Efficacy and safety are the most important factors limiting the development of GnRH peptide vaccines. Romagnoli S et al. (WSAVA guidelines for the control of reproduction in dogs and cats[J].The Journal of Small Animal Practice, 2024.) pointed out that the development of gonadotropin-releasing hormone (GnRH) immunocontraceptive vaccines has problems because GnRH itself is not naturally immunogenic. Vaccine formulations aim to improve the antigenicity and safety of GnRH-conjugates. Research on GnRH vaccines for dogs and cats dates back thirty years, and dozens of vaccines designed for research trials have never been commercialized. Currently, two GnRH vaccines are commercially available, but serious safety issues remain. For example, the binding of GnRH to keyhole hemocyanin (KLH) can induce local inflammatory responses and durable immune responses, as well as significant injection site reactions. In male dogs, all three vaccinated dogs experienced severe injection site reactions throughout the study period. For female dogs: 70% of vaccinated female dogs developed focal to severe diffuse chronic granulomatous myositis or diffuse coagulative necrosis at the injection site.

[0008] To improve the safety of veterinary vaccines, the use of safe and effective adjuvants is crucial for reducing adverse reactions while enhancing immune responses. Certain peptide vaccine sequences are also closely associated with adverse immune responses, such as local inflammation or autoimmune reactions, especially against self-antigens. This may limit the safety of these vaccines, necessitating the minimization of harmful side effects while enhancing immunogenicity. These risks must be carefully balanced when selecting adjuvants and designing peptide sequences.

[0009] To address these challenges, there is an urgent need for a new approach that combines the advantages of immunotherapy with targeted inhibition of androgens, while optimizing the manufacturing process and ensuring cost-effectiveness. Summary of the Invention

[0010] The purpose of this invention is to overcome the deficiencies of the prior art, provide a new GnRH polypeptide antigen, and select commercially available adjuvants to achieve a safe, efficient, and applicable drug for controlling animal sexual characteristics in dogs and cats.

[0011] The present invention is based on peptide-based vaccines that utilize synthetic peptides to mimic specific regions of male hormones or their receptors, thereby generating a selectively neutralizing immune response to the target hormone. The formulation uses specific broad-spectrum T helper peptides to overcome immune system tolerance to self-antigens such as GnRH, resulting in a more effective and durable immune response. Furthermore, safe adjuvants are screened to further enhance the efficacy and safety of the pharmaceutical composition, thus achieving a balanced and controlled immunosuppression application suitable for pets.

[0012] Based on this, the present invention provides a GnRH polypeptide antigen, wherein the amino acid sequence structure of the GnRH polypeptide antigen is Th+(J+GnRH)n or Th+(J+Th)n+(J+GnRH)n, wherein the amino acid sequence of GnRH is as shown in SEQ ID No. 28, Th is an artificial helper T cell epitope, J is a linker sequence connecting Th and GnRH, and n is 1, 2 or 3.

[0013] In a preferred embodiment, the amino acid sequence structure of the GnRH polypeptide antigen is Th+(J+GnRH)n, Th+J+Th+(J+GnRH)n, or Th+J+Th+J+Th+(J-GnRH)n.

[0014] In this invention, the amino acid sequence of the Th is selected from any of the amino acid sequences shown in SEQ ID No. 29-31. When the GnRH polypeptide antigen contains multiple Th, the multiple Th may be the same or different from each other.

[0015] Particularly preferably, Th is selected from a peptide having one of the following amino acid sequences:

[0016] Ala-Gln-Ile-Thr-Ala-Gly-Ile-Ala-Leu-His-Gln-Ser-Asn-Leu-Asn

[0017] Leu-Ser-Glu-Ile-Lys-Gly-Val-Ile-Val-His-Arg-Leu-Glu-Gly-Val

[0018] {D-Ala}-Lys-{CHA}-Val-Ala-Ala-Trp-Thr-Leu-Lys-Ala-Ala-{D-ALA}

[0019] Preferably, the amino acid sequence of J is selected from -AA-, -GG-, -AG-, -GK-, or -EE-.

