Composition that induces an immune response to sperm and method of use to decrease conception

Abstract

A peptide consisting essentially of the Izumo1 domain and the Ig-like domain of the Izumo1 (Izumo sperm-egg fusion protein 1) peptide; a nucleic acid encoding same; a vector comprising the nucleic acid; a host cell comprising the vector; a method of producing the peptide; a composition comprising the peptide, vector or host cell; methods of inducing an immune response to Izumo1.

Description

70624-02 COMPOSITION THAT INDUCES AN IMMUNE RESPONSE TO SPERM AND METHOD OF USE TO DECREASE CONCEPTION CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. provisional patent application no.63 / 573,214, which was filed April 2, 2024, and which is hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0002] This disclosure relates to a peptide consisting essentially of the Izumo1 domain and the Ig-like domain of the Izumo1 (Izumo sperm-egg fusion protein 1) protein; a nucleic acid encoding same; a vector comprising the nucleic acid; a host cell comprising the vector; a method of producing the peptide; a composition comprising the peptide, vector or host cell; methods of inducing an immune response to Izumo1. SEQUENCE LISTING

[0003] A computer-readable form (CRF) of the Sequence Listing is submitted with this application. The sequence listing is entitled 70624-02_SEQ_LISTING.xml, was generated on March 24, 2025, and is 47000 bytes in size. The entire content of the sequence listing is incorporated herein by reference in its entirety. BACKGROUND

[0004] Overpopulation of wild animals is an increasing problem caused by inadvertent or intentional introduction of animals in places where they do not have natural predators (e.g., rabbits and horses in Australia), removal of natural predators (e.g., white-tailed deer in North America) and a reduction or fragmentation of natural habitat resulting from human encroachment. Tools are needed to help control the animal populations in a nonlethal and humane manner.

[0005] One such tool is a vaccine that induces long-term reversible infertility. There currently are two types of contraceptive vaccines. One is based on the gonadotropic releasing hormone (GnRH) and consists of a protein conjugated with multiple GnRH peptides and a water-in-oil emulsion adjuvant (AdjuVac). The other is based on a porcine zona pellucida protein (pZP)70624-02 formulation with modified Freund’s adjuvant (see, also, Isojima et al., USPN 5,348,866, which discloses the use of a pZP-4α or -4β polypeptide as a contraceptive composition; Grimes et al., USPN 4,795,634, which discloses a composition comprising anti-ZP anti-idiotypic antibodies; Dunbar, USPN 4,996,297, which discloses a composition comprising rabbit ZP protein; Dunbar, USPN 6,455,041, which discloses the use of ZP1 peptides to induce antibodies that bind mammalian zona pellucida; Dean, USPN 5,626,846, which discloses alloimmunization with ZP, e.g., mouse ZP2 aa 114-129; Dunbar et al., USPN 5,637,300, which discloses the use of recombinant rabbit 55kD ZP protein with altered glycosylation; Brown et al., USPN 5,736,141 and US RE37,224, which discloses the use of ZP-derived antigen and Freund’s adjuvant encapsulated in a liposome, a single dose of which resulted in anti-ZP antibodies being detected in seals for up to 22 months; Harris, USPNs 5,837,497 and 6,001,599, which disclose mammalian ZP proteins for contraception; Habenicht et al., USPAPN 2002 / 004479, which discloses the use of zona pellucida peptides for contraception; and Samoylova et al., USPAPN 2011 / 0311565, which discloses the use of ZP binding peptides / polypeptides to immunize against conception). Both vaccines induce an immune response against self-proteins, which requires a breakdown of immune tolerance and the use of potent adjuvants, and have been used with modest success [1, 2]. The vaccines reduce the likelihood of conception in immunized animals, but the effect does not last long. Furthermore, both vaccines are associated with reactions at the injection site, attributed to the adjuvant, ranging from swelling to abscessation. Since the GnRH vaccine targets hormones, it may affect the secretion of sex hormones, the development and function of the gonads, and cause changes in behavior. Since the pZP-based vaccine is prepared from ovaries collected at slaughterhouses, it presents a risk of supply interruption when infectious diseases break out and inadvertent transmission of infectious agents. In addition, the pZP-based vaccine is rather crude, can vary from one batch to the next, and can damage the ovaries.

