Characterisation of antigenic / immunogenic peptides for the development of contraceptive vaccines for the purpose of limiting the proliferation of the rodent arvicola terrestris scherman

Abstract

The present invention relates to the definition of a pool of antigenic sperm peptides with high species specificity for regulating populations of water vole (Arvicola terrestris scherman) using a vaccine approach.

Description

[0001] CHARACTERIZATION OF ANTIGENIC / IMMUNOGENIC PEPTIDES FOR THE DEVELOPMENT OF CONTRACEPTIVE VACCINES TO LIMIT THE PULLULATIONS OF THE RODENT ARVICOLA TERRESTRIS SCHERMAN DESCRIPTION Technical field of the invention The present invention relates to the regulation of populations of the common vole Arvicola terrestris scherman (ATS) by a vaccination approach. In the description below, the references in parentheses [ ] refer to the list of references presented at the end of the text. State of the art The common vole Arvicola terrestris Scherman (ATS) is an endemic rodent that lives in underground galleries in mid-mountain regions generally located between 400 and 1500 m altitude [1]. The proliferation of this rodent shows cyclical variations over a period of approximately 5 to 6 years, with densities ranging from 50-100 animals / hectare to more than 1000 during peak outbreaks [2,3].The excessive cyclical proliferation and expansion of ATS colonization areas, since the 1970s, in mid-mountain agronomic and tourist ecosystems, constitutes an increasingly important problem with strong environmental and economic repercussions [4-6]. At high densities, ATS affects the botanical composition of grasslands, causing the regression of legumes and the increase of poor quality grasses and unwanted plants. On cultivated plots, ATS can damage cereal crops, orchards, vegetable gardens, vineyards, forests and ornamental gardens [7,8]. One of the consequences of ATS activity on grasslands is the presence of earth mounds (tumuli) resulting from burrowing activity that facilitate the ingestion of soil by grazing animals. It.

[0002] results in an alteration of milk quality, characterized by a reduction in protein content and contamination by butyric acid bacteria. ATS outbreaks also constitute a risk for public health because they are vectors of zoonoses. There is thus a strong relationship between ATS density and the risk factor for alveolar echinococcosis [9]. Human alveolar echinococcosis is a serious parasitic disease caused by the larva of a flatworm, Echinococcus multilocularis, which is characterized mainly by hepatic development in the form of tumors, and for which there is currently no treatment. Current strategies for controlling ATS populations are dominated by lethal procedures, such as physical trapping and chemical poisoning with baits (wheat, carrot) soaked in the anticoagulant bromadiolone, or more recently Ratron

[0010] .Although these approaches are effective, they are no longer economically viable (trapping) or environmentally acceptable (chemical control). Indeed, the non-specific nature of chemical control means that collateral damage to non-target wildlife (animals with a similar diet or predators of ATS, such as wild boars, foxes, stoats, weasels, birds of prey, and even cats and dogs) is significant [11,12]. In addition, although chemical control is carried out in a reasoned manner (surveillance of outbreaks, treatment by administrative order subject to prefectural decree, controlled distribution of baits, abandonment of treatments during periods or in areas of high density), the contamination of ecosystems (groundwater, rivers, treatment plants, etc.) by bromadiolone constitutes a new health risk for the population and wildlife

[0013] .Recently, in France, the French National Agency for Food, Environmental, and Occupational Health and Safety (ANSES) declared that the use of this chemical product does not exclude "an unacceptable risk for aquatic organisms, terrestrial vertebrates, and contamination of groundwater" (AMM No. 9800526). This situation encourages the development of alternative strategies to combat outbreaks of this rodent, which is harmful to crops and ecosystems. In this context, immunocontraception may be an interesting option to limit the reproduction of ATS. Immunocontraception, or vaccine-based contraception, involves the administration of an antigen that induces an immune response.

[0003] adaptive, resulting in subfertility or even sterility. Contraceptive vaccines have been used and proven effective in many situations to control fertility in captive and / or semi-captive wild or domestic animal populations, including horses

[0014] , deer [15,16], squirrels

[0017] , marsupials

[0018] and African elephants

[0019] . In brief, three strategies can be used to achieve vaccine contraception: a "post-fertilization" strategy consisting of interfering with fetal implantation through an anti-GnRH (gonadotropin-releasing hormone) vaccine and two "pre-fertilization" strategies aimed at limiting gametic interaction by interfering with either the female or male gamete.Anti-GnRH and anti-ZP3 vaccines, ZP3 being one of the proteins of the zona pellucida surrounding the oocyte in mammals, have been shown to be effective in inducing vaccine-mediated contraception [20,21]. However, these two immunocontraceptive approaches have a major pitfall in the context of interest to us, namely that they do not allow species specificity given the good conservation of the sequence of the GnRH hormone and the ZP3 protein in mammals. Only a vaccine strategy directed against sperm antigens allows a high level of species specificity to be envisaged due to the large number of potential sperm antigenic targets and inter-species sequence polymorphism [22-24].While it is rather intuitive that sperm antigens might be recognized as “non-self” by the immune system of female animals, they should theoretically also trigger the production of anti-sperm antibodies in males. Indeed, sperm are produced at puberty, well after the “self” repertoire has been established. They are therefore considered “non-self” even for the male that produces them. That sperm antigens are good triggers of immune responses has been clearly demonstrated by the observation that 70% of men who have undergone vasectomy (ligation of the vas deferens) develop anti-sperm antibodies or ASA [25-27], which often compromises the restoration of fertility in the case of vasovasostomy (vasectomy reversal).These ASAs result from the presentation of sperm antigens to the host immune system due to the increased permeability of the blood-testis-epididymis barriers, a consequence of vasectomy

[0028] . ASAs have been identified in some cases of male infertility [29,30], for which.

