GBD-SSTAD-SSTAD RECOMBINANT PROTEIN AND METHOD FOR PRODUCING AND USING SAME

Abstract

The present invention relates to genetic engineering, biotechnology, immunology, and microbiology. Recombinant GBD-SSTad-SSTad protein and a method for preparation thereof on glucan are described, including the binding of GBD-SSTad-SSTad protein in cell extracts of E. coli BL21 [pGBD-SSTad-SSTad] strain to an alpha-glycan-containing sorbent due to affinity interaction during the incubation procedure, subsequent washing from unbound bacterial proteins and isolation of the desired product. The invention also relates to an immunogenic composition and a kit comprising said peptide. The invention also relates to the use of the obtained protein to increase the number of maturing follicles and improve the quality of sperm. This technical result, an increase in the number of maturing follicles and an improvement in the quality of sperm is based on the use of the KNFFWKTFTS peptide, which is part of a recombinant protein that is capable of eliciting autoantibodies to somatostatin.

Claims

1. Recombinant GBD-SSTad-SSTad protein having a molecular weight of 39.5 kDa, comprising 2 fragments of the SST protein having the sequence SEQ ID NO 1, a spacer having the sequence SEQ ID NO 2 and a spacer having the sequence SEQ ID NO 3, alpha-glucan binding gene domain from Streptococcus mutans having the sequence SEQ ID NO 4, which protein is encoded by the nucleotide sequence of the GBD-SSTad-SSTad gene SEQ ID NO 5.

2. A method for preparation of recombinant GBD-SSTad-SSTad protein according to claim 1 on glucan, which method comprises: binding of GBD-SSTad-SSTad protein in cell extracts of E. coli BL21 [pGBD-SSTad-SSTad] strain to an alpha-glycan-containing sorbent due to affinity interaction during the incubation procedure, subsequent washing from unbound bacterial proteins and isolation of the desired product.

3. A pharmaceutical composition in the form of an injectable preparation for enhancing folliculogenesis and spermatogenesis in mammals, birds and humans, which composition comprises recombinant GBD-SSTad-SSTad protein according to claim 1 and an adjuvant suitable for injection.

4. The pharmaceutical composition according to claim 3, wherein said composition is comprised in one or more containers enclosed in a package with leaflet containing directions for use.

5. The pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is a vaccine.

6. A method of increasing folliculogenesis and spermatogenesis in mammals, birds and humans, which method comprises administering subcutaneous or intramuscular injections of a preparation comprising recombinant GBD-SSTad-SSTad protein according to claim 1, at a dose of 5-50 μg of said protein per kilogram of body weight of a mammal or bird.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0039] FIG. 1 Cysteine bridge between cysteines in the third and fourteenth positions.

[0040] FIG. 2 U-shaped structure of somatostatin.

DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention relates to a compound, comprising the antigenic determinant of somatostatin KNFFWKTFTS (SEQ ID NO: 1) as part of fusion recombinant proteins to accelerate the onset of growth of resting follicles, folliculogenesis and spermatogenesis in mammals, birds and humans for the treatment and prevention of infertility.

[0042] In the preferred embodiment, the invention relates to the compound comprising the antigenic determinant of somatostatin KNFFWKTFTS (SEQ ID NO: 1) as part of fusion recombinant proteins to accelerate the onset of growth of resting follicles, folliculogenesis and spermatogenesis in mammals, birds and humans, for the treatment and prevention of infertility, when active component thereof is administered to mammals, birds or humans.

[0043] In the more preferred embodiment, the invention relates to the compound comprising the antigenic determinant of somatostatin KNFFWKTFTS (SEQ ID NO: 1) as part of fusion recombinant proteins to accelerate the onset of growth of resting follicles, folliculogenesis and spermatogenesis in mammals, birds and humans undergoing assisted reproductive treatment for the therapy and prevention of infertility.

[0044] The present invention provides an immunogenic composition of compound, comprising the antigenic determinant of somatostatin KNFFWKTFTS (SEQ ID NO: 1) and its analogs, and methods for treatment infertility in patients (mammals, birds and humans) which require such treatment option.

[0045] In one embodiment, the invention provides novel polypeptides and polynucleotides encoding thereof, including SSTad polypeptide, fused to a carrier protein through a functionally optimized spacer. The fusion polypeptides of the invention provide highly effective and inexpensive materials for use in the treatment of infertility. A special role in this invention, not disclosed in the above-mentioned analogs, is played by a carrier protein (GBD), which performs a number of functions: a carrier protein for protecting low molecular weight SSTad from proteases, an affinity domain for purifying a fusion protein on a polysaccharide sorbent and an immobilizing domain on a high molecular weight polysaccharide for enchancing the antigen and increasing its immunogenicity.

[0046] The spacer sequences used are optimized in length and composition to ensure efficient expression of SSTad fused to the carrier protein in various microorganisms, in particular in E. coli. The new spacer sequences provide increased protease resistance and an optimal effect of SSTad on the patient's immune system. They promote the formation of the native U-shaped structure of the SSTad, which the SST has between the two cysteines, and expose it to the outside in the fusion protein, thereby forming an immunodominant epitope.

[0047] In another embodiment, the present invention provides novel adjuvant compositions used in the treatment of patients with infertility. In one particular application, the SSTad compound can be combined with novel adjuvants and used in the treatment of infertility or oocyte deficiency conditions or conditions such as sperm deficiency in males or for the prevention of these diseases. The adjuvants in this application are intended for optimal use in mammals, animals, birds, and in particular humans. The adjuvants in this application provide increased immunogenicity of the antigen, allowing lower amounts of antigen to be included in vaccines and higher titers of SST specific antibodies to be reached.

[0048] In another embodiment of the invention, pharmacological compositions are provided that result in immunogenicity against SSTad, which initiates the production of specific antibodies, binding of antibodies to SST and a decrease in its content in the blood. This eliminates the inhibition that SST exerts on follicular release and maturation as well as on spermatogenesis. The vaccine formulation options in the present invention are optimized for both safety and achieving the desired effect for high immunogenicity of the SSTad construct in safe and highly effective adjuvant formulations. Vaccines of the present invention require lower amounts of antigen (compared to the concentration of antigen in traditional subunit vaccines), have an increased shelf life and lower cost.

[0049] The present invention is focused on the treatment of infertility in humans and other vertebrates. These include mammals and birds in which it is required to restore the number of maturing follicles and the quality of sperm.

[0050] The following definitions are provided to facilitate understanding of certain terms often used in the present invention and are not intended to limit the scope of the present disclosure.

Definitions

[0051] “Patient” refers to a vertebrate warm-blooded animal, usually a mammal or bird, in need of a composition and/or method of the present invention, for example, a human (female) in need of recovery or an increase in the number of maturing oocytes, or a human (male) in need of recovery or an increase in sperm production and its quality.

[0052] “Treatment” refers to the improvement in a patient's condition compared to an untreated patient in a relatively identical or baseline situation. Treatment usually indicates that the desired pharmacological and/or physiological effect has been achieved using the compositions and methods of use thereof of the present invention. Treatment may include prophylactic use of the invention results.

[0053] As used herein, the term “infertility treatment” refers to the treatment of a disease or condition, related to the health of the female reproductive system, selected from the group of symptoms, consisting of ovarian failure, premature ovarian failure, infertility, anovulation, infertility characterized by “poor ovarian response” to gonadotropin therapy, delayed puberty, infertility associated with elevated FSH levels, pretreatment with IVF and ART (assisted reproductive technology), spontaneous premature ovarian failure (early menopause), polycystic ovarian disease (fewer growing follicles), and low response (poor response) to COS (controlled ovarian stimulation).

[0054] The term “infertility treatment” also refers to the treatment of diseases, associated with the health of the male reproductive system, selected from the group of parenchymal infertility associated with the density and motility of sperm below normal, secretory disorders of spermatogenesis, primary or secondary failure.

[0055] The term “ovarian reserve” or “ovarian follicular reserve” is used to describe the number of primary follicles remaining in the ovaries.

