CHARACTERIZATION OF ANTIGENIC / IMMUNOGENIC PEPTIDES FOR THE DEVELOPMENT OF CONTRACEPTIVE VACCINES TO LIMIT THE PULLULATIONS OF THE RODENT ARVICOLA TERRESTRIS SCHERMAN

Abstract

The present invention relates to the definition of a pool of antigenic sperm peptides of high species specificity for the regulation of water vole (Arvicola terrestris scherman) populations by a vaccine approach. Figure 1

Description

Title of the invention: CHARACTERIZATION OF ANTIGENIC / IMMUNOGENIC PEPTIDES FOR THE DEVELOPMENT OF CONTRACEPTIVE VACCINES IN ORDER TO LIMIT THE OUTBREAKS OF THE RODENT Al? V / COLA TERRESTRIS SCHERMAN Technical field of the invention

[0001] The present invention relates to the regulation of populations of the field vole Arvicola terrestris scherman by a vaccination approach.

[0002] 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

[0003] 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 above sea level [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 meadows, causing the regression of legumes and the increase of poor quality grasses and undesirable plants.In cultivated fields, Y ATS can damage cereal crops, orchards, vegetable gardens, vineyards, forests and ornamental gardens [7,8]. One of the consequences of ATS activity in grasslands is the presence of earth mounds (tumuli) resulting from burrowing activity that facilitate the ingestion of soil by grazing animals. This results in an alteration of milk quality, characterized by a reduction in protein content and contamination by butyric acid bacteria. ATS outbreaks also pose a risk to 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 mainly characterized . by hepatic development in tumor form, and for which there is currently no treatment.

[0004] 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

[10] . 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].Furthermore, 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 (water tables, rivers, treatment plants, etc.) by bromadiolone constitutes a new health risk for the population and wildlife

[13] . Recently, in France, the National Agency for Food, Environmental and Occupational Health Safety (ANSES) declared that the use of this chemical product did not exclude "an unacceptable risk for aquatic organisms, terrestrial vertebrates and the contamination of groundwater" (AMM n° 9800526). This situation encourages the development of alternative strategies to combat outbreaks of this rodent, which is harmful to crops and ecosystems.

[0005] In this context, immunocontraception may be an interesting option to limit the reproduction of ATS. Immunocontraception or vaccine contraception involves the administration of an antigen that induces an adaptive immune response, resulting in subfertility or even sterility. Contraceptive vaccines have been used and proven effective in many situations to control the fertility of captive and / or semi-captive wild or domestic animal populations, including horses

[14] , deer [15,16], squirrels

[17] , marsupials

[18] and African elephants

[19] .In brief, three strategies can be used to achieve vaccine-based 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 the male gamete. Both anti-GnRH and anti-ZP3 vaccines, ZP3 being one of the proteins in the zona pellucida surrounding the oocyte in mammals, have been shown to be effective in inducing vaccine-based contraception [20,21]. However, these two immunocontraceptive approaches have a major pitfall in the context that . interests 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 us to envisage a high level of species specificity due to the large number of potential sperm antigenic targets and inter-species sequence polymorphism [22-24].

[0006] While it is rather intuitive that sperm antigens can 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 who 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 increased permeability of the blood-testis-epididymis barriers, a consequence of vasectomy

[28] . ASAs have been identified in some cases of male infertility [29,30], where they have been shown to cause infertility by inhibiting sperm / oocyte binding, reducing sperm motility, reducing sperm penetration into cervical mucus, and altering capacitation and / or the acrosome reaction.

[0007] In this context, immunocontraception studies carried out to date, on different models, have used either whole spermatozoa or selected sperm antigens used individually. As examples, the injection of whole spermatozoa as immunogens has been carried out in the Tammar wallaby

[31] , the mouse

[32] , the rabbit [33,34] and the dog

[35] . 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) has not been analyzed. Only the production of antibodies and the effects on fertility have been described. In rodent models, several sperm antigens (proteins and / or peptides) have been studied separately as potential targets for immunocontraception.Effective immune responses and reduced fertility have been obtained, for example, using the sperm antigens FAI

[36] , CRISP1

[37] , SPAG9

[38] or tNASP

[39] .

