MXPA99005693A - Vaccine for the reversible immunocastration of mammals - Google Patents

Abstract

The invention relates to the field of genetic engineering and biotechnology and particularly to the use of a peptide derived from GnRH for the immunocastration of mammals. Vaccinations tests have been carried out in mammals with the gonadotropine release hormone (GnRH), in order to ellicit and immune response which neutralizes the activity of said hormone. The present invention relates to the use of a GnRH variant of mammals wherein a glycin residue has been substituted by a prolin residue in position 6:Glu-His-Trp-Ser-Tyr-Pro-Leu-Arg-Pro-Gly. This substitution or change has induced an immune response which is superior than that of natural GnRH, when both are coupled to a same carrier protein, in experiments carried out in pigs. This same result can be expected in any other mammal since this hormone is present in all species.

Description

PREPARED VACCINATION FOR THE IMMUNE-REVERSIBLE CASTRATION OF MAMMALS TECHNICAL SECTOR The present invention is related to the field of Genetic Engineering and Biotechnology and in particular to the use of a peptide derived from GnRH for mammalian immunocastration.

ANTERIOF TECHNIQUE In domestic animals, such as cats, pigs and cattle, the advisability of preventing repr ction is frequent. The most frequently used methods are castration and the administration of steroids. Both methods have drawbacks, the first requires specialized personnel to be carried out and is irreversible and the second causes side effects. For these reasons, the immuno-castration of animals has been tested with the use of peptides analogous to GnRH conjugated with proteins, with the objective of raising antibodies that neutralize the function of GnRH.

DISCLOSURE OF THE INVENTION The amino acid sequence of GnRH from a large number of species is known. All mammalian sequences reported to date represent the following amino acid sequence: PGlu-His-Trp-Ser-Tyr-Gly-LeuArg-Pro-Gly. This is why it is referred to in the literature as mammalian GnRH. In most known sequences including sequences of birds, fish and reptiles, in position 6 = e they find an amino acid residue glycine. Glycine is the smallest of DS amino acids and causes flexibility in the polypeptide chain. The substitution of glycine for proline, a residue that confers rigidity, can influence the increase of the immune response. Furthermore, this change would cause this peptide to be foreign to the immune system, stimulating the production of antibodies. The production of antibody against GuRH is achieved by conjugating GnRH to an appropriate carrier protein. This conjugation can be done by chemical or genetic engineering methods. In our cases we have selected for conjugation two carrier proteins widely used for these purposes, the BSA protein and an epitope of tetanus toxoid. The BSA is conjugated by a known chemical method, the tetanus toxoid epitope and the GnRH variants are synthesized in a single peptide using two glycine residues as spacers. In this case chemical synthesis is used because the size of the peptide allows it, in case the carrier protein was large, the most feasible way would be Genetic Engineering, inserting variants of GnRH part of its amino acid sequence. This method is possible to do practically with any protein, hence it is necessary that the present invention be protected against any protein that may include the peptide sequence in question, whether obtained by chemical synthesis or by genetic manipulation.

NOVELTY AND ADVANTAGES OF THE PROPOSED SOLUTION Previously c? Warm in position 6 of the GnRh introducing D-amino acids and compounds,: > s analogues to amino acids (Rivier et al., U.S. Patent 4,215,038, Vale, Jr. and others, US Patent 4,410,514). The novelty of this invention consists of the substitution of the amino acid glycine in the sixth position of GnRH by an L-amino acid proline, that is an amino acid that is naturally found in proteins. This variant has been shown to be more effective than natural GnRH when both are conjugated to the same protein. The fact of introducing an amino acid that occurs naturally also gives us the possibility of including this variant in genetic constructions, something impossible in variants that include D-amino acids or analogous compounds.