[0006] In contrast, immunization of female animals with proteins that are selectively expressed on sperm cells can induce a more robust immune response because they are recognized as foreign by the female immune system. Antibodies against sperm surface proteins can prevent binding of sperm to the oocyte and block fertilization. One of the best characterized sperm proteins is IZUMO1 [3-5]. Naturally occurring anti-IZUMO1 antibodies are associated with infertility in women [6]. Mouse IZUMO1 has a 21 amino acid (aa) signal sequence followed by70624-02 a 22-319 aa extracellular domain. The latter contains an Izumo domain (aa 22-166) and an Ig- like domain (aa 167-251) (Fig.1).

[0007] Immunization of mice with a bacterially expressed recombinant IZUMO1 protein with Freund’s adjuvant (four intraperitoneal injections of 25 μg protein) induced an anti-IZUMO1 antibody response but failed to decrease the fertility [7]. Another study showed that three subcutaneous and intramuscular injections of 50 μg of a bacterially expressed peptide fragment of the Ig-like domain of IZUMO1 (aa 154-265) induced an antibody response and modestly reduced fertility (4 / 8 pregnant mice vs.8 / 8 in the control group), whereas immunizations with the Izumo domain or a membrane proximal domain peptide (aa 262-395) had no effect [8]. A subsequent study from the same group of investigators reported that three injections with 50 μg of a single peptide (aa 196-201) with Freund’s adjuvant induced a 50% reduction of the number of pregnant mice [9]. However, others have shown that the IZUMO1 aa 148-160 segment is directly involved in fusion with the oocyte based on structural and mutational analysis [5], and that the antibodies binding to the Izumo domain inhibit fertilization in vitro

[0010] . In yet another study, mice were immunized three different IZUMO1 peptides derived from the Ig-like and membrane proximal domains of IZUMO1. In this study, four intramuscular injections with 150 μg / peptide in Freund’s adjuvant reduced fertility by about 40%

[0011] . Thus, it is not clear which sequence of IZUMO1 is required to induce antibodies that prevent fertilization. Furthermore, all previous studies have been performed with Freund’s adjuvant, a potent adjuvant, but one that can cause a severe reaction at the injection site.

[0008] Beck et al. (USPN 4,585,651) disclose the vaginal administration of microparticles containing antibody or antigen for active or passive immunization of the internal reproductive organs. The microparticles range from 20 to 70 μm. The matrix of the microparticles is polylactic acid, polyglycolic acid, or a mixture thereof. The antigen can be a sperm antigen. In other embodiments, the antigen can be a bacterial or viral antigen, an enzyme, or a hormone (USPN 4,756,907).

[0009] Beck et al. (USPN 4,732,763) disclose the vaginal administration of microparticles containing anti-B-HCG (anti-β subunit of human chorionic gonadotrophin hormone) antibodies. The microparticles range from 10 to 100 μm. The matrix of the microparticles is polylactic acid, polyglycolic acid, or copolymers of glycolic and lactic acids. The microparticles can further contain a hormone, such as estradiol or progesterone.70624-02

[0010] Talwar (USPN 4,780,312) discloses a polyvalent vaccine comprising a β subunit of HCG and a sperm antigen.

[0011] Nudelman et al. (USPN 5,227,160) discloses the monoclonal antibody designated NUH2, which inactivates human sperm motility, and an isolated antigen that can bind to NUH2.

[0012] Herr et al. (USPNs 5,436,157 and 5,602,005) discloses an intra-acrosomal human sperm antigen and its use as a contraceptive vaccine.