[0020] In a preferred embodiment, the amino acid sequence of the GnRH polypeptide antigen is selected from any of the amino acid sequences shown in SEQ ID No. 1-27, i.e.:

[0021] SEQNo1:{D-Ala}K{CHA}VAAWTLKAA{D-Ala}GGEHWSYGLRPG

[0022] SEQNo2:LSEIKGVIVHRLEGVGGEHWSYGLRPG

[0023] SEQNo3:AQITAGIALHQSNLNGGEHWSYGLRPG

[0024] SEQNo4:AQITAGIALHQSNLNGGLSEIKGVIVHRLEGVGGEHWSYGLRPG

[0025] SEQNo5:LSEIKGVIVHRLEGVGG{D-Ala}K{CHA}VAAWTLKAA{D-Ala}GGEHWSYGLRPG

[0026] SEQNo6:{D-Ala}K{CHA}VAAWTLKAA{D-Ala}GGAQITAGIALHQSNLNGGEHWSYGLRPG

[0027] SEQNo7:AQITAGIALHQSNLNGG{D-Ala}K{CHA}VAAWTLKAA{D-Ala}GGEHWSYGLRPG

[0028] SEQNo8:D-Ala}K{CHA}VAAWTLKAA{D-Ala}GGEHWSYGLRPGGEHWSYGLRPG

[0029] SEQNo9:LSEIKGVIVHRLEGVGGAQITAGIALHQSNLNGGEHWSYGLRPG

[0030] SEQNo10:{{D-Ala}K{CHA}VAAWTLKAA{D-Ala}GGLSEIKGVIVHRLEGVGGEHWSYGLRPG

[0031] SEQNo11:LSEIKGVIVHRLEGVGGEHWSYGLRPGGGEHWSYGLRPG

[0032] SEQNo12:AQITAGIALHQSNLNGGEHWSYGLRPGGGEHWSYGLRPG

[0033] SEQNo13:{D-Ala}K{CHA}VAAWTLKAA{D-Ala}GGLSEIKGVIVHRLEGVGGEHWSYGLRPGGGEHWSYGLRPG

[0034] SEQNo14:LSEIKGVIVHRLEGVGG{D-Ala}K{CHA}VAAWTLKAA{D-Ala}GGEHWSYGLRPGGGEHWSYGLRPG

[0035] SEQNo15:{D-Ala}K{CHA}VAAWTLKAA{D-Ala}GGAQITAGIALHQSNLNGGEHWSYGLRPGGGEHWSYGLRPG

[0036] SEQNo16:AQITAGIALHQSNLNGG{D-Ala}K{CHA}VAAWTLKAA{D-Ala}GGEHWSYGLRPGGGEHWSYGLRPG

[0037] SEQNo17:

[0038] AQITAGIALHQSNLNGGLSEIKGVIVHRLEGVGGEHWSYGLRPGGGEHWSYGLRPG

[0039] SEQNo18:

[0040] LSEIKGVIVHRLEGVGGAQITAGIALHQSNLNGGEHWSYGLRPGGGEHWSYGLRPGSEQNo19:{D-Ala}K{CHA}VAAWTLKAA{D-Ala}GGEHWSYGLRPGGGEHWSYGLRPGGGEHWSYGLRPG

[0041] SEQNo20:LSEIKGVIVHRLEGVGGEHWSYGLRPGGGEHWSYGLRPGGGEHWSYGLRPG

[0042] SEQNo21:AQITAGIALHQSNLNGGEHWSYGLRPGGEHWSYGLRPGGGEHWSYGLRPG

[0043] SEQNo22:{D-Ala}K{CHA}VAAWTLKAA{D-Ala}GGLSEIKGVIVHRLEGVGGEHWSYGLRPGGGEHWSYGLRPGGGEHWSYGLRPG

[0044] SEQNo23:LSEIKGVIVHRLEGVGG{D-Ala}K{CHA}VAAWTLKAA{D-Ala}GGEHWSYGLRPGGGEHWSYGLRPGGGEHWSYGLRPG

[0045] SEQNo24:{D-Ala}K{CHA}VAAWTLKAA{D-Ala}GGAQITAGIALHQSNLNGGEHWSYGLRPGGGEHWSYGLRPGGGEHWSYGLRPG

[0046] SEQNo25:AQITAGIALHQSNLNGG{D-Ala}K{CHA}VAAWTLKAA{D-Ala}GGEHWSYGLRPGGGEHWSYGLRPGGGEHWSYGLRPG

[0047] SEQNo26:

[0048] AQITAGIALHQSNLNGGLSEIKGVIVHRLEGVGGEHWSYGLRPGGGEHWSYGLRPGGGEHWSYGLRPG

[0049] SEQNo27:

[0050] LSEIKGVIVHRLEGVGGAQITAGIALHQSNLNGGEHWSYGLRPGGEHWSYGLRPGGGEHWSYGLRPG

[0051] Wherein, {D-Ala} represents D-type alanine, i.e., dextrorotatory alanine; and {CHA} represents cyclohexylalanine.