EXAMPLES OF REALIZATION EXAMPLE 1 Synthesis of the peptides: All the peptides were synthesized using the Boc- strategy in solid phase on the 4-methyl-benzhydrylamine resin (MBHA resin from the commercial house BACHEM, Switzerland). The protected amino acids were supplied by BACHEM. The protection of the reactive groups of the side chains of the amino acids was as follows: Arg (Cough), Asp (OBzl), Cys (4-MeBzl), Glu (OBzl), Lys (2-CI-Z), Trp (CHO), Tyr (Cl2-Bzl), Thr (Bzl) and Ser (Bzl). The Asn, the Gln and the Pro were used without protection in the side chains. The synthesis was carried out following the steps listed below: Step times x min Washes with dichloromethane 1 x 1 Deprotection of the Boc group of 1 x 30 amino terminal Washes with dichloromethane 2 x 1 Washes with 2-propanol 2 x 1 Washes with dichloromethane 2 x 1 Neutralization of the amines groups 3 x 2 Washes with dichloromethane 2 x 1 Coupling reaction 1 x 60 Washes with N, N-dimethylformamide 2 x 1 Washes with dichloromethane 1 x 1 The steps are repeated until completing the sequence Elimination of the proteins of the side chains and recovery of the peptide Deprotection of the Boc protecting group was carried out in all cases with 37.5% trifluoroacetic acid (TFA) in dichloromethane (DCM). The neutralization of the free amines was carried out with 5% N, N-ethyldiisopropylamine in DCM. For the residue chain coupling reaction, the activation method with N, N'-diisopropylcarbodiimide (DIC) in situ was used, except for the amino acids Asn and Gln that were activated with DIC and 1-hydroxybenzotriazole (HOBt) in N, N-dimethylformamide (DMF). The elimination of the protections of the side chains and the release of the peptide from the resin were carried out in special equipment for this purpose. The procedure used was known as "Low-High" HF. In the first part of the procedure (Low HF), the protected peptide-resin system was treated with the mixture HF (25%): DMS (65%): p-Cresol (10%) for 120 min. at 0 ° C. In the case of the peptides with Trp in the sequence, the mixture was replaced by HF (25%): DMS (60%): EDT (10%): p-Cresol (5%). After this treatment the peptide-resin was washed with diethyl ether, dichloromethane and 2-propanol several times and dried under vacuum. In the second part (High HF), the peptide-resin was treated with the mixture HF (90%): Anisol (10%) for 60 min. at 0 ° C. The crude product was washed with ether, extracted with a 30% acetic acid solution in water and lyophilized. The peptides were finally characterized by RP-HPLC on a VYDAC C-18 (4.6 x 100) mm column and by mass spectrometry using the FAB as the ionization method in a JEOL HX-110HF. The purification of the peptides was carried out by the same procedure but on a preparative VYDAC C-18 column. The peptides synthesized were: a) QHWSYGLRPG, identified as GnRH sequence corresponding to the gonadotropin releasing hormone, which is structurally conserved in several mammalian species including the pig. b) QHWSYGLRPGGGQYIKANSKFIGITEL, identified as GnRH-TT, where the gonadotropin-releasing hormone is conjugated at the N-terminus to an epitope of tetanus toxin (residues 830-844) (Panina-Bordingnon et al., 1989) using two glycines as separators c) QHWSYPLRPG, a new GnRH analog peptide, identified as GnRHml, where GnRH has a change in the number 6 position of the amino acid glycine by a proline. d) QHWSYPLRPGGGQYIKANSKFIGITEL, identified as GnRHm1-TT. In this case the GnRHml analogue peptide is conjugated at the N-terminus to the same epitope and in the same way as the peptide GnRH-TT. Figure 2 shows the chromatograms of the GnRH peptides (figure 2a), GnRHml (figure 2b), GnRHTT (figure 2c) and GnRHmITT (figure 2d).

PREPARATION OF THE CONJUGATES The conjugation of the peptides to BSA was carried out by the following protocol: weighing the same amount of BSA (bovine serum albumin, Sigma) as of peptide (3 mg). Dissolve the BSA in 0.5 mL of 0.1 M Na2CO3 in test tube. Dissolve apart the 3 mg of peptide in 0.5 mL of 0.1 M Na2C03 in test tube.