[0056] The term “resting primordial follicles” is used to describe dormant follicles or primordial follicles from the ovarian reserve. The present invention also relates to a composition and/or method for use thereof provided in the present invention to accelerate the maturation of follicles from the follicular reserve in the treatment of infertility.

[0057] “Primary follicle (awakened)” is a rounded formation in the mammalian ovary, consisting of a first-order oocyte surrounded by a zona pellucida, several layers of cubic follicular cells and connective tissue. The primary follicle is three to four times larger than the primordial.

[0058] The “secondary follicle” is ten times larger than the primordial one. A cavity with follicular fluid is formed around the oocyte, which reaches a diameter of 2.5 centimeters. Follicular cells form their own complicated, two-layer structure.

[0059] “Amino acid” refers to any of the twenty naturally occurring proteinogenic amino acids as well as any modified amino acids.

[0060] “Protein” and “peptide” are used to mean an amino acid polymer or a set of two or more interacting or linked amino acid polymers.

[0061] An “antigenic determinant” or “epitope” is part of an antigen macromolecule that is recognized by the immune system (antibodies, B lymphocytes, T lymphocytes). The part of an antibody that recognizes an epitope is called a paratope. Although epitopes usually refer to molecules that are foreign to a given organism (proteins, glycoproteins, polysaccharides, etc.), the regions of its own molecules recognized by the immune system are also called epitopes. Most of the epitopes recognized by antibodies or B cells are three-dimensional structures on the surface of antigen molecules (conformational epitopes), which exactly coincide in the shape and spatial arrangement of electric charges and hydrogen bonds with the corresponding antibody paratopes.

[0062] “Isolation” refers to a polynucleotide or polypeptide that has been separated or recovered from at least the degradation products of the cell of the producer strain. Purification of the polypeptide from contaminating cell components and polypeptides can be accomplished using any set of well-known methods, including precipitation with ammonium sulfate or ethanol, anionic or cation exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography.

[0063] “Antibody” refers to a Y-shaped molecule having a pair of antigen binding sites, a hinge region and a constant region.

[0064] “Vaccine” refers to any composition that can stimulate the immune system of a vaccinated subject to generate antibodies to SST for the purposes described herein.

[0065] Somatostatin

[0066] SST is a peptide hormone that inhibits, among other things, the release of growth hormone from the anterior pituitary gland. SST regulates various endocrine functions by interacting with G-protein-coupled SST receptors on target endocrine cells. SST is secreted from areas of the hypothalamus, stomach, intestines, and pancreas. Controlling the SST level in patients is of interest for increasing the productivity of mammals and birds, changing feeding behavior, and increasing folliculogenesis and spermatogenesis. The effect of SST on the reproductive system is described in the present invention.

[0067] In studies, productivity of mammals was optimized by SST vaccination. Overall, SST-immunized farm animals had a weight gain of 8-17%, appetite decreased by 9%, and food efficacy increased by 11%. Animals immunized with SST and their offspring had the correct proportions and the same distribution of animal weight between muscle, bone and fat as in control animals (Reichlin, 1987, Lab Clin Med. 109 (3): 320-326). Thus, these studies indicate that the induction of autoimmunity to SST in the target animal can provide safe and effective results.

[0068] SST is known to exert an inhibitory effect on a large number of hormones involved in growth, assimilation of food by animals, and regulation of the reproductive system. As previously described in U.S. Pat. No. 6,316,004 and U.S. patent application Ser. No. 12/195,979, SST and SST fusion variants can be used to immunize animals to increase daily weight and, if necessary, for milk production. These immunization procedures were performed with traditional adjuvants. SST immunization has also been used to treat endogenous growth hormone deficiency (Haffer EP 2291194 B1). SSTad immunization has not previously been used to treat infertility.

[0069] The present inventors have obtained an unexpected and reproducible result demonstrating that the modified SST antigen, immunogenic compositions comprising SSTad and vaccination can be used to treat diseases or physiological conditions in a patient, and in particular to treat and prevent infertility.

[0070] Advantages

[0071] Embodiments of the present invention provide SSTad based methods for the treatment and prevention of infertility in vertebrates and, in particular, in mammals and birds. Exemplary embodiments of the invention are directed to the treatment of humans, mammals and birds. Humans, other mammals and birds are treated with the vaccines of the present invention (see below) to limit or inhibit the effects of endogenous SST on the reproductive system. Vaccines of the present invention will result in additional folliculogenesis and spermatogenesis. Vaccines comprising SSTad and adjuvants are optimized for use in vertebrates and, in particular, for the treatment of human diseases that cause infertility. Since SST is a highly conserved hormone in vertebrates, embodiments of the present invention are useful for eliciting an immune response in all target vertebrates vaccinated using the methods and compositions described herein. A significant advantage of the present invention is that in vaccinated patients it can take several weeks to several months between booster immunizations, which allows the patient's immune system to bind SST, reduce its concentration and/or remove it from the body for a long time.

[0072] The technical aspects of the present invention facilitate all stages of the production and use of the SSTad vaccine, providing a simple and effective technique for obtaining the SSTad substance and producing highly immunogenic composition for use in the prevention and treatment of infertility. This compound for SSTad based immunization was optimized both for expression in producer cells, for the technique of purification and the formation of the native SST conformation, and for the formulation of an immunogenic composition.

[0073] In all variants of genetically engineered constructs, SSTad is expressed as codon-optimized chimeric carrier protein, spacer, and SSTad. The carrier protein, due to its binding to polysaccharide sorbents, provides a simple and effective technique of purification from other bacterial proteins, lipopolysaccharides and DNA of the producer strain. Antigens based on the SSTad according to the present invention, due to the binding of the carrier protein to high-polymer soluble polysaccharides, provides the formation of molecular complexes of a larger size (20-40 nm), providing immunogenicity and resistance to degradation by proteases. Thus, the SSTad of the present invention is present in the form of the immunogenic composition in the tissues of the patient for a longer time, causing a greater effect on the patient's immune system. As will be described in more detail below, the present invention also provides a maximized immune response due to optimized adjuvants.

[0074] New Vaccine Options for Use in Infertility Treatment

[0075] Somatostatin has two active forms that are produced by alternative cleavage of the propeptide, somatostatin-28 and somatostatin-14. However, the amino acid sequence of the tetradecapeptide that binds to the SST receptors is the shorter peptides KNFFWKTFTS (SEQ ID NO: 1), NFFWKTFT (SEQ ID NO: 7), FFWKTF (SEQ ID NO: 8), and FWKT (SEQ ID NO: 9), as confirmed by numerous synthetic SST analogues (Heidarpour et al., 2019, J Res Med Sci. 26: 24-29). FWKT (SEQ ID NO: 9) is a key sequence required for specific receptor binding. Therefore, FWKT (SEQ ID NO: 9) is the minimum key epitope for neutralization of the interaction of SST with the receptor by antibodies. However, using recombinant protein technology, it is very difficult to reproduce the U-shaped conformation of such a short peptide (FIG. 2).

[0076] The KNFFWKTFTS (SEQ ID NO: 1) sequence is highly conserved among vertebrates (Lin et al., Comp. 1998, Biochem. Physiol. C. Pharmacol. Toxicol. Endocrinol. 119 (3): 375-388) and, as shown in this invention, is optimal for establishing a specific immune response.

[0077] The polysaccharide binding domain can be attached to the SSTad through a variable length spacer. The spacer is necessary to ensure the presentation of the encoded somatostatin on the surface of the protein molecule, as well as optimal presentation to the patient's immune system. Spacer variants used within the present invention provide efficient formation of the U-shaped SST conformation, increased protease resistance and optimal effect on the epitope, and have shown unexpected additional improvement over constructs, lacking a spacer sequence and/or comprising other spacer sequences different from those provided herein.

[0078] Spacer variants have been optimized in length and composition to ensure efficient expression of SSTad fused with the carrier protein in a variety of microorganisms, in particular E. coli. As noted above, these new spacer sequences provide increased resistance to proteases (thus providing increased antibody production over that of the constructs disclosed in U.S. Pat. No. 6,316,004) and optimal effects on the patient's immune system. Said combination of SSTad, attached to the polysaccharide binding domain using optimally configured spacer, shows an unexpected improvement in immunization of target patients to increase folliculogenesis and spermatogenesis, compared to immunization with Sat-Som. These constructs are intended to be used as antigens comprising SSTad in the treatment of infertility.