[0052] In particular, GnRH polypeptide antigens having the amino acid sequence shown in SEQ ID No. 4, 8 or 10 have better efficacy, especially better effectiveness and safety.

[0053] The present invention also provides the application of the above-mentioned GnRH polypeptide antigen in the preparation of veterinary vaccines.

[0054] The present invention also provides a pharmaceutical composition containing the above-mentioned GnRH polypeptide antigen, wherein the pharmaceutical composition contains a pharmaceutically active adjuvant.

[0055] In this invention, the pharmaceutically active adjuvant can be an oil adjuvant, a water-in-oil-in-water adjuvant, a water-in-oil adjuvant, or an aqueous adjuvant manufactured or commercially available according to existing technology. Examples include SEPPIC 61VG oil adjuvant, SEPPIC 201VG water-in-oil-in-water adjuvant, SEPPIC 15A water-in-oil adjuvant, and SEPPIC 1313VG NST aqueous adjuvant.

[0056] As a preferred embodiment, the pharmaceutically active adjuvant is MONTANIDE ISA 61VG or MONTANIDE ISA 15A VG, particularly preferably MONTANIDE ISA 61VG.

[0057] In this invention, the amount of the pharmaceutically active adjuvant used is 10% to 70% of the total volume of the pharmaceutical composition, preferably 25% to 70%. For example, GnRH polypeptide antigen is prepared into an antigen solution with a concentration of 100 to 1000 nmol / ml, sterilized by filtration through a 0.22 μm filter, and used as the aqueous phase; a sterile adjuvant is used as the oil phase; the oil phase and aqueous phase are mixed and emulsified by a vaccine emulsifier to obtain an emulsified vaccine or an aqueous vaccine. The adjuvant used can be SEPPIC 61VG oil adjuvant, SEPPIC 201VG water-in-oil-in-water adjuvant, SEPPIC 15A water-in-oil adjuvant, and SEPPIC 1313VG NST nanoparticle aqueous adjuvant, etc.

[0058] The present invention also provides the use of the above-described pharmaceutical composition in the preparation of animal sex control vaccines.

[0059] This invention provides a GnRH polypeptide antigen and its application in the preparation of drugs for controlling animal sexual characteristics. This invention achieves total synthesis of intact, natural GnRH with potent Th epitopes, forming a polypeptide antigen with moderate molecular weight and significantly enhanced immunogenicity. Based on this, and with the addition of a validated and preferred adjuvant, a simple vaccine preparation method is formed, overcoming the problems of high antigen production costs, complex processes, excessive steric hindrance of large molecular weight carrier proteins, difficulty in quality control of chemical coupling, significant side effects, low immunogenicity, and limited adjuvant selection in existing technologies. The vaccine prepared by this invention features simple production process, low cost, safety in animal use, and good immunogenicity, and has broad application prospects in the field of animal sexual characteristic control. Detailed Implementation

[0060] The following examples are used to explain the technical solutions of the present invention in a non-limiting manner.

[0061] In this invention, unless otherwise specified, "%" used to explain concentration refers to mass percentage, and ":" refers to mass ratio.

[0062] Example 1: Preparation and Validation of GnRH Peptide Antigen

[0063] Following standard procedures, weigh the resin and add it to the peptide synthesis reactor. Add DMF to clean the resin. Vacuum dry, add an appropriate amount of DCM, stir to swell, and vacuum dry again. Weigh the amino acid to be coupled and add it to the reactor, along with DCM and DIEA. Start the stirring condensation reaction for 150 minutes. Add DCM, DIEA, and methanol to the reactor for sealing. Take a small amount of peptide resin for ninhydrin testing; if the peptide resin turns pale yellow, proceed to the next step.

[0064] Remove the reaction solution and wash 5 times with DMF. Add 20% Pip / DMF solution to the reactor for deprotection for 30 minutes. After deprotection, remove the solution and wash 7 times with DMF.

[0065] Weigh the amino acid to be coupled and add it to the reactor. Add HOBT and DIC solutions, and start the stirring condensation reaction for 150 minutes. Take a small amount of peptide resin for ninhydrin detection. If the peptide resin turns pale yellow, add DCM, DIEA, and methanol to the reactor for sealing. Remove the reaction solution and wash five times with DMF.