Add with stirring slowly and little by little 3 μL of Giutaraldehyde at 25% (grade I, Sigma) to the resuspended BSA and subsequently the peptide resuspended drop by drop. Take up to 1.5 mL with 0.1 M Na2CO3. - Mix with slow stirring and protected from light during 12 hours at 25 ° C. Dialyse against PBS 1X for 12 hours at 4 ° C. Bring the volume up to 3 mL with 1X PBS. Distribute 1 mL (1 mg of conjugated peptide) for each 5 mL syringe. Add 1 mL of Freund's Complete Adjuvant (Sigma) in the first immunization and 1 mL of Incomplete Adjuvant for the second. Mix with agitation for 30 minutes at room temperature. - Store the immunogen at 4 ° C until used.

EXAMPLE 2 Formulation of the vaccine and vaccination For the preparation of the vaccines, 1 mg of peptide resuspended in 1 mL of 1X PBS and emulsified with 1 mL of the Freund Complete Adjuvant (CFA) was used in each case. One mg of the peptides GnRH-TT and GnRHml -TT equals 0.4 mg of GnRH and GnRHml respectively. The stable water-in-oil emulsion was achieved by agitation with the use of an impeller stirrer. In all cases immunogens were prepared just before vaccination. The second vaccination performed 8 weeks after the first one had the same composition but this time emulsified with Adjuvante Incomplete. Animals: a total of fifteen hybrid male pigs from different baits were randomly assigned to five groups of three animals each for the treatment described below. Vaccination: the five groups were vaccinated the first time between 11 and 12 weeks of age and received a revaccination 8 weeks later. On both occasions each animal was injected intramuscularly in the neck with 2 mL of vaccine. All animals were sacrificed 8 weeks after the second immunization. The first group of animals was only vaccinated with 1X PBS and CFA and used as negative control of the experiment. A second and third group were vaccinated with GnRH-BSA and with GnRH-TT respectively and were used as positive controls considering that GnRH is the endogenous hormone. A fourth and a fifth group were vaccinated with the new peptide GnRHml conjugated to BSA (GnRHml -BSA) and to the epitope of the tetanus toxin (GnRHml -TT) respectively. The vaccination scheme lasted four months (January-April 1996).

EXAMPLE 3 Evaluations carried out during the vaccination schedule Starting from day 0 and every 4 weeks until moments before the sacrifice, the testicles were measured and the weight of each animal was verified. At the same time serum samples were taken by retro-orbital sinus puncture for the determination of the anti-GNRH testes. In addition, in week 16, the development of the primary reflexes of copulation was checked over three days: approaching, mounting, erection and ejaculation. The stimulation of the boars was carried out, in some cases with a sow in natural estrus and in others with a sow to which estrus was induced with serum gonadotropin. a) Table 1. Measurement of the testicles: nd- not determined by inability to locate the testicle. D e l - right and left testicles respectively. b) Table 2. Body weight of pigs to nd- not determined. c) Table 3. Increase of the body weight of each group during the time of the experiment. d) Table 4. Results of the verification of the development of the primary reflexes of copulation in pigs immunized. a) reflex of approach: - B (good), - B (*), means aggressive approach, - R (regular), M (bad, means that there was no approach, b) reflection of riding: - b (good), means good reflection, - R (regular), means that the attempt to ride was poor, - M (bad), 'means that there was no ride.

EXAMPLE 4 Evaluations carried out after the sacrifice. After sacrifice, the testicles excised and separated from the epididymides were measured and weighed, while the epididymides were only heavy. Two samples of testicular and epididymal tissue were fixed for histology. After having fixed the middle part of each transversal and sagittal section of these tissues, we proceeded to the inclusion in paraffin and the sectioning of 5 μm thick sections. The staining was performed by the hematoxylin and eosin method. a) Table 5. Measurement of the testicles and weighing of the epididymides and testes.

From the results of the previous table we can calculate the weight ratio of the testicles / weight of the animal for each testicle, obtaining the following values and their means.