[0079] Above-described SSTad recombinant fusion proteins exhibit high storage stability. In addition, the SSTad-based antigens according to the present invention provide a deposition function and a longer half-life in a patient taking into account the increased resistance of these materials to degradation. It is noted that other carrier polypeptides can replace the polysaccharide binding domain for attachment to the SSTad. For example, SSTad can be combined with KLH, tetanus toxoid, CRM 197, chloramphenicol acetyltransferase, or other protein carriers.

[0080] Embodiments of the invention also provide novel adjuvant compositions for enhancing the induction of humoral immunity in a target patient. These adjuvant compositions provide a significant improvement over traditional (aluminum hydroxide, oil) for the induction of a humoral response and are safe for use in humans, mammals and birds. Adjuvant compositions are used herein with SSTad based antigens to prepare vaccines of the present invention.

[0081] In one embodiment of the invention, the immunological adjuvant comprises dextran-500 to bind a carrier protein and form high-polymer, polysaccharide-soluble molecular complexes of larger size (20-40 nm) that provide immunogenicity and resistance to protease degradation. DEAE-dextran-500 binds CpG oligonucleotides and provides their resistance to nuclease degradation. In more detail, the complex is prepared using CpG oligonucleotide 1585 for animals and CpG oligonucleotide 2216 for humans. CpG oligonucleotides are prepared by oligonucleotide synthesis. In some embodiments of the invention, monophosphoryl lipid A, muramyldipeptide, polymuramil, or other ligands of toll-like receptors can be used, but the use of CpG oligonucleotide is more preferable in the treatment of infertility. It is possible to use the immunogenic composition without CpG oligonucleotide, but achieving a comparable result in the treatment of infertility will require significantly higher amounts of the SSTad fusion protein and a longer immunization schedule. CpG oligonucleotides are dissolved in saline. Adjuvant compositions are combined with the recombinant SSTad fusion protein to prepare vaccines of the invention (Example 5).

[0082] In yet another embodiment, the immunological adjuvant comprises Montanide™ (Examples 6, 7, 8) or aluminum hydroxide (Example 9). Adjuvants are combined with the SSTad fusion polypeptides to produce vaccines of the invention. The adjuvants used in the present invention are safe and effective for use in mammals, birds and humans, and are free from animal products and carcinogenic compounds.

[0083] The specific combination of adjuvants and concentrations are shown below.

[0084] Vectors and Host Cells

[0085] The present invention also relates to vectors, containing the polynucleotide molecules of the invention, as well as host cells, transformed with such vectors. Any of the polynucleotide molecules within the invention can be linked to a vector, which typically comprises a selectable marker and replication site, for the corresponding producer cell. Producer cells are genetically engineered to replicate these vectors and thereby express the proteins of the invention. Typically vectors in the examples provided comprise polynucleotide molecules of the present invention, operably linked to suitable transcriptional or translational regulatory sequences, such as sequences for bacterial or viral host cells. Examples of regulatory sequences include transcriptional promoters, operators, mRNA ribosome binding sites, and corresponding sequences that control transcription and translation. Nucleotide sequences are operably linked when the regulatory sequences in the present invention are operably related to polynucleotides encoding the fusion polypeptide of the present invention.

[0086] Typical vectors include plasmids, yeast shuttle vectors, baculovirus, modified adenovirus, etc. In one embodiment, the carrier is a modified pET30 plasmid. Host cells for use in the present invention include bacteria such as E. coli, yeast, SF-9 insect cells, mammalian cells, plants, etc.

[0087] In one embodiment, the regulatory sequences include a T7lac, Trp or T5 promoter for expressing the fusion proteins of the invention in E. coli or other prokaryotic cells. These regulatory sequences are widely reported and used in appropriate and distinct conditions. Various plasmids of the present invention have been designed to express the fusion proteins of the present invention using regulatory sequences. Plasmids with the T7lac promoter are preferred.

[0088] Host cells for the expression of the targeted fusion proteins include prokaryotic, yeast, and higher eukaryotic cells. Illustrative prokaryotic hosts include cells from bacteria of the genera Escherichia, Salmonella and Bacillus, as well as the genera Pseudomonas and Streptomyces. In an exemplary embodiment, the host cell belongs to the genus Escherichia and may be Escherichia coli (E. coli). As shown in the examples below, the constructs of the invention provide optimal expression of the SSTad, which is spacer coupled to the polysaccharide-binding domain under a variety of conditions. These constructs are especially effective in terms of expression in prokaryotic host cells and in particular in bacteria of the genus Escherichia.

[0089] In one embodiment, E. coli cells are transformed with pET plasmid, containing the SSTad fusion protein gene, having suitable regulatory sequences for expression in E. coli cells. In some cases, fermentation of approximately ten liters of these cells results in at least 100 grams of total biomass, which then gives approximately 10 grams of total protein. By staining with silver and Coomassie blue, a quarter of the protein mass is the target protein.

[0090] Purification of GBD-SSTad-SSTad Fusion Protein

[0091] The fusion protein can be purified in accordance with routine protein purification technologies, including, for example, lysis of bacterial cells with the enzyme lysozyme, DNA disruption in French-press modules, using ultrasound or DNAse, subsequent differential centrifugation of inclusion bodies, dissolution of inclusion bodies in guanidine chloride or urea, refolding procedures, column chromatography on affinity and ion exchange columns, and the like.

[0092] Aspects of the present invention include the production of an endotoxin-free immunogenic fusion protein. In some embodiments, the fusion protein is produced in a substantially endotoxin-free state. Additional purification completely removes or reduces the concentration of endotoxin to acceptable levels for human use in accordance with pharmacopoeia standards. As such, some of the embodiments of the technique disclosed herein allow achieving the production of substantially endotoxin-free fusion proteins for use in vaccines. In some embodiments, endotoxin levels are at or below 1 EU/ml, and in other embodiments, endotoxin levels are eliminated, that is, the fusion proteins of the present invention are substantially free of endotoxins.

[0093] The concentration of the purified fusion protein ranges from 1 to 8 mg/ml and typically from 4 to 6 mg/ml. In some instances, the substantially endotoxin-free chimeric protein is used in the vaccine compositions at a dose of about (1.5 to 5 mg) per 2 ml, and more typically at a dose of (2.0 to 3.5 mg) per 2 ml.

[0094] Vaccines

[0095] Vaccines in this invention are combinations of immunological adjuvants, as described herein, and antigens, necessary for the prevention or treatment of a patient's condition associated with infertility.

[0096] A pharmaceutical dosage for an embodiment of the vaccine as used herein comprises 1-5 mg of the recombinant fusion protein. In all embodiments, the vaccine must be sterile, stable under the conditions of manufacture and storage. Prevention of the growth of the number of microorganisms can be achieved by the addition of various antibacterial and antifungal agents, for example, benzyl alcohol, parabens, chlorobutanol, sorbic acid, thiomersal, and the like.

[0097] The adjuvants of the present invention are combined with the SSTad fusion polypeptide to provide a vaccine useful for the treatment of diseases and/or conditions associated with infertility.

[0098] Vaccines of the invention typically comprise the SSTad antigen in an amount of recombinant protein from 1 mg/ml to 10 mg/ml dose.

[0099] Dextran 500 from 1 mg/ml to 10 mg/ml dose,

[0100] DEAE-dextran 500 from 0.2 mg/ml to 2 mg/ml dose, Monophosphoryl lipid A, muramyldipeptide and/or CpG oligonucleotide 1585 from 0.02 mg/ml to 0.2 mg/ml.

[0101] Vaccine solutions of the invention are prepared by mixing materials in the required quantities and volumes (antigen, adjuvant, other ingredients), and final sterilization using ultrafiltration. Alternatively, the vaccine solutions of this invention can be prepared using individually sterilized components prior to final formulation.