[0004] They have been shown to cause infertility by inhibiting sperm / oocyte binding, reducing sperm motility, reducing sperm penetration into cervical mucus, and impairing capacitation and / or the acrosome reaction. In this context, immunocontraception studies conducted to date, in different models, have used either whole sperm or selected sperm antigens used individually. As examples, the injection of whole spermatozoa as immunogens has been carried out in a homologous system (i.e. a mammal of one species reacting against the sperm antigens of a mammal of the same species) in the Tammar wallaby

[0031] , the mouse

[0032] , the rabbit

[0033] and the dog

[0035] or in a heterologous system (i.e. a mammal of one species (e.g. rabbit) reacting against sperm antigens of a mammal of another species (e.g. ATS))

[0034] .The aim of this "pioneering" work was to show that immune responses against sperm antigens could be induced in mammals. However, in these studies, the determination of sperm antigens leading to the triggering of the immune response (i.e., the appearance of specific anti-sperm antigen antibodies) was not analyzed. Only antibody production and effects on fertility were described. In rodent models, several sperm antigens (proteins and / or peptides) were studied separately as potential targets for immunocontraception. Effective immune responses and reduced fertility were, for example, obtained using the sperm antigens FA1

[0036] , CRISP1

[0037] , SPAG9

[0038] , or tNASP

[0039] .Recently, the inventors used a whole sperm immunization strategy to identify via a no-priori approach a set of sperm antigens (in the final form of immunogenic peptides) with the best possible specificity for the targeted species (ATS) with the aim of constituting a bank of antigenic epitopes for the development of contraceptive vaccines

[0040] . Among the approximately 120 sperm antigens identified that can cause the appearance of anti-sperm antibodies and that could be used to provoke a homologous anti-sperm immune response (i.e. within the same species), only 17 sperm antigens (CHDH, ZPBP1, CRISP2, AKAP3, GK2, PGK2, CRISP4, PRSS21, CFAP44, INSL6, CATSPER1, ACR, GAPDHS, ZPBP2, NUP210L, IZUMO1, ZP3R) were retained. The selection was based on a.

[0005] multi-criteria screen focusing on: the level of the ATS antibody response after immunization with each of these antigens; the nature of the antibody response (systemic and mucosal); the tissue distribution of the antigens concerned; their role in the reproductive function; and finally, the presence within these antigens of epitopes whose primary sequence presented a maximum level of species specificity. Description of the invention The present invention consisted, from the sperm antigens previously identified in 2021

[0040] , in the selection and characterization of immunogenic peptide epitopes (= antigenic sperm peptides) strictly specific to the European field vole (ATS) with the aim of using them in a vaccination strategy targeting males and females of the species to limit the anarchic outbreaks of this harmful rodent in mid-mountain areas without producing any collateral effect on non-targeted fauna.This strategy is proposed as an alternative or in addition to the chemical control commonly used over the last 30 years but subject to a ban at the European level due to the collateral damage inflicted on non-target wildlife and the potential associated health risks for the human population. To do this, the Inventors parenterally immunized male and female ATS rodents with ATS spermatozoa (homologous system), and identified by immunoprecipitation techniques, proteomics coupled with mass spectrometry and via in silico approaches, the sperm proteins (= antigens) having caused the appearance of anti-sperm antibodies in the bloodstream (serum IgG).This made it possible to identify (after a long process of proteomics, mass spectrometry, bioinformatics) 121 proteins in total likely to initiate an antibody response in ATS, and a first multi-criteria screen of these potential sperm antigenic targets was carried out in order to retain only antigens presenting the greatest sequence specificity for the species of interest (ATS), leaving aside those that could be found in other mammals (based on the sequencing data available to date in mammals).

[0006] This approach made it possible to reduce the list of potential candidates to 17 proteins, all of which were different from the proteins identified in previous experiments in a heterologous system, thus confirming the importance of a study in a homologous system on the ATS itself, whose immune system does not react in the same way as that of another mammal (e.g., rabbit) to antigens from its own spermatozoa. For each targeted protein, several potentially immunogenic peptides (i.e., those with an amino acid composition and primary sequence configuration theoretically likely to provoke an immune response, i.e., the production of antibodies) were preselected.Thus, from the peptide sequence of these 17 proteins, using classical computer tools for antigenic prediction and always keeping the specificity of the ATS species, 33 potentially antigenic sperm peptides were defined. These 33 immunogenic peptides of ATS sperm antigens are derived from the following 17 proteins: CHDH, ZPBP2, IZUMO1, ZP3R, NUP210L, INSL6, GSK2, PRSS21, PGK2, CFAP44, ACR, AKAP3, ZPBP1, GAPDHS, CRISP2, CRISP4, CATSPER1). Based on these 33 peptides, and for each of these 17 ATS sperm proteins, a peptide specific to the ATS species (i.e. not presenting any risk of cross-immunization on species either predators of ATS, or sharing its ecosystem - if these species were to be confronted with ATS vaccine antigens) was selected, chemically synthesized and tested for its capacity to provoke a humoral (IgG) and mucosal (IgA) immune response in ATS.ATS males were immunized with the immunogenicity of the 17 peptides and a sufficient immune response. The proteins listed above were individually tested for their ability after immunization to: - provoke a systemic and mucosal immune response, - target the male reproductive organs in which sperm are stored, transit or are in contact with the secretions of these organs in the semen (testicle, epididymis, seminal vesicle, prostate),