[0066] Repeat the above operation, performing coupling cycles sequentially according to the amino acid sequence of the polypeptide shown in SEQ ID No. 1-27, until the last amino acid is completed.

[0067] After condensation, once all amino acids at all sites are coupled in the peptide resin, deprotection is achieved by adding 20% ​​Pip / DMF solution. A small amount of the peptide resin is tested for ninhydrin; if the resin turns purple, it indicates that Fmoc has been removed. After deprotection, the solution is drained, washed with DMF, and then subjected to lipid shrinkage treatment with methanol. After shrinkage, the resin is dried, sealed in packaging, labeled, and stored at 2–8°C.

[0068] Separation of peptides from the solid support: Prepare a lysis buffer solution at a ratio of 5–10 ml per gram of peptide resin. The peptide resin lysis buffer is prepared as follows: trifluoroacetic acid (TFA) / 1,2-ethylenedithiol (EDT) / triisopropylsilane (TIS) / water = 90 / 4 / 2 / 4. Add the pre-cooled (-20°C) lysis buffer to the lysis flask, stir, and slowly add the peptide resin for lysis. After lysis for 1.5–3 hours, concentrate the lysate by distillation. Add 5 times the volume of pre-cooled (-80°C) methyl tert-butyl ether to the flask, and slowly add the concentrated distillate (peptide resin) to the methyl tert-butyl ether while stirring. After a white solid precipitates, filter and wash with methyl tert-butyl ether. Dry the crude product and store at 2–8°C.

[0069] Purification, salt transfer, and desalting of the synthetic peptide antigen: Dissolve the crude synthetic peptide in methanol with stirring to obtain a synthetic peptide solution with a concentration of 0.6–2.0 mg / mL. Purify the peptide antigen using ultrafiltration membrane or reverse-phase preparative chromatography. Alternatively, the synthetic peptide solution can be slowly passed through an ion exchange resin for purification, salt transfer, and desalting. Collect and store the eluent. Filter the eluent through a 0.22 μm filter for sterilization and then store.

[0070] Example 2: Vaccine Preparation

[0071] The polypeptide antigens P600-RH (SEQ ID No. 4), P750-RH (SEQ ID No. 8), and P800-RH (SEQ ID No. 10) obtained in Example 1 were dissolved in ultrapure water for injection to form a solution of 500 nmol / ml, and then filtered for sterilization using a 0.22-micron filter.

[0072] SEPPIC ISA 61VG, SEPPIC 201VG, SEPPIC 15A or MONTANIDE IMS1313VGNST were sterilized at 121 degrees Celsius for 30 minutes.

[0073] Using a high-speed stirring emulsifier, the high-temperature sterilized adjuvant and the filtered sterilized polypeptide antigen were emulsified under aseptic conditions according to the proportions and final concentrations recorded in Table 1-2 to prepare vaccines, which were labeled as R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, G1, G2, G3, G4, G5, G6, G7, G8 and G9, respectively.

[0074] Table 1. Rat test groups and vaccine components

[0075]

[0076]

[0077] Table 2. Beagle test groups and vaccine components

[0078]

[0079] Rat experiment: Sixty-five 11-week-old male Sprague-Dawley rats were divided into 13 groups, including 12 immunization groups and 1 control group, with 5 rats in each group. The vaccines listed in Table 1 were administered via intramuscular injection in the leg twice, with each rat receiving 0.25 ml per injection, 21 days apart. Blood samples were collected on the day of the first immunization and again two weeks after the second immunization. Serum was separated, and serum testosterone concentration was measured. When the rat's testosterone level was below 0.4 ng / mL, the animal was considered to have suppressed testosterone secretion (Suprelorin: EPAR - Summary for the public - Scientific Discussion).

[0080] (https: / / www.ema.europa.eu / en / medicines / veterinary / EPAR / suprelorin#authorisation-details-section).

[0081] Beagle Experiment: Fifty male beagles aged 8 months and older were divided into 10 groups: 9 immunization groups and 1 control group, with 5 dogs in each group. The beagles were immunized twice via subcutaneous injection in the neck using the vaccine listed in Table 2, with 0.25 ml injected each time, 28 days apart. Blood samples were collected on the day of the first immunization and in the second week after the second immunization. Serum was separated, and serum testosterone concentration was measured. Animals with testosterone levels below 0.4 ng / mL were considered to have suppressed testosterone secretion. Furthermore, testicular volume was measured on the day of the second immunization and in the third week after the second immunization. The percentage decrease in testicular volume was calculated and compared. Animals with a testicular volume decrease greater than or equal to 5% were considered to have experienced testicular atrophy.