[0102] Vaccines of this invention can be prepared in the form of sterile lyophilized preparations comprising CpG (Example No. 5), oil emulsion with Montanide™ (Examples No. 6, 7, 8) or suspension with aluminum hydroxide (Example No. 9). These preparations are stable under the conditions of manufacture and storage. Embodiments of vaccines in accordance with the invention may further comprise dispersing or wetting agents, suspending agents, or other similar materials.

[0103] Method for Treatment of the Patient's Diseases

[0104] The described invention provides pharmaceutical grade vaccines, comprising the SSTad fusion proteins and the adjuvants within the invention. Such vaccines can be administered to patients with infertility.

[0105] The vaccines of the present invention are intended for the treatment of infertile patients. In one embodiment, the vaccine of the present invention was applied 2 times, however, the number of injections may be increased to 3-5 boosters upon the discretion of the medical professional or veterinarian. Typical amounts of the vaccine antigen are 50-100 μg/ml of fusion recombinant protein per kg of patient weight. The vaccines can be administered by routine methods. In one embodiment, the vaccine is administered by subcutaneous injection (Examples No. 5, 6, 7, 8). In another embodiment, the vaccine is administered by intramuscular injection (Example No. 9).

[0106] The progress of treatment in patients, receiving the vaccine formulation according to the invention should be monitored with additional administrations provided upon necessity. The elevation of the growth hormone and anti-SST antibodies levels are target indicators for monitoring treatment efficacy. Based on individual patient's observation of, additional vaccine injections may be administered with adjustable quantity of the SST antigen in accordance with the present invention. In addition, alternative adjuvant combinations may be used to alter the response of a particular patient to vaccination as determined by the medical professional or veterinarian.

[0107] The proposed treatment options may be combined with other conventional infertility treatments. For example, the vaccination contemplated by the invention may be combined with conventional hormone therapy strategies.

[0108] In general, the described invention will be easier to assess by referring to the following examples.

EXAMPLES

[0109] The solution to the problem of cloning, production and purification of recombinant GBD-SSTad-SSTad protein is provided by the following means and methods.

[0110] Recombinant protein having a molecular weight of 39.5 kDa, comprises 2 fragments of the antigenic determinant of somatostatin (SSTad) with the sequence SEQ ID NO 1, spacer 1 Gly-Ser with the sequence SEQ ID NO 2, spacer 2 aiming the formation of the U-shaped conformation of SST with the sequence SEQ ID NO 3, the alpha-glucan binding domain (GBD) of a gene from Streptococcus mutans with the sequence SEQ ID NO 4. This recombinant protein is encoded by the nucleotide sequence of the GBD-SSTad-SSTad gene SEQ ID NO 5.

[0111] A method for producing recombinant GBD-SSTad-SSTad protein on glucan comprises: [0112] growing E. coli cells, expressing the GBD-SSTad-SSTad gene; [0113] binding of GBD-SSTad-SSTad protein in the composition of cell extracts of E. coli BL21 strain to a glucan-containing sorbent due to affinity interaction during the incubation procedure; [0114] subsequent washing from unbound bacterial proteins and isolation of the desired product.

[0115] Recombinant GBD-SSTad-SSTad protein comprises protein sequence of the glucan binding domain, which determines the ability of this protein to bind to the glucan-containing sorbent, which allows to achieve the one-stage concentration, purification, and immobilization of the protein product on glucan. Immobilization on glucan is provided due to the presence of glucan-binding domain from the alpha-glucan-binding domain of the gene from Streptococcus mutans in the recombinant protein/. The described domain demonstrates high affinity for alpha-glucans (pullulan, glycogen, dextran, starch) and provides irreversible binding to the carrier in a wide range of pH values 6.0-9.0 and salt concentrations 0-3 M NaCl.

[0116] Since E. coli cells lack proteins that bind to alpha-glucan, the recombinant GBD-SSTad-SSTad protein synthesized in cells is the only protein of the producer strain that strongly binds to alpha-glucan. This provides the means for a one-step production of a highly purified recombinant protein preparation immobilized on a glucan-containing sorbent.

[0117] An injectable preparation for enhancing folliculogenesis and spermatogenesis in mammals, birds and humans comprises recombinant GBD-SSTad-SSTad protein, as described above, suspended in a medium, consisting of a glucan-containing sorbent in a liquid carrier suitable for injection. The method of increasing the folliculogenesis and spermatogenesis in mammals, birds and humans comprises: two time, with an interval of 14-20 days, subcutaneous or intramuscular injections of a preparation, containing recombinant GBD-SSTad-SSTad protein suspended in the medium, consisting of a glucan-containing sorbent in a liquid carrier suitable for injection, at a dose of 5-200 μg of the described protein per kilogram of the patient's body weight.

[0118] Therefore, the provided bifunctional recombinant GBD-SSTad-SSTad protein is, capable to bind spontaneously to a glucan-containing sorbent, forming a highly immunogenic composition in the form of polyantigen, to induce the synthesis of specific autoantibodies to SST when administered to patients and consequently stimulating folliculogenesis and spermatogenesis.

Example 1. Preparation of Recombinant GBD-SSTad-SSTad Fusion Protein

[0119] At the first stage, the gene of the antigenic determinant of somatostatin is obtained, followed by its cloning. The SSTad gene was obtained by a chemical-enzymatic method. An oligonucleotide duplex was obtained, encoding the corresponding gene, and optimized for expression in E. coli. Then GBD-SSTad-SSTad plasmid was obtained comprising the sequences encoding somatostatin, spacer and glucan binding domain (GBD).

Example 2. Preparation of E. coli Strain Producing Recombinant Somatostatin Antigen Coupled to Glucan Binding Domain

[0120] To obtain E. coli strain producing recombinant GBD-SSTad-SSTad protein, E. coli BL21 cells were transformed by the pGBD-SSTad-SSTad plasmid. 3 μl of 0.1 M solution of isopropyl-beta-O-thiogalactopyranoside (IPTG) was added to the culture and mixture was incubated for 3 hours at 37° C. When comparing the range of proteins synthesized by cells of the E. coli BL21 [pGBD-SSTad-SSTad] strain, an additional protein band was found. The molecular weight, corresponding to the additional band was consistent with the 39.5 kDa expected for recombinant GBD-SSTad-SSTad protein. The level of protein synthesis in E. coli was determined by comparing the intensity of staining of the band of the recombinant protein with the band of the corresponding protein standard molecular weight. It was shown that recombinant GBD-SSTad-SSTad protein is synthesized in E. coli cells in an insoluble form as inclusion bodies.

Example 3. Obtaining of Recombinant GBD-SSTad-SSTad Protein Immobilized on Alpha-Glucan

[0121] To obtain the recombinant protein, E. coli BL21 [pGBD-SSTad-SSTad] cell culture was grown in 1000 ml of LB medium (Luria broth) with ampicillin (100 μg/ml) at 37° C. to an optical density corresponding to 1 unit absorption at a wavelength of 550 nm. 15 μl of 0.1 M IPTG solution was added to the medium and incubated for 3 hours. The cells were sedimented by centrifugation at 5000 g for 15 minutes.

[0122] The pellet was resuspended in phosphate buffer containing lysozyme. Additionally, the suspension was sonicated. After centrifugation at 6000 g, insoluble GBD-SSTad-SSTad protein remained in the sediment. The precipitate was suspended in 8 M urea, centrifuged at 12000 g for 30 minutes, and the supernatant was collected. To immobilize recombinant GBD-SSTad-SSTad protein on the sorbent, the supernatant was diluted four times with phosphate buffer, 1/10 of the volume of the alpha-glucan suspension was added, and incubated at 25° C. for 2 hours. The mixture was centrifuged at 8000 g, the pellet was resuspended in phosphate buffer; and alpha-glucan washing was repeated 3 times. The GBD-SSTad-SSTad antigen immobilized on alpha-glucan is a suspension of the sorbent with the protein adsorbed thereon. The purity of the preparation was at least 95%. The preparation was preserved by adding benzyl alcohol to a final concentration of 0.1% (by volume).