TABLE 6 Ratio testicle weight / animal weight When calculating the probabilities associated with the Student test for each pair of group of values, we obtain the following probability matrix: TABLE 7 Probabilities associated with the Student test for the values in Table 6 Note: there is a significant difference when the value of the associated probability is less than 0.05. Noting that there are no statistically significant differences between the placebo group and the two variants of GnRH conjugated to the BSA. On the contrary, between any of these three groups and the groups conjugated to the epitope of the tetanus toxin if there are statistically significant differences. And when comparing these last two groups, we also find significant differences, with the average of the GnRHml variant being the smallest among all the groups and therefore the one that has the greatest effect in reducing the size of the testicles. We do the same for the epididymis, obtaining: TABLE 8 Ratio weights of the epididymis / weight of the animal As in the case of the testicles, we obtain the following probability matrix: TABLE 9 Probabilities associated with the Student test for the values in Table 8 In the case of the epididymis, the same result is repeated as with the testicles. There are no statistically significant differences between the placebo groups and the BSA conjugated groups, but if between these and the two groups conjugated to the tetanus toxin peptide and the two groups associated with the tetanus toxin, there are significant differences between them (see figure). 2), which implies that the glycine residue change. As can be seen, the results obtained with the GnRH and GnRHml peptides conjugated to BSA are very similar to those obtained for the negative control group or placebo. This could be explained taking into account that the homology between bo albumin and porcine is very high (80%), so it would be recognized as proper by pigs. When comparing the GnRHml -TT group with the placebo a reduction in the weight of the epididymis is observed 2.3 times, a reduction of 5.0 times in the weight of the testicles, and a total reduction of 3.8 times. When comparing the results of the GnRH-TT group with placebo, a reduction in the weight of the epididymis was observed 1.6 times, a reduction of 2.8 times in the weight of the testicles, and a total reduction of 2.4 times. By way of conclusion we can say that we are in the presence of a new peptide analog of GnRH which turns out to be between 1.4 and 1.8 times more effective than the endogenous hormone itself being both conjugated to the universal epitope of the tetanus toxin.

DESCRIPTION OF THE FIGURES Figure 1: testicles from the placebo groups (left), GnRH-TT (center) and GnRHml -TT (right). The effect produced in the two groups conjugated to the tetanus toxin is evident. Between these two groups one can also observe a greater effect of the GnRHml -TT variant. Figure 2. Chromatograms of the peptides a) GnRH, b) GnRHml, c) GnRHTT and d) GnRHmITT. In all cases, a reverse phase columan C18 VYDAC (4.6 x 100 mm) with a gradient of 5-60% acetonitrile (0.05% TFA) in water (0.1% TFA) was used in 35 min. The peptides were detected at a wavelength of 226 nm.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: CENTER OF GENETIC ENGINEERING AND BIOTECHNOLOGY (B) ADDRESS: Ave. 31 between 158 and 190, Beach (C) CITY: City of Havana (D) PROE: City of Havana (E) COUNTRY: Cuba (F) ZIP CODE (ZIP): 10600 ( G) TELEPHONE: 53 7 218466 (H) TELEFAX: 53 7 218070/336008 (¡i) TITLE OF THE INVENTION: PREPARED VACCINATION FOR THE IMMUNE-REVERSIBLE CASTRATION OF MAMMALS, (iii) NUMBER OF SEQUENCES: 1 (iv) FORM OF COMPUTED READING: (A) TYPE OF MEDIUM: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (EPO) ( vi) PRIORITY APPLICATION DATA: (A) APPLICATION NUMBER: CU 120/96 (B) APPLICATION DATE: DEC 17, 1996 (2) INFORMATION FOR SEQ ID NO. 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETIC: NO ( iv) ANIT-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Mutant gonadotropin-containing hormone. (C) ISOLATION: Mammals (xi) DESCRIPTION OF SEQ ID NO. 1: pGlu His Trp Ser Tyr Pro Leu Arg Pro Gly 1 5 10

Claims (2)

NOVELTY OF THE INVENTION CLAIMS

1. - A vaccine preparation for immunocastration of mammals, consisting of the peptide: pGlu-His-Trp-Ser-Tyr-Pro-Leu-Arg-Pro-Gly in combination with a carrier protein.

2. A vaccine preparation according to claim 1, further characterized in that the carrier protein is the Th epitope of tetanus toxoid (residues 830-844).