[0008] 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

[40] . 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 multi-criteria screen covering: 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

[0009] The present invention consisted, from the sperm antigens previously identified, in the characterization of immunogenic peptide epitopes (= antigenic sperm peptides) specific to the European field vole (EV) with the aim of using them in a vaccination strategy targeting males and females of the species to limit the uncontrolled outbreaks of this harmful rodent in mid-mountain areas without producing any collateral effects on non-target fauna. This strategy is proposed as an alternative or in addition to the chemical control commonly used over the last 30 years but banned on a European scale due to the collateral damage inflicted on non-target fauna and the potential associated health risks for the human population.

[0010] To do this, the inventors parenterally immunized male and female ATS rodents with ATS spermatozoa, 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 121 proteins in total, and a first screening of these sperm antigenic targets was carried out so as to retain only antigens having 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).

[0011] This approach made it possible to reduce the list of potential candidates to 17 proteins. From the peptide sequence of these 17 proteins, using classical computer antigen prediction tools and always keeping the ATS species specificity, 33 potentially antigenic sperm peptides were defined. These 33 immunogenic peptides of ATS sperm antigens concern the following 17 proteins: CHDH, ZPBP2, IZUM01, ZP3R, NUP210L, INSL6, GSK2, PRSS21, PGK2, CFAP44, ACR, AKAP3, ZPBP1, GAPDHS, CRISP2, CRISP4, CATSPER1). For each of these 17 ATS sperm proteins, a species-specific peptide was selected, chemically synthesized and tested for its ability to elicit a humoral (IgG) and mucosal (IgA) immune response in ATS. Males were immunized parenterally (subcutaneous injections) initially to verify the immunogenicity of the 17 selected peptides.

[0012] Thus, all peptides (17) were individually tested for their capacity 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), - target the sperm, - cause notable physiological effects (in particular an alteration of spermatogenesis, and / or a phenotype at the level of the male genital tract).

[0013] 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" (cf. [Fig.l]).

[0014] Following this evaluation, 12 antigenic peptides (included in the first two categories "good and average immunogenic") were retained to constitute the pool of spermatic ATS immunogenic peptides. The CATSPER1 antigen (as well as all the immunogenic peptides which may result from it) already being the subject of protection for its use as an antigen for immunocontraceptive purposes, was excluded.

[0015] The subject of the present invention is therefore a vaccine composition for contraceptive purposes comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 immunogenic sperm peptide(s) chosen from the peptides of the following sequences: CHDH (SEQ ID No.: 1; AWAVLGQSRYPCS); CRISP2 (SEQ ID NO: 2; MEWSVETTG); AKAP3 (SEQ ID NO: 3; SESKAQGVKEY); PGK2 (SEQ ID No.: 4; YNPAKVEAFR); ZPBP2 (SEQ ID No.: 5; YTLQGNRQINI); GK2 (SEQ ID NO: 6; YEVEKLAREV); CFAP44 (SEQ ID No.: 7; VYSQFIAEKIA); INSL6 (SEQ ID No.: 8; WEPWVPPDYQFEKSNLLPEKTEEFSSRDVHS); GAPDHS (SEQ ID No.: 9; DLEIQVYQWLGAP); NUP210L (SEQ ID NO: 10; KIMFQFLKY); ZP3R (SEQ ID NO: 11; YLPVLMEPQ); and a pharmaceutically acceptable excipient.

[0016] 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.

[0017] 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,

[41] ).

[0018] The present invention also relates to a vaccine composition contraceptive 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.).

[0019] The present invention also relates to the immunogenic peptides of sequences SEQ ID NOs: 1-11. BRIEF DESCRIPTION OF THE FIGURES

[0020] [Fig.l] represents the classification of the immunogenicity of the selected immunogenic sperm peptides in ATS.

[0021] [Fig.2] represents a simplified diagram of the strategy for identifying antigenic sperm proteins of ATS.