[0007] - target spermatozoa, - cause notable physiological effects (in particular an alteration of spermatogenesis, and / or a phenotype in the male genital tract). Depending on the level of the immune response obtained (evaluated by the antibody titer generated after immunizations of male ATS under similar dose conditions) the 17 peptides were categorized as “good immunogenic”, “medium immunogenic” and “weak immunogenic” (see Figure 1). During immunization tests with the 17 peptides, it was also verified that the produced ATS anti-sperm antibodies were not able to recognize spermatozoa from other species such as humans, rats and bulls. Otherwise, the peptides were considered unusable.Following this evaluation, 12 antigenic peptides (included in the first two categories "good and average immunogenic") were selected to constitute the pool of sperm ATS immunogenic peptides. Each of these peptides was named using the name of the protein from which it was derived (e.g. the peptide derived from the CHDH protein was named CHDH, hereinafter identified under the sequence SEQ ID NO: 1). The CATSPER1 antigen (as well as all immunogenic peptides that could be derived from it) already protected for its use as an antigen for immunocontraceptive purposes, was excluded. Consequently, 11 peptides derived from 11 different proteins were selected which, although they do not present a unity of structure, do present a unity of function due to their ability to trigger a specific immune reaction in ATS that has never been described before in this species.The introduction of a donc to obtain a vaccine composition in a contraceptive view at a time of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 peptide(s) sperm immunogen(s) choose from the peptides from the suivantes: CHDH (SEQ ID N. O : 1 ; AWAVLGQSRYPCS); CRISP2 (SEQ ID N O : 2 ; MEWSVETTG) ;

[0008] AKAP3 (SEQ ID N O : 3 ; SESKAQGVKEY) ; PGK2 (SEQ ID N O : 4 ; YNPAKVEAFR); ZPBP2 (SEQ ID N O : 5 ; YTLQGNRQINI); GK2 (SEQ ID N O : 6 ; YEVEKLAREV) ; CFAP44 (SEQ ID N O : 7 ; VYSQFIAEKIA); INSL6 (SEQ ID N O : 8 ; WEPWVPPDYQFEKSNLLPEKTEEFSSRDVHS) ; GAPDHS (SEQ ID N O : 9 ; DLEIQVYQWLGAP); NUP210L (SEQ ID N O : 10 ; KIMFQFLKY); ZP3R (SEQ ID N O: 11; YLPVLMEPQ); and a pharmaceutically acceptable excipient. According to a particular embodiment of the present invention, the vaccine composition may comprise the 11 immunogenic peptides of sequences SEQ ID NOs: 1-11, preferably the 6 peptides of sequences SEQ ID NOs: 1-6 (for immunodominance criteria and search for maximum species specificity), preferentially the 3 peptides of sequences SEQ ID NOs: 1, 8 and 10. According to a particular embodiment of the present invention, the contraceptive vaccine composition further comprises an antigenic peptide of amino acid sequence AKFVAAWTLKAAA (SEQ ID NO: 12) as a response activator (PADRE activator for Pan HLA DR-binding epitope,

[0041] ). Each peptide can therefore appear in two forms: alone or associated with this particular PADRE sequence.The present invention also relates to a contraceptive vaccine composition according to the present invention, for use as a medicament. In particular, said medicament is a contraceptive intended to regulate the populations of the field vole. For example, said medicament is intended for oral administration (e.g. in the form of tablets, granules, gummies, etc.).

[0009] The present invention also relates to the immunogenic peptides of sequences SEQ ID NOs: 1-11. Brief description of the figures [Figure 1] represents the classification of the immunogenicity of the immunogenic sperm peptides selected in ATS. [Figure 2] represents a simplified diagram of the strategy for identifying antigenic sperm proteins of ATS. [Figure 3] represents a simplified diagram of the strategy for identifying antigenic sperm peptides of ATS with high species specificity and immunogenic potential. EXAMPLES EXAMPLE 1: IDENTIFICATION OF IMMUNOGENIC SPERM PEPTIDES (see figures 1 and 2) Male and female ATS were collected in the open field in the department of Puy-de-Dôme (063, France): Perpezat (45°68'33''North - 2°78'33''East); Nébouzat (45°42'59''North - 2°54'19''East); Saint-Julien-Puy-Lavèze (45°39'58''North - 2°40'25''East).In the immediate vicinity of the freshly made molehills, galleries were detected using a probe and a trap was placed inside. Only animals of reproductive age, i.e., weighing more than 70 grams, were kept alive and brought back to the animal facility where they were housed in a controlled environment (23°C, 12 hours of light / 12 hours of darkness). The animals were fed carrots ad libitum. All procedures used on the ATS were approved by the Auvergne Animal Experimentation Ethics Committee (C2E2A) and the French Ministry of Research (APAFIS authorization # 10653-2017071016422159 v5). When necessary, animals were anesthetized with 4% isoflurane (Isovet) in inspired air for injections and blood sampling. They were then killed by cervical dislocation before tissue collection.