Example 4. Biological Effects of Recombinant GBD-SSTad-SSTad Protein

[0123] In a preferred embodiment of the invention, the preparation comprises recombinant GBD-SSTad-SSTad protein, lyophilized with the necessary excipients and CpG oligonucleotide or suspended in water oil suspension of Montanide™ (50% by weight) or aluminum hydroxide, and administered by subcutaneous or intramuscular injection of the preparation twice with an interval between injections of 20 days at a dose of 5-200 μg of recombinant protein per kilogram of body weight of an animal or bird. The mechanism of action of the drug is based on temporary blocking of the activity of endogenous SST by autoantibodies.

[0124] The following examples illustrate the efficacy of the use of the preparation for enhancing the activity of folliculogenesis and spermatogenesis in mammals, birds and humans.

Example 5

[0125] To describe the in vivo action of the injectable preparation with the SST antigenic determinant and to determine its effect on the development of the primary follicle, the patent applicants conducted studies on an in vivo mouse model. The close similarity of the reproductive system, especially regarding ovarian morphology, physiology and endocrinology of the ovaries of rodents and women, makes the mouse a useful model for studying the regulation of ovarian function, fertility aneurysm and ovarian reserve (Dixon et al., 2014, J Toxicol Pathol, 27 (3-4 Suppl); Cora et al., 2015, Toxicol Pathol., 43 (6): 776-793). According to the results of the above study, the applicants unexpectedly found that by inducing the synthesis of specific autoantibodies to SST, it is possible to suppress the responses of the SST receptors, which leads to a pharmacological effect on the ovarian reserve of the ovaries and, as a consequence, accelerates the onset of growth of resting follicles. This effect contributed to an increase in the number of primary and secondary follicles in comparison with those in the control group of animals.

[0126] The biological activity of the resulting immunogenic composition was investigated using subcutaneous injections twice with an interval of 20 days at a dose of 50 μg of recombinant protein per animal. The lyophilized vaccine preparation was suspended in saline and injected subcutaneously.

[0127] Table 1 shows the results of the enzyme-linked immunosorbent assay (ELISA) of blood in the form of antibody titers. In this case, a comparison was made of the obtained antibody titers in blood samples of mice after immunization with four vaccine compositions using the recombinant GBD-SSTad-SSTad as antigen, with various adjuvants in each case. Vaccines differed in the absence or presence of molecular adjuvants, which were monophosphoryl lipid A, muramyldipeptide and CpG oligonucleotide 1585. A group of 5 mice was allocated for each composition. According to the results obtained in each group, the average values of the ELISA dilution parameters (titer) were derived. The highest value, equal to 6×10.sup.4, was obtained in the group of mice immunized with the composition comprising the CpG oligonucleotide. The lowest value, equal to 2×10.sup.3, was found in the group of mice immunized with the composition comprising only dextran. Based on the data obtained, it can be concluded that the most striking immune response is elicited by the composition using the CpG oligonucleotide as an adjuvant.

TABLE-US-00001 TABLE 1 ELISA dilution parameters (antibody titers) Antigen The value of the dilution parameters in ELISA ELISA composition Number of Method of (titer) average components immunizations administration mouse 1 mouse 2 mouse 3 mouse 4 mouse 5 (titer) GBD-SSTad- 3 subcutaneously 1 × 10.sup.3 1 × 10.sup.3 2 × 10.sup.3 1 × 10.sup.3 2 × 10.sup.3 2 × 10.sup.3 SSTad GBD-SSTad- 3 subcutaneously 1 × 10.sup.4 2 × 10.sup.4 1 × 10.sup.4 1 × 10.sup.4 1 × 10.sup.4 1 × 10.sup.4 SSTad, MPLA GBD-SSTad- 3 subcutaneously 2 × 10.sup.4 1 × 10.sup.4 1 × 10.sup.4 2 × 10.sup.4 1 × 10.sup.4 1 × 10.sup.4 SSTad, MDP GBD-SSTad- 3 subcutaneously 4 × 10.sup.4 5 × 10.sup.4 6 × 10.sup.4 7 × 10.sup.4 8 × 10.sup.4 6 × 10.sup.4 SSTad, CpG

[0128] GBD-SSTad-SSTad is recombinant protein, Dextran 500, DEAE is Dextran 500, MPLA is monophosphoryl lipid A, MDP is muramyldipeptide, CpG is oligonucleotide.

[0129] Results of Histological Examination

[0130] Despite the differences in the values of the ELISA dilution parameters when using compositions with various kinds of adjuvants, the follicular response was approximately at the same level in all groups of mice studied.

[0131] According to the results of a histological examination of the ovaries of mice immunized with GBD-SSTad-SSTad using the previously studied compositions, a significant increase in the number of follicles was found from the mean value in the control group, which is equal to from 11.1 to 18.8 in the group of vaccinated mice corresponding to 69% increase. This parameter includes the total number of all follicles: from primordial to vesicular and Graaf vesicles and characterizes an increase in the rate of their differentiation, which result can be considered as a positive effect on folliculogenesis.

[0132] The total number of structural and functional elements also increased from an average value of 25.8 in the control group to 43.1 in the groups after vaccination. The percentage of growth is 67. This value includes, in addition to follicles, also atretic elements and corpora lutea. The latter are formed in the luteal phase after the release of the oocyte, in extremely rare cases luteinization of unovulated follicles was observed, and respectively, this parameter can be interpreted as the number of mature oocytes released. In the control group, the average number of corpora lutea was 8.2. In the test groups, this value was 13.3 (an increase of 62%), which indicates a positive effect of immunoneutralization of endogenous SST on an increase in oocytes release during ovulation.

[0133] Atretic elements, which are formed at different stages of folliculogenesis, are follicles that have not reached the stage of a mature Graaf bubble and follicles undergoing destructive changes. Increasing in the number of such elements is also observed after immunoneutralization, from 6.5 in the control to 11.0 in the test groups (an increase of 69%), which fits into the value of an increase in follicles and a general increase in the number of structural and functional elements of the ovaries.

TABLE-US-00002 TABLE 2 Morphofunctional assessment of the effect of the GBD-SSTad-SSTad preparation on the ovary Increase GBD-SSTad- compared to Parameter Control SSTad CpG control (%) Primary follicles 1.8:(0.5-3.1) 4.3:(2.6-6.0) 80 Secondary follicles 1.8:(1.4-2.2) 2.8:(1.1-4.5) 56 Tertiary (early atrial 3.2:(2.3-4.1) 4.2:(2.9-5.5) 31 follicle) Vesicular 1.1:(0.7-1.5) 2.8:(1.5-4.1) 154 follicle with and without cumulus oophorus, with and without oocyte, and with little fluid Follicles (from 11.1:(9.4-12.8)  18.8:(15.4-22.2) 69 primordial to quaternary) Corpora lutea  8.2:(4.8-11.6)  13.3:(11.6-15.0) 62 Interstitial glands 6.5:(2.7-7.8) 11.0:(8.0-14.0) 69 (corpora atretica) and atretic follicles Atretic follicle and  14.7:(11.7-17.7)  24.3:(20.9-27.7) 65 corpora lutea Total number of  25.8:(23.0-28.6)  43.1:(37.9-48.3) 67 structural and functional elements

[0134] Close values of the studied parameters were obtained in works and patents (Gougeon, 2011, Gynecol Obstet Fertil. 39 (9): 511-3; Gougeon et al., 2010, Endocrinology 151 (3): 1299-309; Gougeon and Loumaye EP 20040791488 08.10.2004; Gougeon US 20070155659 A1 5.7.2007; Tran and Gotteland EP2835136A1 11.02.2015) using SST antagonist analogs.

Example 6

[0135] A study of the GBD-SSTad-SSTad preparation was conducted to restore the sexual cyclicity and fertility of dairy cows with ovarian hypofunction.

[0136] One of the reasons that hinder the maximum realization of the reproductive and productive potential of highly productive dairy cows is postpartum ovariopathy, the main form of which is ovarian hypofunction, characterized by depression of folliculogenesis and ovulation. Postpartum ovarian dysfunction refers to hypothalamic-pituitary regulation diseases associated with functional shifts in the neuroendocrine system.