[0013] In view of the above, there remains a need for a composition that can induce an immune response that results in a reduction in the likelihood of conception yet does not suffer from the disadvantages of currently available vaccines. It is an object of the present disclosure to provide such a composition. This and other objects and advantages, as well as inventive features, will be apparent from the detailed description provided herein. SUMMARY

[0014] An isolated or purified peptide consisting essentially of the Izumo1 domain and the Ig- like domain of the Izumo1 (Izumo sperm-egg fusion protein 1) protein is provided. In some embodiments, the Izumo1 domain and the Ig-like domain are at least 90% identical to the Izumo1 domain and the Ig-like domain of the Izumo1 peptide of Mus musculus, Homo sapiens, Rattus norvegicus, Capra hircus, Ovis aries, Bos taurus, Sus scrofa, Equus caballus, Oryctolagus cuniculus, Canis familiaris, or Felis catus. In other embodiments, the Izumo1 domain and the Ig-like domain are at least 95% identical to the Izumo1 domain and the Ig-like domain of the Izumo1 peptide of Mus musculus, Homo sapiens, Rattus norvegicus, Capra hircus, Ovis aries, Bos taurus, Sus scrofa, Equus caballus, Oryctolagus cuniculus, Canis familiaris, or Felis catus. In yet other embodiments, the Izumo1 domain and the Ig-like domain are identical to the Izumo1 domain and the Ig-like domain of the Izumo1 peptide of Mus musculus, Homo sapiens, Rattus norvegicus, Capra hircus, Ovis aries, Bos taurus, Sus scrofa, Equus caballus, Oryctolagus cuniculus, Canis familiaris, or Felis catus. In certain embodiments, the peptide comprises one or more of the following separately contiguous amino acid sequences (a)-(d): (a) KRIT-X-SD [SEQ ID NO: 18], wherein X is D or E, and which can further comprise N-terminal to the K the contiguous amino acid sequence WSFLKDL [SEQ ID NO: 19] or LKDL [SEQ ID NO: 20] and / or which can further comprise C-terminal to the D (i) the contiguous amino acid sequence X-KGELF-X-KEL [SEQ ID NO: 21], wherein the first X is L, V or I and70624-02 the second X is I or V or (ii) the contiguous amino acid sequence X-KGELF [SEQ ID NO: 22], wherein X is L, A or V; (b) CPNKCG [SEQ ID NO: 23], which can further comprise N-terminal to the C the contiguous amino acid sequence FQKEV-X [SEQ ID NO: 24], wherein X is L, Y or F, and / or which can further comprise C-terminal to the G (i) the contiguous amino acid sequence VM-X- QTLISWC [SEQ ID NO: 25], wherein X is S or L, (ii) the contiguous amino acid sequence X- MSQTLIWC-X-KCEKQ [SEQ ID NO: 26], wherein the first X is V or T and the second X is L or N, (iii) the contiguous amino acid sequence LM, TM or VM, or (iv) the amino acid L, T or V; (c) either: GLTDYSFYRVW [SEQ ID NO: 27], which can further comprise N-terminal to the G (i) the contiguous amino acid sequence LSWH-X-AS-X [SEQ ID NO: 28] or (ii) the contiguous amino acid sequence WH-X-AS-X [SEQ ID NO: 29], wherein the first X is R or H and the second X is K or E, and / or which can further comprise C-terminal to the W the contiguous amino acid sequence X-NSSETL [SEQ ID NO: 30], wherein X is E or G; or GATRYSFYRVW [SEQ ID NO: 31], which can further comprise N-terminal to the G (i) the contiguous amino acid sequence LDWHKIAT [SEQ ID NO: 32] and / or which can further comprise C-terminal to the W the contiguous amino acid sequence GSKADSL [SEQ ID NO: 33]; and / or (d) LTK-X-MVGPED-X-G-X-YRC [SEQ ID NO: 34], wherein the first X is S, P or T, the second X is A or Q, and the third X is N, S or T, and which can further comprise N-terminal to the L the contiguous amino acid sequence KGKEPY [SEQ ID NO: 35], KGKEPT [SEQ ID NO: 36] or KGKEAT [SEQ ID NO: 37], wherein: (a) is N-terminal to (b), (c), and / or (d) when present, (b) is C-terminal to (a) and N-terminal to (c) and / or (d) when present, (c) is C-terminal to (a) and / or (b) and N-terminal to (d) when present, and (d) is C-terminal to (a), (b) and / or (c) when present. In certain embodiments, the peptide comprises (i) CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31]. In embodiments, the peptide has the amino acid sequence of amino acids 1-255 of any one of SEQ ID NOS: 2, 4, 6, 7, 8, 9, 10, 11, 12, or 13. In70624-02 embodiments, the peptide has an amino acid sequence that is at least 90% identical to the amino acid sequence of amino acids 1-255 of any one of SEQ ID NOS: 2, 4, 6, 7, 8, 9, 10, 11, 12, or 13. In embodiments, the amino acid sequence of the Izumo1 domain and the amino acid sequence of the Ig-like domain are identical to the amino acid sequence of the Izumo1 domain and the amino acid sequence of the Ig-like domain, respectively, of any one of SEQ ID NOS: 2, 4, 6, 7, 8, 9, 10, 11, 12, or 13. In embodiments thereof, the peptide comprises (i) CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31]. In embodiments, the peptide has an amino acid sequence that is at least 95% identical to the amino acid sequence of amino acids 1-255 of any one of SEQ ID NOS: 2, 4, 6, 7, 8, 9, 10, 11, 12, or 13. In embodiments, the amino acid sequence of the Izumo1 domain and the amino acid sequence of the Ig-like domain are identical to the amino acid sequence of the Izumo1 domain and the amino acid sequence of the Ig-like domain, respectively, of any one of SEQ ID NOS: 2, 4, 6, 7, 8, 9, 10, 11, 12, or 13. In embodiments thereof, the peptide comprises (i) CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31].