[0010] Whole spermatozoa from ATS were used to immunize animals and generate anti-sperm antibodies(15.10 7spz / injection in a total volume of 300 µL, corresponding to an immunogenic dose based on protein content, approximately 100 mg). For all experiments, sperm were collected by squeezing the cauda epididymis into 500 µL of Whitten's-HEPES buffer (WH: 100 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.5 mM glucose, 1 mM pyruvic acid, 4.8 mM lactic acid, 20 mM HEPES, pH 7.4). After 30 min at 37°C to release a maximum of gametes, sperm were recovered. An aliquot was diluted (1:50 in WH buffer) and counted using a Malassez hemacytometer. The cell suspensions were finally washed three times by centrifugation in PBS buffer (500g, 5 min, room temperature) and the final pellet was frozen and stored at -20°C until use. Immunizations were performed subcutaneously between the two shoulder blades of male and female animals.Before sperm injection, a blood sample was collected from the tail vein to recover preimmune sera. Five males and six females were simultaneously immunized. The immunization procedure lasted 6 weeks: on the first day (primary injection), sperm were injected with an equal volume of complete Freund's adjuvant (Sigma). After three weeks, the same amount of sperm was injected (booster), diluted in an equal volume of incomplete Freund's adjuvant (Sigma). The immunization procedure was stopped after six weeks and blood was collected by intracardiac puncture under anesthesia to recover immune sera. The animals were then sacrificed. The recovered immune sera (and preimmune sera used as controls) were then used to identify the ATS sperm proteins (antigens) that elicited an immune response in the immunized individuals by an immunoprecipitation strategy.Briefly, spermatozoa from ATS epididymal caudae were collected as previously described, and 10 x 10. 7 Cells were incubated in 1.2 ml of RIPA buffer with protease and phosphatase inhibitors (2 h, room temperature). After sonication, the lysates were centrifuged (12000 g, 10 min, 4°C). One hundred µL of supernatants were incubated with protein A / G magnetic beads (30 min, 4°C, Dynabeads Protein G, ThermoScientific, USA) for pre-cleavage. Then,

[0011] This supernatant was incubated with 25 µL of immune or pre-immune serum under gentle rotation (7 rpm) overnight at 4°C (to limit proteolysis). The supernatants (now containing the antibody-antigen complexes) were incubated with protein A / G magnetic beads (4.5 mg / mL, ThermoScientific) under gentle rotation (2 h, room temperature). The bead-antibody-antigen complexes were eluted with 35 µL of elution buffer (50 mM glycine, pH 2.8) for 20 min at room temperature to detach the beads from the Ab-Ag complexes. Protein A / G magnetic beads (4.5 mg / mL) were again added to separate the antibodies and antigens. The supernatants containing the antigens were mixed with 4X Laemmli buffer and boiled at 95°C for 10 minutes. Magnetic beads were washed three times with 200 µl of PBS and boiled at 95°C for 10 minutes in 25 µl of 4X Laemmli buffer before performing Western blots.Proteins obtained by immunoprecipitation migrated in a polyacrylamide gel (12%) and the 1D bands (protein-gel) of interest extracted from the SDS-PAGE gel (fragments of about 5 mm in length) stained with blue G250 (ThermoScientific) were reduced with 200 µl of 10 mM dithiothreitol (Euromedex) in 50 mM ammonium bicarbonate buffer (Sigma) at 56°C for 1 hour. Then, alkylation was performed with 55 mM iodoacetamide (Sigma) in the same buffer at room temperature for 30 minutes in the dark. Protein gels were then destained with 100 µL of 5% acetonitrile (15 min), then 50% (30 min) successively in 25 mM ammonium bicarbonate buffer. They were then dehydrated by incubation in 200 μL of 100% acetonitrile for 10 min. The protein gel was digested by overnight incubation with 100 μL of trypsin solution (10ng / μL, Promega) in 25 mM ammonium bicarbonate buffer at 37°C.The resulting peptide mixtures were extracted with 50% acetonitrile in 20 mM ammonium bicarbonate buffer. Finally, the peptides were concentrated by desiccation. The volumes were adjusted to 15 µL with 98% H2O / 2% acetonitrile / 0.05% trifluoroacetic acid solution, if the volume was less than 15 µL for further LC-MS / MS analysis. The trypsin-hydrolyzed proteins were then subjected to LC-MS / MS analysis. Six µL of each hydrolysate was injected into a nanoHPLC (Ultimate 3000, Dionex) on a concentration column to retain the peptides and remove contaminants that could interfere with mass spectrometry analysis. The peptides were then separated according to their.

[0012] hydrophobicity on an analytical column (Acclaim 75µm, 15 cm pepmap C18, 2micro 100 A PN16453, SN10702989). The NanoHPLC was coupled to a nanoESI source, a Q-Orbitrap HFX mass spectrometer (ThermoScientific) which operates in data-dependent mode. At the end of the LC-MS / MS analysis, the samples were analyzed in the PROGENESIS QI software (v 4.0, Nonlinear Dynamics). This software allows both the detection and quantification of all peptide ions detected during the analysis, then the MS / MS analyses carried out in the mass spectrometer are interrogated in the Microtus ochrogaster (meadow vole) database due to its phylogenetic proximity (ATS having no database available). For the identification of the proteins concerned, the search engines Mascot (V. 2.5.1, internally licensed version) and Peaks (v10, internally licensed version) were used.Protein abundance was calculated from the sum of the normalized surface abundances of each peptide for a specific protein for each series. Proteins with a high confidence level (FDR <0.01) were considered positively identified. The maximum fold change (mFC) of proteins detected with male or female immunized sera was compared to the mFC of proteins detected for male or female pre-immunized sera. Statistical analyses were performed using a Mann & Whitney test performed using R software. This approach led to the identification in ATS immune sera of 121 sperm protein antigens that elicited the appearance of anti-sperm antibodies secondary to immunizations. These proteomic experiments and analyses were performed on the Plateforme d'Exploration du Métabolisme, Composante Protéomice (PFEMcp, INRAE, Theix, France).In parallel, the search for sperm ATS antigens was also performed using a conventional two-dimensional electrophoresis technique after isoelectric focusing, followed by Western blot analysis with the immune sera as primary antibody. The spots corresponding to the recognized proteins were then collected on Coomassie blue-stained gels and analyzed by mass spectrometry as described above. Briefly, isoelectric focusing (IEF) was performed on 11 cm long IPG strips (Biorad) (pH 3-10) using 200 μg of protein. IEF was performed using a PROTEAN IEF cell (Biorad) according to the following scheme: passive rehydration (10 h), active rehydration at 50V.