[0137] The experiment was carried out on 40 infertile cows with clinically pronounced signs of ovarian hypofunction. In animals, persistent anaphrodisia was noted (no resumption of sexual cyclicity for 2.5-3.0 months after childbirth). Transrectal ultrasound examination showed that the ovaries are reduced in size, and the diameter of the growing follicles reaches 6-10 mm.

[0138] The experiment was performed on 40 cows, which were divided into 2 groups of 20 cows each. The animals from the first group were injected with GBD-SSTad-SSTad at a dose of 50 μg per 1 kg of live weight (dose selection for large animals is given in Example 3). Recombinant GBD-SSTad-SSTad protein suspended in the medium consisting of alpha-glycan (50% by weight), water oil suspension Montanide™ (50% by weight) was injected twice subcutaneously with an interval of 20 days. Individuals from the second (intact) group were chosen as controls. They were monitored for 3 months.

[0139] Within 3 months, in cows of the control group, fertility recovered only in 26.7% of cases, while in animals received the preparation with GBD-SSTad-SSTad protein, fertility recovered in 85.7% of cases (Table 3). The duration of infertility per one cow in the experiment decreased by 1.8 times when using GBD-SSTad-SSTad.

TABLE-US-00003 TABLE 3 Efficacy of the preparation comprising GBD-SSTad- SSTad protein in cow's ovarian hypofunction GBD-SSTad- Control Parameter SSTad group group Number of animals, head 20 20 Restored sexual cycling and were inseminated number of animals 19 14 % 95.4 74.2 The period from the beginning of 15.0 ± 2.0 78.0 ± 18.0 observation until the onset of the sexual cycle and insemination, days Fertilized number of animals 17 6 % 85.7 26.7 Period from the beginning of 22.7 ± 4.6 78.2 ± 20.7 observation to fertilization, days Average number of days of infertility 38.3 90.2 per cow over the observation period

[0140] Therefore, when administered to cows with ovarian hypofunction, GBD-SSTad-SSTad specifically acts on the pituitary-gonadal system, leveling postpartum ovariopathy, characterized by 35uppression of folliculogenesis and ovulation.

Example 7

[0141] The efficacy of the preparation in increasing spermatogenesis in stud farm animals at the stage of physiological maturity disconfirmed by the following examples. To select the optimal amount of the drug (dose selection), the GBD-SSTad-SSTad preparation was injected twice with an interval of 20 days to three groups of boars of Large White breed at the dose of 25 or 50 or 100 μg of recombinant protein per 1 kg of live weight. Recombinant GBD-SSTad-SSTad protein was suspended in the medium consisting of alpha-glycan (50% by weight), water-oil suspension Montanide™ (50% by weight) and was injected subcutaneously. Before and after the use of the preparation, sperm was taken from the stud boars, according to the established procedure and the volume of ejaculate, the sperm concentration in the ejaculate and the number of semen doses were determined. The parameters of sperm production in boars when using the drug at a dose of 25 μg per 1 kg of live weight are shown in Table 4. The drug had a positive effect on boar sperm production.

TABLE-US-00004 TABLE 4 Parameters of sperm production in boars of Large White breed when using the GBD-SSTad-SSTad preparation at a dose of 25 μg of recombinant protein per 1 kg of live weight (n = 5) Values 30 Values 60 Values 90 Sperm days after days after days after quality Baseline the second the second the second parameters values injection injection injection Ejaculate 215.7 ± 8.9  221.9 ± 19.3 230.6 ± 16.5 229.6 ± 17.3 volume, ml Sperm 180.6 ± 15.3 190.5 ± 19.3 195.5 ± 18.5 194.1 ± 15.8 concentration, million/ml Number of 14 16 18 18 semen doses received from one animal

[0142] According to the data presented in Table 4, the GBD-SSTad-SSTad preparation at a dose of 25 μg of active ingredient per kilogram of live weight resulted a slight increase in sperm production compared to baseline values in boars of Large White breed. The volume of ejaculates increased, depending on the time of control of this parameter (30, 60, 90 days after the second injection of the drug), by 5.9-8.0%. The concentration of sperm in ejaculates increased by 2.8-5.8%, which led to an increase in the number of semen doses received from one animal by 15.4-16.7%.

[0143] Sperm production parameters in boars of Large White breed when using the GBD-SSTad-SSTad preparation at a dose of 50 μs of recombinant protein per kilogram of live weight are shown in Table 5.

[0144] As follows from the data presented in Table 5, the administration of the GBD-SSTad-SSTad preparation at a dose of 50 μg of active ingredient per kilogram of live weight resulted in a significant increase in sperm production compared to baseline values in boars of Large White breed. The volume of ejaculates of animals increased, depending on the time of control of this parameter (30, 60, 90 days after the second injection of the drug), by 13.9-20.0%.

TABLE-US-00005 TABLE 5 Parameters of sperm production in boars of Large White breed when using the GBD-SSTad-SSTad preparation at a dose of 50 μg of recombinant protein per 1 kg of live weight (n = 3) Values 30 Values 60 Values 90 Sperm days after days after days after quality Baseline the second the second the second parameters values injection injection injection Ejaculate 215.9 ± 5.8  242.5 ± 18.2 254.9 ± 15.5 253.5 ± 15.1 volume, ml Sperm 180.8 ± 15.5 205.5 ± 17.7 213.3 ± 15.6 214.4 ± 14.6 concentration, million/ml Number of 14 18 20 20 semen doses received from one animal

[0145] The sperm concentration in the obtained ejaculates of animals increased by 13.2-15.0%, which led to an increase in the number of semen doses obtained from one animal by 26.8-35.6%.

[0146] Sperm production parameters in boars of Large White breed when using the GBD-SSTad-SSTad preparation at a dose of 100 μg of recombinant protein per kilogram of live weight are shown in Table 6.

[0147] As follows from the data presented in Table 6, administration of the GBD-SSTad-SSTad preparation to animals at a dose of 100 μg of active ingredient (recombinant protein) per kilogram of live weight resulted in an increase in sperm production compared to baseline values in boars of Large White breed. The volume of ejaculates of animals statistically and significantly increased, depending on the time of control of this parameter (30, 60, 90 days after the second injection of the drug), by 14.8-19.3%. The sperm concentration in the ejaculates of animals increased by 8.3-10.7%, which led to an increase in the number of semen doses received from one animal by 21.5-33.0%.

TABLE-US-00006 TABLE 6 Parameters of sperm production in boars of Large White breed when using the GBD-SSTad-SSTad preparation at a dose of 100 μg of recombinant protein per 1 kg of live weight (n = 3) Values 30 Values 60 Values 90 Sperm days after days after days after quality Baseline the second the second the second parameters values injection injection injection Ejaculate 214.5 ± 9.7  243.3 ± 14.7 253.2 ± 13.5 254.5 ± 15.6 volume, ml Sperm 183.5 ± 15.6 200.6 ± 16.5 211.6 ± 16.7 210.8 ± 18.6 concentration, million/ml Number of 15 17 19 20 semen doses received from one animal

[0148] Therefore, the experiments carried out in the conditions of an industrial pig-breeding farm to substantiate the most effective dose of the GBD-SSTad-SSTad preparation confirmed pharmacological activity thereof and promising perspective of its use for stimulating spermatogenesis in boars of Large White breed.

[0149] When the GBD-SSTad-SSTad preparation is used twice, 50 and 100 μg per kilogram of animal weight, approximately the same biological effect is observed. Therefore, it is advisable to use a dose of GBD-SSTad-SSTad equal to 50 μg/kg of animal weight.

[0150] Analysis of boar spermograms showed an increase in spermatogenesis after the administration of the preparation at a dose of 50 μg of protein per kilogram of live weight (Table 7). During the entire observation period, boars showed an increase in the total volume of ejaculates, the sperm concentration and motility, which resulted in a statistically and significantly increased number of semen doses suitable for insemination of breeding sows and young gilts by 25.2% compared to baseline value. In the ejaculates of the animals of the experimental group, 60 days after the administration of the GBD-SSTad-SSTad preparation, an increase in the absolute survival of spermatozoa by 10% from the baseline value was observed.