[0022] [Fig.3] represents a simplified diagram of the strategy for identifying antigenic sperm peptides of AFS with high species specificity and immunogenic potential. EXAMPLES

[0023] EXAMPLE 1: IDENTIFICATION OF IMMUNOGENIC SPERMATIC PEPTIDES (see figures 1 and 2)

[0024] Male and female ATS were collected in open fields in the Puy-de-Dôme department (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 sounder and a trap was placed inside. Only animals of reproductive age, i.e., weighing more than 70 grams, were kept alive and returned 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.

[0025] Whole dATS spermatozoa were used to immunize animals and generate anti-sperm antibodies (15.107 spz / injection in a total volume of 300 pL, corresponding to an immunogenic dose based on protein content, approximately 100 mg). For all experiments, spermatozoa were collected by pressing 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 rnM lactic acid, 20 mM HEPES, pH 7.4). After 30 min at 37°C to release a maximum of gametes, spermatozoa 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.

[0026] Immunizations were performed subcutaneously between the two shoulder blades of male and female animals. Before sperm injection, a fraction of blood was taken from the tail vein to recover the pre-immune sera. Five males and six females were simultaneously immunized. The immunization procedure lasted 6 weeks: on the first day (primary injection), the 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 the immune sera. The animals were then sacrificed.

[0027] The recovered immune sera (and pre-immune sera used as control sera) were then used to identify the ATS sperm proteins (antigens) that provoked an immune response in the immunized individuals by an immunoprecipitation strategy.

[0028] Briefly, spermatozoa from ATS epididymal caudae were collected as previously described, and 10 x 107 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 (12,000 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, 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 added again to separate the antibodies and antigens. The supernatants containing the antigens were mixed with 4X Laemmli buffer and boiled at 95°C for 10 min. The magnetic beads were washed three times with 200 μL of PBS and boiled at 95°C for 10 min in 25 μL of 4X Laemmli buffer before performing Western blots.

[0029] The 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 a 50 mM ammonium bicarbonate buffer (Sigma) at 56°C for 1 hour. Then, alkylation was carried out with 55 mM iodoacetamide (Sigma) in the same buffer at room temperature for 30 minutes in the dark. The protein gels were then destained with 100 μL of 5% acetonitrile (15 min), then 50% (30 min) successively in a 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 (10 μg / 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 pL with 98% H2O / 2% solution. acetonitrile / 0.05% trifluoroacetic acid, if the volume was less than 15 pL for further analysis by LC-MS / MS.

[0030] The trypsin-hydrolyzed proteins were then subjected to LC-MS / MS analysis. Six μL of each hydrolyzate were injected into a nanoHPLC (Ultimate 3000, Dionex) on a concentration column to retain the peptides and eliminate contaminants that could interfere with mass spectrometry analysis. The peptides were then separated according to their hydrophobicity on an analytical column (Acclaim 75pm, 15 cm pepmap Cl8, 2micro 100 A PN16453, SN10702989). The NanoHPLC was coupled to a nanoESI source, a Q-Orbitrap HFX mass spectrometer (ThermoScientific) that operates in data-dependent mode. At the end of the LC-MS / MS analysis, the samples were analyzed in 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 performed 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 used Mascot (V. 2.5.1, version under internal license) and Peaks (vlO, version under internal license) 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 as 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 Metabolic Exploration Platform, Proteomic Component (PFEMcp, INRAE, Theix, France).

[0031] In parallel, the search for ATS sperm antigens was also carried out using a conventional two-dimensional electrophoresis technique after isoelectrofocusing, followed by Western blot analysis with the immune sera as primary antibody. The spots corresponding to the recognized proteins were then taken from 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 pg of protein. IEF was performed using a PROTEAN IEF cell (Biorad) according to the following scheme: passive rehydration (10 h), active rehydration at 50V (12 h), and migration (250 V for 15 min, 8000 V for 2.5 h, and a final phase of 8000 V up to a maximum of 35,000 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 llcm+lwell gels precast, 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).

[0032] 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 [Fig.2]). The selection criteria used were: the intensity of the immune response obtained; the biological function of the protein, in particular 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, in particular, 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 immunogenic sperm antigens identified (see summary diagram of the identification strategy in [Fig.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 already been 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.