[0013] (12 h), and migration (250 V for 15 min, 8000 V for 2 h 30 min, and a final phase of 8000 V up to a maximum of 35000 V / h). After IEF, the focused strips were rotated in 135 mM dithiothreitol (Euromedex) in equilibration buffer (6 M urea; 2% SDS; 0.375 M Tris-HCl pH 8.8; 20% glycerol, 10 mL for 15 min) and then in 135 mM iodoacetamide (Sigma) in equilibration buffer (10 mL for 15 min). Second dimension gels (1 mm thick, 4-15% acrylamide gradient, Criterion TGX - IPG 11cm+1well precast gels, Biorad) were run (5 h, 80 V) using the Criterion cell (Biorad). Proteins were then transferred to activated PVDF membranes and blocked with TBS-T, 5% w / v BSA.Membranes were hybridized with pre-immune or immune serum (1:1000, overnight, 4°C), anti-beta-Actin (1:2500, Sigma) or anti-GAPDH (1:10000, Sigma) and secondary antibodies were applied after three washing steps in TBS-T: HRP-conjugated rabbit anti-ATS IgG antibody (1:1000, 1 h, RT) or HRP-conjugated anti-rabbit IgG antibody (BI 2407, 1:1000 for beta-actin and 1:5000 for GAPDH, Abliance). Proteins were finally visualized by the ECL technique (Clarity Western ECL Substrate, Biorad) on a ChemiDoc MP Imaging system (Biorad). The pool of these 121 sperm protein antigens was then screened in order to select a pool of relevant antigens (see summary diagram of the identification strategy in Figure 2).The selection criteria used were: the intensity of the immune response obtained; the biological function of the protein, including its involvement in the reproductive function; the tissue and cellular localization, or even the subcellular localization of the protein (membrane, cytosolic, nuclear) when these were known, including, if possible, its restriction to the male genital tract, or even to spermatozoa. This first screen, which ensures part of the targeting specificity (functional targeting), led to the retention of 17 / 121 (14%) of the identified immunogenic sperm antigens (see summary diagram of the identification strategy in Figure 3). Sixteen of these sperm protein antigens are original in the sense that they have not yet been described as being able to initiate an anti-sperm immune response.Only the CATSPER1 antigen has been previously described as being able to induce an anti-sperm immune response and protected for this purpose. We therefore excluded it from the pool of ATS sperm protein antigens leading to the production of anti-sperm antibodies.

[0014] On these 16 identified and selected sperm ATS protein antigens, an in silico analysis of immunogenic epitope prediction was then carried out (software: IEDB and SVMTrip) targeting the search for epitopes preferentially stimulating the B response (antibody response) and on the accessibility of epitopes within the identified proteins (favoring antigenic presentation). In order to search for the best possible species specificity, these selected sperm ATS peptide sequences were then aligned (software: Blastp NCBI) with all known sequences (i.e. present in databases) of homologous proteins in non-target species. A particular focus was made on species sharing the ecosystem (common predators; domestic and livestock animals; humans).Peptide targeting was then performed on ATS peptides with the least primary sequence identity with proteins of non-target species. Confirmation of the immunogenicity of the selected peptides. The peptide epitopes selected using the approach described above were individually tested by parenteral (subcutaneous) immunization of ATS males under the conditions described above (1 primary injection of 300µg followed by a booster at 2-week intervals) to confirm their ability to elicit an antibody response consistent with that obtained in animals with the corresponding whole sperm protein. The presence of serum IgG and mucosal IgA antibodies post-immunization served as confirmation of the immunogenicity of the selected epitopes. Protocols: IgG detection: The detection of serum IgG was performed using a slot-blot technique.Different amounts of the peptide used for immunization (1.25 – 2.5 and 5 µg) or of a non-relevant peptide (negative control in the same amounts) were deposited on a nitrocellulose membrane previously hydrated in PBS. The membrane was then saturated by incubation for 1 h at room temperature in a 5% TBS-T-BSA solution. The membrane was then incubated overnight at 4°C with the serum of a male immunized against the peptide concerned (after dilution to 1 / 250). ème in PBS). After three 5-minute washes at