TABLE-US-00007 TABLE 7 Effect of the use of the GBD-SSTad-SSTad preparation on the parameters of sperm production in boars when administering 50 μg of recombinant protein per 1 kg of live weight of animals (n = 5) Values 30 days Values 60 days Sperm quality Baseline after the second afterv the second parameters value injection injection Ejaculate 210.67 ± 7.5  221.33 ± 4.4  225.54 ± 6.7  volume, ml Sperm motility, 8.5 8.7 8.9 score Sperm 303.45 ± 15.6 307.84 ± 15.6 306.54 ± 16.7 concentration, million/ml Number of 14 18 20 semen doses received Absolute sperm 1160 1250 1290 survival at 16- 18° C., RU (relative units)

[0151] When breeding sows were inseminated with ejaculates obtained from studs received the GBD-SSTad-SSTad preparation, an increase in the fertilizing ability of sperm was found. In the groups of breeding sows inseminated with the sperm of the boar received the preparation, there was a tendency to an increase in multiple pregnancies, including an increase in the number of live newborn piglets by 10%.

Example 8

[0152] The preparation comprising recombinant GBD-SSTad-SSTad protein suspended in the medium consisting of alpha-glycan (50% by weight), water oil suspension Montanide™ (50% by weight) was injected to dairy bulls twice subcutaneously with an interval of 20 days at the rate of 50 μg of recombinant protein per 1 kg of live weight of animals. The parameters of sperm production in animals on days 30, 60, and 90 after the second injection of the preparation are presented in Table 8.

TABLE-US-00008 TABLE 8 Parameters of sperm production in dairy bulls when using the GBD-SSTad-SSTad preparation at a dose of 50 μg of recombinant protein per 1 kg of live weight (n = 3) Values 30 Values 60 Values 90 Sperm days after days after days after quality Baseline the second the second the second parameters values injection injection injection Ejaculate 3.80 ± 0.18 4.25 ± 0.16 4.56 ± 0.16 5.15 ± 0.16 volume, ml Sperm 1.22 ± 0.14 1.22 ± 0.12 1.20 ± 0.12 1.21 ± 0.13 concentration, million/ml Number of 616 940 969 980 semen doses received from one animal

[0153] The results of administration of the GBD-SSTad-SSTad preparation indicate its positive effect on sperm production and the quality of bovine sperm. In stud dairy bulls, the volume of ejaculates during the observation period statistically and significantly increased by 12%, the number of semen doses received from one animal increased by 40%, while the percentage of sperm rejects decreased by two. The sperm concentration in the ejaculates of dairy bulls during the observation period remained at the same level.

Example 9

[0154] The efficacy of the GBD-SSTad-SSTad preparation for increasing spermatogenesis in roosters at the stage of physiological maturity is illustrated by the following example.

[0155] The study of the effect of the preparation on the productive qualities of stud roosters (two-line hybrid of Plymouth Rock and Cornish) was carried out at poultry farms.

[0156] The preparation was used at a dose of 50 μg/kg of recombinant GBD-SSTad-SSTad protein suspended in the medium consisting of alpha-glycan, aluminum hydroxide suspension, twice with an interval of 20 days at the rate of 50 μg of recombinant protein per 1 kg of live weight of birds. Before and after the administration of the preparation, sperm samples were obtained from the stud roosters, and the volume of ejaculate, the concentration of sperm in the ejaculate and the number of received semen doses were determined. The parameters of sperm production in roosters when using the preparation at a dose of 50 μg per 1 kg of live weight are shown in Table 9.

TABLE-US-00009 TABLE 9 Parameters of sperm production in roosters when using the GBD-SSTad-SSTad preparation at a dose of 50 μg per kg of live weight (n = 5) Values 30 Values 60 Values 90 Sperm days after days after days after quality Baseline the second the second the second parameters values injection injection injection Ejaculate 0.53 ± 0.05 0.59 ± 0.02 0.74 ± 0.03 0.76 ± 0.03 volume, ml Sperm  2.1 ± 0.05 1.98 ± 0.06  2.1 ± 0.06  2.2 ± 0.04 concentration, million/ml Number of 13 15 19 18 semen doses received from one animal

[0157] The results of the experiment confirm the stimulation of sperm production in breeding roosters resulting from the use of the preparation. So the volume of ejaculate taken from roosters 90 days after the first injection increased by 40%. During the period of observation, the quality parameters of the collected sperm in terms of sperm survival improved. The use of the GBD-SSTad-SSTad preparation in stud roosters resulted in an increase in the number of received semen doses by 40% and a decrease in the percentage of sperm rejects based on biological indicators.

[0158] The above examples of the implementation of the invention are not limiting. Other embodiments are possible within the scope of the patent claims.

[0159] However, according to the studies conducted and attempts to carry out the invention, the present examples demonstrate that an essential feature of the invention are new spacers specially designed to solve the problem of obtaining a highly immunogenic protein for effective vaccination in the framework of the treatment and/or prevention of infertility, improving the quality of oocytes and/or sperm.

[0160] During the implementation of the invention, attempts have been made to produce a recombinant protein comprising somatostatin antigenic determinants using spacers other than those of sequence 2 and 3. For example, attempts have been made to use the known spacer ProGlySerGlySerGlySerGlySerGlySerAla (SEQ ID NO: 10). These attempts, both using the specified spacer in combination with one of the spacers according to the invention, and using only the specified spacer, did not lead to the formation of the correct three-dimensional structure of the recombinant protein, which precluded solving the problem. The desired efficacy could not be achieved correspondingly with the technical results indicated in this application, which showed the importance of the optimal selection of the spacer for the recombinant protein to solve the assigned task. Only the spacers developed according to the invention resulted in the formation of U-shaped SSTad structure (which is identical to the somatostatin fragment between two cysteines), as well as exposing this structure to the outside of the fusion protein. Thus, an immunodominant epitope was formed resulting in the development of the preparation based on the recombinant protein, capable to induce a high immune response against somatostatin. In addition, the spacers of the invention provide sufficient proteolytic protection for the antigenic determinant, as illustrated by the examples. All the effects of the invention can only be achieved using the spacers of the invention.

[0161] In addition, during the implementation of the invention, the importance of using exactly 2 antigenic determinants was confirmed. The prior patents could not suggest the use of 2 distinct antigenic determinants of somatostatin, since the single one was always used in the previous patents. This technique is also new to the authors of the present invention in relation to other immunogenic antigenic determinants, which shows the originality and novelty of this approach. It should be emphasized that in the process of working on the invention, a recombinant protein was obtained with only one antigenic determinant. Such a recombinant protein did not show the effects characteristic of the claimed group of inventions. Likewise, the use of a determinant different from that used according to the claimed invention did not succeed in solving the assigned tasks.

[0162] To summarize the abovementioned facts, we can conclude that the essential features of the invention are the sequences of spacer 1 and spacer 2 according to the invention and the use of two antigenic determinants having the sequence KNFFWKTFTS (SEQ ID NO: 1) to obtain a recombinant protein for the treatment and/or prevention of infertility, as well as increasing folliculogenesis and spermatogenesis in mammals, birds and humans.