[0082] The serum testosterone concentration was determined using the iodine-125 radioimmunoassay. According to the instructions for the iodine [125I]-testosterone radioimmunoassay kit, the procedure was as follows: Place the required reagents and serum samples at room temperature (for at least 30 minutes). Number the 60×12mm polystyrene tubes as follows: T tube, NSB tube, A-G tubes, test serum tubes (1-n), and control serum 1 and 2. Following the reagent usage and procedure table, add the following reagents to each tube sequentially: Add 100 μl of T standard A to the NSB tube (Non-Specific Binding); add 100 μl each of T standards A-G to tubes A-G respectively; add 100 μl of test serum to each of the test serum tubes (1-n) (all test samples were tested in duplicate); add 100 μl each of control serum 1 and 2 to control serum tubes 1 and 2 respectively (all control serum samples were tested in duplicate). Add 200 μl of 125I-T label to all tubes. Except for the T tubes and NSB tubes, add 200 μl of anti-T serum to each of the remaining tubes, mix thoroughly, and incubate at 37°C for 1.5 hours. Except for the T tubes, add 500 μl of separating agent to all tubes, mix thoroughly, incubate at room temperature for 5–10 minutes, then centrifuge at 3500 rpm / min for 20 minutes and discard the supernatant. Count the testosterone levels in each tube using a gamma-ray radioimmunoassay counter (CPM 1 minute). The gamma counter is connected to a computer; the computer system automatically collects data and generates a standard curve, from which the test serum and control serum testosterone concentrations are read.

[0083] The results of testosterone measurements before and after immunization in each rat group are shown in Table 3.

[0084] Table 3 Testosterone concentrations in rats before and after immunization

[0085]

[0086]

[0087] The results showed that the peptides of the present invention, whether formulated with the potent adjuvant MONTANIDE ISA 61VG or the relatively weak adjuvant MONTANIDE IMS1313VG NST, effectively induced an immune response and suppressed testosterone secretion in immunized animals after two immunizations. Vaccines formulated with peptide P800-RH and any of four different types of adjuvants suppressed testosterone secretion in 5 / 5 of rats two weeks after the second immunization. Vaccines formulated with peptide P600-RH and either W / O adjuvant (MONTANIDE ISA 61) or O / W adjuvant (MONTANIDE ISA 15A) suppressed testosterone secretion in 5 / 5 of rats after immunization. Vaccines formulated with peptide P600-RH and either W / O / W adjuvant (MONTANIDE ISA 201) or nanoparticle water adjuvant (MONTANIDE IMS1313) suppressed testosterone secretion in 4 / 5 and 3 / 5 of rats, respectively, two weeks after the second immunization. Vaccines formulated with peptide P750-RH and W / O, O / W, or W / O / W adjuvants suppressed testosterone secretion in 4 / 5 of rats in the second week after secondary immunization. Vaccines formulated with peptide P750-RH and nanoparticle water adjuvant suppressed testosterone secretion in 2 / 5 of rats within two weeks after the second immunization.

[0088] Table 4 shows the results of testosterone measurements and changes in testicular volume before and after immunization in each group of beagle dogs.

[0089] Table 4. Testosterone concentration and testicular volume in Beagle dogs before and after immunization.

[0090]

[0091]

[0092] The results of the beagle dog test showed that when the antigenic peptide of the present invention was formulated with W / O, O / W or nanoparticle type water adjuvant, it could induce a good immune response, inhibiting testosterone secretion in 2 / 5 or more of the immunized dogs, or reducing testicular volume in 3 / 5 or more of the immunized dogs, as detailed in Table 5.

[0093] Table 5 Summary of testosterone secretion and testicular volume in beagle dogs after immunization.

[0094]

[0095] When the antigenic polypeptide P600-RH of this invention is formulated with a w / o type adjuvant (MONTANIDE ISA 61VG), after two immunizations, testosterone secretion is suppressed in 5 / 5 of the beagle dogs, and testicular volume is reduced in 5 / 5 of the beagle dogs. When the antigenic polypeptide P600-RH is formulated with an w / o type adjuvant MONTANIDE ISA 15A VG or a nanoparticle type water adjuvant (MONTANIDE IMS1313), after two immunizations, testosterone secretion is suppressed in 2 / 5 and 1 / 5 of the beagle dogs, respectively, but testicular volume is reduced in 3 / 5 of the beagle dogs in both cases.