[0015] After incubation at room temperature in TBS-T, the membrane was incubated with HRP-conjugated rabbit anti-ATS IgG antibody (1:10000 in TBS-T-BSA 2%, 1 h, RT). The final revelation was performed by the ECL technique (Clarity Western ECL Substrate, Biorad) on a ChemiDoc MP Imaging system (Biorad). IgA detection IgA detection on spermatozoa extracted from the cauda epididymis: Before the experiments, the slides (Superfrost, Thermoscientific) were treated with acetone (45 min, RT) and stored in absolute ethanol (-20°C) until use. The cauda epididymis of immunized and non-immunized ATS were squeezed with forceps to expel sperm through the vas deferens, then punctured with a 26G needle in WH buffer and incubated for 30 minutes at room temperature. Sperm were collected by centrifugation (500g, 5 min), washed three times with PBS, spread on slides, and allowed to air dry (1 h, RT).Slides were then washed twice for 5 minutes in PBS without agitation. Saturation was performed with PBS containing 0.1% BSA (1h, RT) and slides were incubated with a goat anti-mouse IgA antibody that recognizes ATS IgA (Abcam ab97235 at 1:20, 1h RT) in a PBS-0.1% BSA solution. Slides were incubated with an AlexaFluor 555-coupled goat anti-IgA antibody (Invitrogen, 1:1000, in PBS 0.1% BSA, 1h, RT). After two washes, nuclei were stained with Hoechst 33342 (1µg / mL in PBS, 5min in the dark, Invitrogen). The slides were finally mounted in an anti-fading solution Citifluor AF100 and Citifluor Tris-MWL 4-88 (ratio 1:9 respectively, Electron Microscopy Sciences) and stored at +4°C until observation by epifluorescence microscopy (Zeiss Imager M2 with Apotome).Detection of IgA in male genital tract tissues: The search for secretory IgA in these tissues was carried out on 5µm thick paraffin sections (testes, epididymides) or on 7µm thick cryosections (prostate and seminal vesicles). The paraffin sections were deparaffinized and subjected to antigen retrieval by immersion in boiling Tris-EDTA buffer for 20 min and then allowed to cool for 30 min at RT. The slides were then washed 2x5 min at RT in water and 1x5 min at RT in.

[0016] PBS. Cryosections were thawed and dried for 30 min at RT before being fixed in a 4% paraformaldehyde solution in PBS for 15 min at RT, then washed 2x5 min in PBS. Both types of slides were then processed according to the same protocol. Endogenous peroxidases were inhibited by incubation for 30 min at RT in a solution of 0.3% H2O2 in PBS and then washed by passage for 5 min at RT in PBS. Tissues were then saturated in a PBS-2.5% horse serum (HS) solution for 1 h at RT and incubated with the HRP-coupled secretory anti-IgA antibody (Abcam Ab17921, mouse anti-human 1:100 in PBS-0.25% HS) overnight at 4°C. After two 5-min washes at RT (Streptavidin and Alexa Fluor™ 555 Tyramide” according to the supplier’s instructions (Invitrogen™ Molecular Probes™). Nuclei were stained with Hoechst 33342 (1µg / mL in PBS, 5 min in the dark, Invitrogen).The slides were finally mounted in an anti-fading solution Citifluor AF100 and Citifluor Tris-MWL 4-88 (ratio 1:9, respectively, Electron Microscopy Sciences) and stored at +4°C until observation by epifluorescence microscopy (Zeiss Imager M2 with Apotome). EXAMPLE 2: CONTRACEPTIVE EFFECT OF IMMUNOGENIC SPERM PEPTIDES The contraceptive effect per se of the peptides according to the invention administered orally could not be tested in an animal facility because ATS do not reproduce in captivity. Therefore, oral immunizations were performed with either individual peptides or a cocktail of peptides. The effects on the quantity and mobility of spermatozoa present in the epididymides of ATS were evaluated, in order to indirectly prove the relevance of the contraceptive strategy using the peptides according to the invention.Indeed, the success of fertilization is closely dependent on the sperm count in the ejaculates and the ability of the spermatozoa to move (motility).

[0017] Peptide(s) tested Form ingested Doses ingested and duration Effects noted post-immunization Control not 12 million spzs* and immunized 38% total mobility SEQ ID NO: 12 1 tablet 1X100mg – 7 weeks 4.8 million spzs and coupled with SEQ ID NO: 15% total mobility 9 2X1 tablet 2X100mg – 7 weeks 1 dissolved tablet 1X100mg – 7 weeks No spermatozoa and given by gavage detected The 11 peptides 1 single oral dose 100mg / peptide – 7 5.5 million spzs and together, each by gavage weeks 21% total mobility coupled with SEQ ID NO: 12 The 11 peptides 1 single oral dose 200mg / peptide – 7 1.7 million spzs and together,each by gavage weeks 17% total motility coupled with SEQ ID NO: 12 *spzs = spermatozoa The various tests reported in the table above show that one or more peptides according to the invention administered orally caused a very significant decrease in both the total quantity of spermatozoa collected in the epididymis (accessory organ of the male genital tract in the terminal part of which spermatozoa are stored between two ejaculations), but also in the percentage of motile spermatozoa. In two cases, no more spermatozoa were detectable. These data demonstrate, if not a total contraceptive effect, at least a very significant decrease in the quantity and motility of spermatozoa, two key criteria for reproductive success.