TABLE-US-00010 ---> List of sequences: Amino acid sequence of the antigenic determinant of somatostatin <210> 3 <211> 11 <212> PRT <213> Homo sapience <400> 3 SEQ ID NO 1 Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser 1 5 10 Spacer 1 amino acid sequence <210> 3 <211> 11 <212> PRT <213> Artifical Sequence <400> 3 SEQ ID NO 2 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 Ala Gly Gly Gly Gly Ser Arg 20 Spacer 2 amino acid sequence <210> 3 <211> 11 <212> PRT <213> Artifical Sequence SEQ ID NO 3 Ser Gly Thr Gly Ser Gly Glu Ile Ala Ala Leu Glu Gln Glu Ile Ala 1 5 10 15 Ala Leu Glu Lys Glu Asn Ala Ala Leu Glu Trp Glu Ile Ala Ala Leu 20 25 30 Glu Gln Gly Gly Pro Gly Thr Gly-Gly Thr Gly Thr Gly Ser Gly Ala 35 40 45 Lys Ile Ala Ala Leu Lys Gln Lys Ile Ala Ala Leu Lys Tyr Lys Asn 50 55 60 Ala Ala Leu Lys Lys Lys Ile Ala Ala Leu Lys Gln Gly Gly Gly Thr 65 70 75 80 Gly Ser Gly Thr Arg Amino acid sequence of the alpha-glucan binding domain from Streptococcus mutans <210> 3 <211> 11 <212> PRT <213> Streptococcus mutans <400> 3 SEQ ID NO 4 Met Gly Ser Thr Asn Gln Tyr Tyr Gln Leu Ala Asp Gly Lys Tyr Met 1 5 10 15 Leu Leu Asp Asp Ser Gly Arg Ala Lys Thr Gly Phe Val Leu Gln Asp 20 25 30 Gly Val Leu Arg Tyr Phe Asp Gln Asn Gly Glu Gln Val Lys Asp Ala 35 40 45 Ile Ile Val Asp Pro Asp Thr Asn Leu Ser Tyr Tyr Phe Asn Ala Thr 50 55 60 Gln Gly Val Ala Val Lys Asn Asp Tyr Phe Glu Tyr Gln Gly Asn Trp 65 70 75 80 Tyr Leu Thr Asp Ala Asn Tyr Gln Leu Ile Lys Gly Phe Lys Ala Val 85 90 95 Asp Asp Ser Leu Gln His Phe Asp Glu Val Thr Gly Val Gln Thr Lys 100 105 110 Asp Ser Ala Leu Ile Ser Ala Gln Gly Lys Val Tyr Gln Phe Asp Asn 115 120 125 Asn Gly Asn Ala Val Ser Ala Arg 130 135 Nucleotide sequence of the GBD-SSTad-SSTad gene SEQ ID NO 5 CTCGAGAAAT CATAAAAAAT TTATTTGCTT TGTGAGCGGA TAACAATTAT AATAGATTCA 60 ATTGTGAGCG GATAACAATT TCACACAGAA TTCATTAAAG AGGAGAAATT AACT      114 ATG GGC TCC ACC AAT CAA TAT TAT CAA CTT GCC GAT GGC AAA TAT ATG  162 Met Gly Ser Thr Asn Gln Tyr Tyr Gln Leu Ala Asp Gly Lys Tyr Met 1 5 10 15 CTT CTT GAT GAT TCC GGC AGG GCC AAA ACC GGC TTT GTT CTT CAA GAT  210 Leu Leu Asp Asp Ser Gly Arg Ala Lys Thr Gly Phe Val Leu Gln Asp 20 25 30 GGC GTT CTT AGG TAT TTT GAT CAA AAT GGC GAA CAA GTT AAA GAT GCC  258 Gly Val Leu Arg Tyr Phe Asp Gln Asn Gly Glu Gln Val Lys Asp Ala 35 40 45 ATT ATT GTT GAT CCC GAT ACC AAT CTT TCC TAT TAT TTT AAT GCC ACC  306 Ile Ile Val Asp Pro Asp ThrAsn Leu Ser Tyr Tyr Phe Asn Ala Thr 50 55 60 CAA GGC GTT GCC GTT AAA AAT GAT TAT TTT GAA TAT CAA GGC AAT TGG  354 Gln Gly Val Ala Val Lys Asn Asp Tyr Phe Glu Tyr Gln Gly Asn Trp 65 70 75 80 TAT CTT ACC GAT GCC AAT TAT CAA CTT ATT AAA GGC TTT AAA GCC GTT  402 Tyr Leu Thr Asp Ala Asn Tyr Gln Leu Ile Lys Gly Phe Lys Ala Val 85 90 95 GAT GAT TCC CTT CAA CAT TTT GAT GAA GTT ACC GGC GTT CAA ACC AAA  450 Asp Asp Ser Leu Gln His Phe Asp Glu Val Thr Gly Val Gln Thr Lys 100 105 110 GAT TCC GCC CTT ATT TCC GCC CAA GGC AAA GTT TAT CAA TTT GAT AAT  498 Asp Ser Ala Leu Ile Ser Ala Gln Gly Lys Val Tyr Gln Phe Asp Asn 115 120 125 AAT GGC AAT GCC GTT TCC GCC AGG TCC GGC GGC GGC GGC TCC GGC GGC  546 Asn Gly Asn Ala Val Ser Ala Arg Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 GGC GGC TCC GGC GGC GGC GGC TCC GCC GGC GGC GGC GGC TCC AGG TCC  594 Gly Gly Ser Gly Gly Gly Gly Ser Ala Gly Gly Gly Gly Ser Arg Ser 145 150 155 160 GGC ACC GGC TCC GGC GAAATT GCC GCC CTT GAA CAA GAAATT GCC GCC    642 Gly Thr Gly Ser Gly Glu Ile Ala Ala Leu Glu Gln Glu Ile Ala Ala 165 170 175 CTT GAA AAA GAA AAT GCC GCC CTT GAA TGG GAAATT GCC GCC CTT GAA   690 Leu Glu Lys Glu Asn Ala Ala Leu Glu Trp Glu Ile Ala Ala Leu Glu 180 185 190 CAA GGC GGC CCC GGC ACC GGC AAA AAT TTT TTT TGG AAA ACC TTT ACC  738 Gln Gly Gly Pro Gly Thr Gly Lys Asn Phe Phe Trp Lys Thr Phe Thr 195 200 205 TCC GGC ACC GGC ACC GGC TCC GGC GCC AAA ATT GCC GCC CTT AAA CAA  786 Ser Gly Thr Gly Thr Gly Ser Gly Ala Lys Ile Ala Ala Leu Lys Gln 210 215 220 AAA ATT GCC GCC CTT AAA TAT AAA AAT GCC GCC CTT AAA AAA AAA ATT  834 Lys Ile Ala Ala Leu Lys Tyr Lys Asn Ala Ala Leu Lys Lys Lys Ile 225 230 235 240 GCC GCC CTT AAA CAA GGC GGC GGC ACC GGC TCC GGC ACC AGG TCC GGC  882 Ala Ala Leu Lys Gln Gly Gly Gly Thr Gly Ser Gly ThrArg Ser Gly 245 250 255 ACC GGC TCC GGC GAAATT GCC GCC CTT GAA CAA GAAATT GCC GCC CTT    930 Thr Gly Ser Gly Glu Ile Ala Ala Leu Glu Gln Glu Ile Ala Ala Leu 260 265 270 GAA AAA GAA AAT GCC GCC CTT GAA TGG GAAATT GCC GCC CTT GAA CAA   978 Glu Lys Glu Asn Ala Ala Leu Glu Trp Glu Ile Ala Ala Leu Glu Gln 275 280 285 GGC GGC CCC GGC ACC GGC AAA AAT TTT TTT TGG AAA ACC TTT ACC TCC 1026 Gly Gly Pro Gly Thr Gly Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser 290 295 300 GGC ACC GGC ACC GGC TCC GGC GCC AAA ATT GCC GCC CTT AAA CAA AAA 1074 Gly Thr Gly Thr Gly Ser Gly Ala Lys Ile Ala Ala Leu Lys Gln Lys 305 310 315 320 ATT GCC GCC CTT AAA TAT AAA AAT GCC GCC CTT AAA AAA AAA ATT GCC 1122 Ile Ala Ala Leu Lys Tyr Lys Asn Ala Ala Leu Lys Lys Lys Ile Ala 325 330 335 GCC CTT AAA CAA GGC GGC GGC ACC GGC TCC GGC ACC AGG TCC TAA CCG 1170 Ala Leu Lys Gln Gly Gly Gly Thr Gly Ser Gly Thr Arg Ser Stop 340 345 350 GACTTCGAAG CGTTCGGTTG GGTCCGGAAT TTCGTATGGC AATGAAAGAC GGTGAGCTGG 1230 TGATATGGGA TAGTGTTCAC                                           1250 <---