[0015] An isolated or purified nucleic acid encoding an above-described peptide is also provided. In embodiments, the nucleic acid has the nucleotide sequence of nucleotides 1-765 of any one of SEQ ID NOS: 1, 3, 5, or 14. In embodiments, the nucleic acid has a nucleotide sequence that is at least 90% identical to nucleotides 1-765 of any one of SEQ ID NOS: 1, 3, 5, or 14. In some embodiments of the foregoing, the nucleotide sequence encoding the Izumo1 domain and the nucleotide sequence encoding the Ig-like domain are identical to the nucleotide sequence encoding the Izumo1 domain and the nucleotide sequence encoding the Ig-like domain, respectively, of any one of SEQ ID NOS: 1, 3, 5, or 14. In some embodiments thereof, the nucleotide sequence encodes (i) CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31]. In embodiments, the nucleic acid has a nucleotide sequence that is at least 95% identical to nucleotides 1-765 of any one of SEQ ID NOS: 1, 3, 5, or 14. In some embodiments of the foregoing, the nucleotide sequence encoding the Izumo1 domain and the nucleotide sequence encoding the Ig-like domain are identical to the nucleotide sequence encoding the Izumo1 domain and the nucleotide sequence encoding the Ig- like domain, respectively, of any one of SEQ ID NOS: 1, 3, 5, or 14. In some embodiments thereof, the nucleotide sequence encodes (i) CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31].70624-02

[0016] In view of the above, also provided is an expression vector comprising the isolated or purified nucleic acid operably linked to a promoter. Further provided is a host cell comprising and expressing the expression vector.

[0017] In further view of the above, a method of producing a peptide consisting essentially of the Izumo1 domain and the Ig-like domain of the Izumo1 (Izumo sperm-egg fusion protein 1) peptide is also provided. The method comprises culturing an above-described host cell in a culture medium. The method can further comprise recovering and purifying the expressed polypeptide.