[0033] On these 16 identified and selected spermatic 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 the epitopes within the proteins. identified (favoring antigenic presentation). In order to seek 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. Particular focus was made on species sharing the ecosystem (common predators; domestic and livestock animals; humans). Peptide targeting was then done on ATS peptides showing the least primary sequence identity with proteins of non-target species.

[0034] Confirmation of the immunogenic nature of the selected peptides.

[0035] The peptide epitopes selected via the approach described above were individually tested by parenteral (subcutaneous) immunization of ATS males under the conditions described above (1 primary injection of 300 pg followed by a booster at 2-week intervals) in order to confirm their ability to provoke an antibody response consistent with that obtained in animals with the corresponding whole sperm protein. The presence of post-immunization serum IgG and mucosal IgA antibodies served as confirmation of the immunogenic nature of the selected epitopes.

[0036] Protocols:

[0037] IgG detection:

[0038] Detection of serum IgG was performed by 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 in PBS). After three 5-min washes at room temperature in TBS-T, the membrane was incubated with an HRP-conjugated rabbit anti-ATS IgG antibody (1:10,000 in 2% TBS-T-BSA, 1 h, RT). The revelation was finally carried out by the ECL technique (Clarity Western ECL Substrate, Biorad) on a ChemiDoc MP Imaging system (Biorad).

[0039] IgA detection

[0040] Detection of IgA on spermatozoa extracted from the cauda epididymis:

[0041] 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 pressed with forceps to expel the spermatozoa 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 (1h, 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, 1:1,000, in PBS 0.1% BSA, 1h, RT). After two washes, the nuclei were stained with Hoechst 33342 (1pg / mL in PBS, 5 min in the dark, Invitrogen).The slides were finally mounted in an anti-fading solution Citifluor AF 100 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).

[0042] Detection of IgA on male genital tract tissues:

[0043] The search for secretory IgA on these tissues was carried out on 5 μm thick paraffin sections (testes, epididymides) or on 7 μm thick cryosections (prostate and seminal vesicles). The paraffin sections were deparaffinized and subjected to antigen unmasking by immersion in boiling Tris-EDTA buffer for 20 min and then left to cool for 30 min at RT. The slides were then washed 2x5 min at RT in water and 1x5 min at RT in PBS. The cryosections were thawed and then 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 treated 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 PBS-2.5% horse serum (HS) solution for 1 h at RT and then incubated with HRP-coupled secretory anti-IgA antibody (Abcam Ab 17921, 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 (1pg / mL in PBS, 5 min in the dark, Invitrogen). Slides were finally mounted in an “anti-fading” solution Citifluor AF 100 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). List of references .

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Claims

Claims

1. Contraceptive vaccine composition comprising at least one immunogenic peptide chosen from the peptides of the following sequences: CHDH (SEQID No.: 1; AWAVLGQSRYPCS); CRISP2 (SEQ ID NO: 2; MEWSVETTG); AKAP3 (SEQ ID NO: 3; SESKAQGVKEY); PGK2 (SEQ ID No.: 4; YNPAKVEAFR); ZPBP2 (SEQ ID No.: 5; YTLQGNRQINI); GK2 (SEQ ID NO: 6; YEVEKLAREV); CFAP44 (SEQ ID No.: 7; VYSQFIAEKIA); INSL6 (SEQ ID No.: 8; WEPWVPPDYQFEKSNLLPEKTEEFSS RD VHS); GAPDHS (SEQ ID No.: 9; DLEIQVYQWLGAP); NUP210L (SEQ ID NO: 10; KIMFQFLKY); ZP3R (SEQ ID NO: 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 activator.

5. Vaccine composition for contraceptive purposes 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 water vole Arvicola terrestris scherman.

7. A contraceptive vaccine composition for use according to claim 5 or 6, wherein said medicament is in the form of tablets, granules, or gummies.

8. Immunogenic peptide selected from the peptides of sequences SEQ ID NOs: 1-11.

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