[0096] When the antigenic polypeptide P750-RH or antigenic polypeptide P800-RH of the present invention is prepared with the W / O type adjuvant MONTANIDE ISA61VG, after two immunizations, testosterone secretion is inhibited in 3 / 5 of the beagle dogs, and testicular volume is reduced in 4 / 5 and 3 / 5 of the beagle dogs, respectively.

[0097] The antigenic polypeptide P750-RH or antigenic polypeptide P800-RH, when formulated with the O / W type adjuvant MONTANIDE ISA15AVG, inhibited testosterone secretion in 2 / 5 and 1 / 5 of beagle dogs after two immunizations, and reduced testicular volume in 2 / 5 and 3 / 5 of beagle dogs, respectively.

[0098] After the antigenic polypeptide P750-RH or antigenic polypeptide P800-RH was formulated with a nanoparticle-type water adjuvant (MONTANIDEIMS1313), the testosterone secretion of 3 / 5 and 0 / 5 of the beagle dogs was inhibited after two immunizations, but the testicular volume of 3 / 5 of the beagle dogs was reduced.

[0099] These results suggest that the combination of the GnRH peptide antigen and adjuvant of the present invention may be a key factor in the effective induction of immune sterilization by the vaccine in target animals.

[0100] Regarding safety, no serious injection site reactions were observed in any of the beagle dog tests, indicating that the antigenic peptides of the present invention are safe even when combined with different types of adjuvants.

[0101] The application of the vaccine in this invention was primarily tested in rat and canine models. Results showed that the GnRH antigen peptide vaccine effectively inhibited sex hormone secretion, induced a reduction in testicular volume, and achieved the effect of controlling sexual characteristics and reproductive capacity. This effectively controlled sex hormone-related indecent behavior in animals and demonstrated good safety. Similar effects are expected in cats and other veterinary-important species. Therefore, the vaccine of this invention is applicable to a wide range of animal species and can be used to control estrus behavior and sexual maturity characteristics, providing an effective solution for sexual characteristic regulation in the veterinary field.

Claims

1. A GnRH polypeptide antigen, wherein the amino acid sequence structure of the GnRH polypeptide antigen is Th+(J+GnRH)n or Th+(J+Th)n+(J+GnRH)n, wherein the amino acid sequence of GnRH is as shown in SEQ ID No. 28, Th is an artificial helper T cell epitope, J is a linker sequence connecting Th and GnRH, and n is 1, 2 or 3; the amino acid sequence of Th is selected from any of the amino acid sequences shown in SEQ ID No. 29-31, and when the GnRH polypeptide antigen contains multiple Th, the multiple Th are the same or different from each other; the amino acid sequence of J is selected from -AA-, -GG-, -AG-, -GK- or -EE-.

2. The GnRH polypeptide antigen according to claim 1, characterized in that... The amino acid sequence of the GnRH polypeptide antigen is selected from any of the amino acid sequences shown in SEQ ID No. 1-27.

3. The GnRH polypeptide antigen according to claim 1, characterized in that... The amino acid sequence of the GnRH polypeptide antigen is selected from the amino acid sequence shown in any of SEQ ID No. 4, 8 or 10.

4. The use of the GnRH polypeptide antigen according to any one of claims 1-3 in the preparation of animal sexual characteristic control drugs.

5. A pharmaceutical composition comprising the GnRH polypeptide antigen as described in any one of claims 1-3, characterized in that... The pharmaceutical composition contains a pharmaceutically active adjuvant.

6. The pharmaceutical composition according to claim 5, characterized in that... The pharmaceutically active adjuvant is an oil adjuvant or a nanoparticle aqueous adjuvant.

7. The pharmaceutical composition according to claim 5, characterized in that... The pharmaceutically active adjuvant is an oil adjuvant, an oil-in-water adjuvant, an oil-in-water adjuvant, or an aqueous adjuvant.

8. The pharmaceutical composition according to claim 5, characterized in that... The amount of the pharmaceutically active adjuvant is 25% to 70% of the total volume of the pharmaceutical composition.

9. The use of the pharmaceutical composition of claim 5 in the preparation of a drug for controlling animal sexual characteristics.

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