[0018] In still further view of the above, provided is a composition comprising (i) the peptide, the vector, or host cell, alone or in further combination with (ii) an adjuvant. In embodiments thereof, the adjuvant is NanoST, alone or in further combination with AddaS03 (Invivogen, San Diego, CA), AS03, MF59 or AddaVax.

[0019] A method of inducing an immune response to sperm expressing Izumo1 is also provided. The method comprises administering to a female, which expresses a receptor (Juno, folate receptor 4, folate receptor δ, or IZUMO1R) that binds Izumo1, with an immune response- inducing amount of the above-described, whereupon an immune response to sperm introduced into the reproductive tract of the female is induced. The female can be a deer, a horse, a dog, a cat, a rabbit, a rat, a mouse, a human, a boar, or a pig, for example. The composition can be administered by injection.

[0020] Another method of inducing an immune response to sperm expressing Izumo1 is also provided. The method comprises administering to a pest population of mammals, the females of which express a receptor (Juno, folate receptor 4, folate receptor δ, or IZUMO1R) that binds Izumo1, an above-described host cell, which is a nonpathogenic bacterium or virus or an attenuated pathogenic bacterium or virus. In embodiments, the peptide expressed by the bacterium or virus is at least 90% homologous to the Izumo1 expressed by the males of the pest population of mammals and induces an immune response to Izumo1. In other embodiments, the peptide expressed by the bacterium or virus is at least 95% homologous to the Izumo1 expressed by the males of the pest population of mammals and induces an immune response to Izumo1. In yet other embodiments, the peptide expressed by the bacterium or virus is identical to the Izumo1 expressed by the males of the pest population of mammals and induces an immune response to Izumo1. The host cell can be administered to the pest population of mammals in food pellets.70624-02 The food pellets further comprise an adjuvant, such as NanoST, alone or in further combination with AddaS03. FIGURES

[0021] Fig.1A is a schematic diagram of the mouse Izumo1 (labeled MIZUMO1) peptide. The arrow indicates the portion of the peptide, amino acids 1-255, that was synthesized.

[0022] Fig.1B shows the nucleotide sequence of the mouse Izumo1 peptide shown in Fig.1C [SEQ ID NO: 1].

[0023] Fig.1C shows the amino acid sequence of the mouse Izumo1 peptide containing amino acids 1-255 with a C-terminal 6-His tag [SEQ ID NO: 2]. The theoretical pI / Mw is 6.48 / 30027.45.

[0024] Fig.2 shows SDS-PAGE and Western blot (anti-His Ab) of purified recombinant mouse IZUMO1 peptide.

[0025] Fig.3A shows the effect of treatment of CD-1 mice with a composition comprising mouse IZUMO1 peptide and NanoST / AddaS03 adjuvant on fertility.

[0026] Fig.3B shows the effect of treatment of CD-1 mice with a composition comprising mouse IZUMO1 peptide and NanoST / AddaS03 adjuvant on anti-Izumo1 antibody titer.

[0027] Figs.4A-4C show anti-Izumo1 antibodies (IgG in Fig.4A; IgG1 in Fig.4B; and IgG2a in Fig.4C) in serum of mice immunized with recombinant mouse Izumo1 in combination with AddaS03, NanoST or NanoST / AddaS03. The antibody titers are presented as the geometric mean and 90% confidence interval of six mice per group. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. DESCRIPTION

[0028] The present disclosure is predicated on the male sperm protein Izumo1 (Izumo sperm-egg fusion protein 1 in humans), which is recognized as foreign by the female immune system. It inhibits the fusion of sperm and oocyte. It has been expressed in Chinese hamster ovary (CHO) cells and is well-defined. A peptide containing the Izumo domain and the Ig-like domain has been combined with an effective and safe adjuvant composed of two or three components, namely Nano-11, which is a plant-derived nanoparticle, ADU-S100, which is a STING (stimulator of interferon genes) agonist, and in some instances AddaS03, which is an emulsion.70624-02

[0029] Female mice, which received two injections of the nanoparticulate composition, developed an antibody response and exhibited decreased conception / fertility. A single, controlled-release formulation can be used to maintain wild animal populations, such as horses, deer, and feral swine, and zoo populations, for example.