[0018] List of references 1. Airoldi, JP; Altrocchi, R.; Meylan, A. The burrowing behavior of the European field vole Arvicola terrestris Scherman Shaw (Mammalia, Rodentia). Rev. Suisse Zool.1976, 83, 282–286. 2. Berthier, K.; Piry, S.; Cosson, J.F.; Giraudoux, P.; Foltête, J.-C.; Defaut, R.; Truchetet, D.; Lambin, X. Dispersal, landscape and traveling waves in cyclic vole populations. Ecol. Lett.2014, 17, 53–64. 3. Saucy, F. Description of the multiannual cycles of Arvicola terrestris scherman in western Switzerland using the time series analysis method. EPPO Bull.1988, 18, 401–413. 4. Fichet-Calvet, E.; Pradier, B.; Quéré, JP; Giraudoux, P.; Delattre, P. Landscape composition and vole outbreaks: Evidence from an eight-year study of Arvicola terrestris. Ecography 2000, 23, 659–668. 5. Giraudoux, P.; Delattre, P.; Habert, M.; Quéré, JP; Deblay, S.; Defaut, R.; Duhamel, R.; Moissenet, MF; Salvi, D.; Truchetet, D.Population dynamics of fossorial water voles (Arvicola terrestris 18cherman): A land use and landscape perspective. Agric. Ecosyst. Environ.1997, 66, 47–60. 6. Saucy, F. Density dependence in time series of the fossorial form of the water vole, Arvicola terrestris. Oïkos 1994, 74, 381–392. 7. Courant, F.; Brunet-Lecomte, P.; Volobouev, V.; Chaline, J.; Queré, J.P.; Nadachowski, A.; Montuire, S.; Bao, G.; Viriot, L.; Rausch, R.; et al. Karyological and dental identification of Microtus limnophilus in a large focus of alveolar echinococcosis (Gansu, China). Comptes Rendus Acad. Sci. III 1999, 322, 473–480. 8. Destrez, A. ; Perrot, E. ; Granger, S. ; Gaillard, C. ; Michelin, Y. Les impacts du campagnol terrestrial sur les systèmes fourragers : Le cas de l'élevage bovin allaitant en Bourgogne. Fourrages 2014, 220, 291–296. 9. Viel, J.F. ; Giraudoux, P. ; Abrial, V. ; Bresson-Hadni, S.Water vole (Arvicola terrestrisschern) density as risk factor for human alveolar echinococcosis. Am.J.Trop. Med. Hyg.1999, 61, 559–565. Int. J. Mol. Sci.2021, 22, 996523 of 24.

[0019] 10. Giraudoux, P.; Tremollières, C.; Barbier, B.; Defaut, R.; Rieffel, D.; Bernard, N.; Lucot, E.; Berny, P. Persistence of bromadiolone anticoagulant rodenticide in Arvicola terrestris populations after field control. Approx. Res.2006, 102, 291–298. 11. Berny, PJ; Buronfosse, T.; Buronfosse, F.; Lamarque, F.; Lorgue, G. Field evidence of secondary poisoning of foxes (Vulpes vulpes) and buzzards (Buteo buteo) by bromadiolone, a 4-year survey. Chemosphere 1997, 35, 1817–1829. 12. Fournier-Chambrillon, J. ; Berny, PJ ; Coiffier, O. ; Barbedienne, P. ; Dassé, B. ; Delas, G. ; Galineau, H. ; Mazet, A. ; Pouzenc, P. ; Rosoux, R. ; et al. Evidence of secondary poisoning of free-ranging riparian mustelids by anticoagulant rodenticides in France: Implications for conservation of European mink (Mustela lutreola). J. Wildl. Dis.2004, 40, 688–695. 13. Ruiz-Suarez, N.; Henriquez-Hernandez, LA; Valerón, PF; Boada, LD; Zumbado, M.; Camacho, M.; González, MA; Luzardo, OPAssessment of anticoagulant rodenticide exposure in six raptor species from the Canary Islands (Spain). Sci. Total Environ.2014, 485–486, 371–376. 14. Liu, I.K.; Bernoco, M.; Feldman, M. Contraception in mares heteroimmunized with pig zonae pellucidae. J. Reprod. Fertil.1989, 85, 19–29. 15. Kirkpatrick, J.F.; Turner, J.W., Jr.; Liu, I.K.; Fayrer-Hosken, R. Applications of pig zona pellucida immunocontraception to wildlife fertility control. J. Reprod. Fertil. Suppl.1996, 50, 183–189. 16. Miller, L.A.; Johns, B.E.; Killian, G.J. Long-term effects of PZP immunization on reproduction in white-tailed deer. Vaccine 1999, 18, 568–574. 17. Moore, H.D.; Jenkins, N.M.; Wong, C. Immunocontraception in rodents: A review of the development of a sperm-based immunocontraceptive vaccine for the grey squirrel (Sciurus carolinensis). Reprod. Fertil. Dev.1997, 9, 125–129. 18. Duckworth, J.A.; Buddle, B.M.; Scobie, S. Fertility of brushtail possums (Trichosurus vulpecula) 19cherman19 against sperm. J.Reprod. Immunol.1998, 37, 125–138. 19. Fayrer-Hosken, R.A.; Grobler, D.; van Altena, J.J.; Bertschinger, H.J.; Kirkpatrick, J.F. Immunocontraception of African elephants. Nature 2000, 407, 149.

[0020] 20. Talwar, G.P. Vaccines for control of fertility and hormone-dependent cancers. Immunol. Cell Biol.1997, 75, 184–189. 21. Aitken, R.J.; Paterson, M.; Koothan, P.T. Contraceptive vaccines. Br. Med. Bull.1993, 49, 88–99. 22. Kerr, L.E.; Paterson, M.; Aitken, R.J. Molecular basis of sperm-egg interaction and the prospects for immunocontraception. J. Reprod. Immunol.1998, 40, 103–118. 23. Naz, R.K. Contraceptive vaccines: Success, status, and future perspective. Am. J. Reprod. Immunol.2011, 66, 2–4. 24. Naz, R.K. Antisperm contraceptive vaccines: Where we are and where we are going? Am. J. Reprod. Immunol.2011, 66, 5– 12. 25. Alexander, N.J.; Tung, K.S. Immunological and morphological effects of vasectomy in the rabbit. Anat. Rec.1977, 188, 339– 350. 26. Ansbacher, R. Sperm-agglutinating and sperm-immobilizing antibodies in vasectomized men. Fertil. Steril.1971, 22, 629–632. 27. Tung, K.S.; Alexander, N.J. Immunopathologic studies on vasectomized guinea pigs. Biol. Reprod.1977, 17, 241–254. 28. Cyr, D.G.; Finsson, K.; Dufresne, J.; Gregory, M. The Epididymis: From Molecules to Clinical Practice; Robaire, B., Hinton, B.T., Eds.; Springer Science & Business Media: Berlin / Heidelberg, Germany, 2002; pp.103–118. 29. McLaughlin, E.A.; Aitken, R.J. Is there a role for immunocontraception? Mol. Cell Endocrinol.2011, 335, 78–88. 30. Primakoff, P.; Lathrop,W.; Bronson, R. Identification of human sperm surface glycoproteins recognized by autoantisera from immune infertile men, women, and vasectomized men. Biol. Reprod.1990, 42, 929–942. 31. Asquith, K.L.; Kitchener, A.L.; Kay, D.J. Immunisation of the male tammar wallaby (Macropus eugenii) with spermatozoa elicits epididymal antigen-specific antibody secretion and compromised fertilization rate. J. Reprod. Immunol.2006, 69, 127–147. 32. Tung, K.S.; Goldberg, E.H.; Goldberg, E. Immunobiological consequence of immunization of female mice with homologous spermatozoa: Induction of infertility. J. Reprod. Immunol.1979, 1, 145–158. 33. Castle, P.E.; Whaley, K.J.; Hoen, T.E.; Moench, T.R.; Cone, R.A. Contraceptive effect of sperm-agglutinating monoclonal antibodies in rabbits. Biol. Reprod.1997, 56, 153–159.