[0030] In view of the above, provided is an isolated or purified peptide consisting essentially of the Izumo1 domain and the Ig-like domain of the Izumo1 (Izumo sperm-egg fusion protein 1) peptide. Desirably, the peptide is substantially free of proteins, polypeptides, and / or peptides that are not Izumo1 peptides, such that the peptide is at least 90%, 95%, or 98% pure by weight. Put another way, the peptide contains no more than 10%, 5%, or 2% by weight of proteins, polypeptides, and / or peptides that are not Izumo1 peptides. Purity can be determined by SDS polyacrylamide gel electrophoresis and immunoblotting (as exemplified herein). The peptide can be synthesized using a commercially available peptide synthesizer.

[0031] In embodiments of the peptide, the Izumo1 domain and the Ig-like domain are at least 90% identical to the Izumo1 domain and the Ig-like domain of the Izumo1 peptide of Mus musculus, Homo sapiens, Rattus norvegicus, Capra hircus, Ovis aries, Bos taurus, Sus scrofa, Equus caballus, Oryctolagus cuniculus, Canis familiaris, or Felis catus. In other embodiments of the peptide, the Izumo1 domain and the Ig-like domain are at least 95% identical to the Izumo1 domain and the Ig-like domain of the Izumo1 peptide of Mus musculus, Homo sapiens, Rattus norvegicus, Capra hircus, Ovis aries, Bos taurus, Sus scrofa, Equus caballus, Oryctolagus cuniculus, Canis familiaris, or Felis catus. In yet other embodiments of the peptide, the Izumo1 domain and the Ig-like domain are identical to the Izumo1 domain and the Ig-like domain of the Izumo1 peptide of Mus musculus, Homo sapiens, Rattus norvegicus, Capra hircus, Ovis aries, Bos taurus, Sus scrofa, Equus caballus, Oryctolagus cuniculus, Canis familiaris, or Felis catus.

[0032] In embodiments of the peptide, the peptide comprises one or more of the following separately contiguous amino acid sequences (a)-(d): (a) KRIT-X-SD [SEQ ID NO: 18], wherein X is D or E, and which can further comprise N-terminal to the K the contiguous amino acid sequence WSFLKDL [SEQ ID NO: 19] or LKDL [SEQ ID NO: 20] and / or which can further comprise C-terminal to the D (i) the contiguous amino acid sequence X-KGELF-X-KEL [SEQ ID NO: 21], wherein the first X is L, V or I and70624-02 the second X is I or V or (ii) the contiguous amino acid sequence X-KGELF [SEQ ID NO: 22], wherein X is L, A or V; (b) CPNKCG [SEQ ID NO: 23], which can further comprise N-terminal to the C the contiguous amino acid sequence FQKEV-X [SEQ ID NO: 24], wherein X is L, Y or F, and / or which can further comprise C-terminal to the G (i) the contiguous amino acid sequence VM-X- QTLISWC [SEQ ID NO: 25], wherein X is S or L, (ii) the contiguous amino acid sequence X- MSQTLIWC-X-KCEKQ [SEQ ID NO: 26], wherein the first X is V or T and the second X is L or N, (iii) the contiguous amino acid sequence LM, TM or VM, or (iv) the amino acid L, T or V; (c) either: GLTDYSFYRVW [SEQ ID NO: 27], which can further comprise N-terminal to the G (i) the contiguous amino acid sequence LSWH-X-AS-X [SEQ ID NO: 28] or (ii) the contiguous amino acid sequence WH-X-AS-X [SEQ ID NO: 29], wherein the first X is R or H and the second X is K or E, and / or which can further comprise C-terminal to the W the contiguous amino acid sequence X-NSSETL [SEQ ID NO: 30], wherein X is E or G; or GATRYSFYRVW [SEQ ID NO: 31], which can further comprise N-terminal to the G (i) the contiguous amino acid sequence LDWHKIAT [SEQ ID NO: 32] and / or which can further comprise C-terminal to the W the contiguous amino acid sequence GSKADSL [SEQ ID NO: 33]; and / or (d) LTK-X-MVGPED-X-G-X-YRC [SEQ ID NO: 34], wherein the first X is S, P or T, the second X is A or Q, and the third X is N, S or T, and which can further comprise N-terminal to the L the contiguous amino acid sequence KGKEPY [SEQ ID NO: 35], KGKEPT [SEQ ID NO: 36] or KGKEAT [SEQ ID NO: 37], wherein: (a) is N-terminal to (b), (c), and / or (d) when present, (b) is C-terminal to (a) and N-terminal to (c) and / or (d) when present, (c) is C-terminal to (a) and / or (b) and N-terminal to (d) when present, and (d) is C-terminal to (a), (b) and / or (c) when present. In certain embodiments, the peptide comprises (i) CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31]. In embodiments thereof, the peptide comprises (i)70624-02 CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31].