[0021] 34. Grignard, E.; Cadet, R.; Saez, F.; Drevet, J.R.; Vernet, P. Identification of sperm antigens as a first step towards the generation of a contraceptive vaccine to decrease fossorial water vole Arvicola terrestris Scherman proliferations. Theriogenology 2007, 68, 779–795. 35. Esmailnejad, A.; Nikahval, B.; Mogheiseh, A.; Karampour, R.; Karami, S. The detection of canine anti-sperm antibody following parenteral immunization of bitches against homogenized whole sperm. Basic Clin. Androl.2020, 30, 1. 36. Naz, R.K.; Phillips, T.M.; Rosenblum, B.B. Characterization of the fertilization antigen 1 for the development of a contraceptive vaccine. Proc. Natl. Acad. Sci. USA 1986, 83, 5713–5717. 37. Ellerman, D.A.; Brantúa, V.S.; Martínez, S.P.; Cohen, D.J.; Conesa, D.; Cuasnicú, P.S. Potential contraceptive use of epididymal proteins: Immunization of male rats with epididymal protein DE inhibits sperm fusion ability. Biol. Reprod.1998, 59, 1029–1036. 38. Jagadish, N.; Rana, R.; Mishra, D.; Garg, M.; Selvi, R.; Suri, A. Characterization of immune response in mice to plasmid DNA encoding human sperm associated antigen 9 (SPAG9). Vaccine 2006, 24, 3695–3703. 39. Wang, M.; Shi, J.L.; Cheng, G.Y.; Hu, Y.Q.; Xu, C. The antibody against a nuclear autoantigenic sperm protein can result in reproductive failure. Asian J. Androl.2009, 11, 183–192. 40. Chorfa et al., Identification of Arvicola terrestris scherman sperm antigens for immune contraceptive purposes. Int. J. Mol. Sci., 2021,22, 99625. 41. Alexander N.J et al. Linear PADRE T Helper Epitope and Carbohydrate B Cell Epitope Conjugates Induce Specific High Titer IgG Antibody Responses. J Immunol 2000, 164 (3), 1625–1633.

Claims

REVENDICATIONS [Revendication 1] Composition of a contraceptive vaccinal compound with the presence of a peptide immunogen specific to the campagnologia Arvicola terrestris scherman selected from the peptides from the sequences suivantes: CHDH (SEQ ID N) O : 1 ; AWAVLGQSRYPCS); CRISP2 (SEQ ID N O : 2 ; MEWSVETTG) ; AKAP3 (SEQ ID N O : 3 ; SESKAQGVKEY) ; PGK2 (SEQ ID N O : 4 ; YNPAKVEAFR); ZPBP2 (SEQ ID N O : 5 ; YTLQGNRQINI); GK2 (SEQ ID N O : 6 ; YEVEKLAREV) ; CFAP44 (SEQ ID N O : 7 ; VYSQFIAEKIA); INSL6 (SEQ ID N O : 8 ; WEPWVPPDYQFEKSNLLPEKTEEFSSRDVHS) ; GAPDHS (SEQ ID N O : 9 ; DLEIQVYQWLGAP); NUP210L (SEQ ID N O : 10 ; KIMFQFLKY); ZP3R (SEQ ID N O: 11; YLPVLMEPQ); and a pharmaceutically acceptable excipient.

2. Contraceptive vaccine composition according to claim 1, comprising the 11 immunogenic peptides of sequences SEQ ID NOs: 1-11.

3. Contraceptive vaccine composition according to claim 1, comprising the 3 immunogenic peptides of sequences SEQ ID NOs: 1, 8 and 10.

4. A contraceptive vaccine composition according to any one of claims 1 to 3, further comprising an antigenic peptide of amino acid sequence AKFVAAWTLKAAA (SEQ ID NO: 12) as a response enhancer.

5. A contraceptive vaccine composition according to any one of claims 1 to 4, for use as a medicament.

6. A contraceptive vaccine composition for use according to claim 5, wherein said medicament is a contraceptive for regulating populations of the common vole Arvicola terrestris scherman.

7. A contraceptive vaccine composition for use according to claim 5 or 6, wherein said medicament is for oral administration.

8. An immunogenic peptide selected from peptides of sequences SEQ ID NOs: 1-11 specific for the common vole Arvicola terrestris scherman.

Images