[0033] In embodiments of the peptide, the peptide has the amino acid sequence of amino acids 1- 255 of any one of SEQ ID NOS: 2, 4, 6, 7, 8, 9, 10, 11, 12, or 13. In other embodiments of the peptide, the peptide has an amino acid sequence that is at least 90% identical the amino acid sequence of amino acids 1-255 of any one of SEQ ID NOS: 2, 4, 6, 7, 8, 9, 10, 11, 12, or 13. In embodiments thereof, the amino acid sequence of the Izumo1 domain and the amino acid sequence of the Ig-like domain are identical to the amino acid sequence of the Izumo1 domain and the amino acid sequence of the Ig-like domain, respectively, of any one of SEQ ID NOS: 2, 4, 6, 7, 8, 9, 10, 11, 12, or 13. In embodiments thereof, the peptide comprises (i) CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31].

[0034] In other embodiments of the peptide, the peptide has an amino acid sequence that is at least 90% identical the amino acid sequence of amino acids 1-255 of any one of SEQ ID NOS: 2, 4, 6, 7, 8, 9, 10, 11, 12, or 13. In embodiments thereof, the amino acid sequence of the Izumo1 domain and the amino acid sequence of the Ig-like domain are identical to the amino acid sequence of the Izumo1 domain and the amino acid sequence of the Ig-like domain, respectively, of any one of SEQ ID NOS: 2, 4, 6, 7, 8, 9, 10, 11, 12, or 13. In embodiments thereof, the peptide comprises (i) CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31].

[0035] Naturally occurring amino acid residues in peptides are abbreviated as recommended by the IUPAC-IUB Biochemical Nomenclature Commission as follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I; Methionine is Met or M; Norleucine is NMe; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; Glycine is Gly or G, and X is any amino acid unless otherwise specifically defined herein.

[0036] The amino acid sequence of the peptide can vary, such as by amino acid substitution (e.g., conservative amino acid substitutions), addition, or deletion, and yet retain sufficient immunogenicity to induce an immune response to sperm expressing Izumo1. The peptide can be70624-02 modified; some such modifications can enhance the immunogenicity or anti-fertility effect of the peptide. Unwanted amino acids can be removed by techniques such as limited proteolytic digestion using enzymes (e.g., trypsin or papain). Alternatively, the peptide can be modified using synthetic procedures.

[0037] Desirably, the peptide can induce an immune response to a sperm expressing Izumo1. The peptide can be used alone or in further combination with an adjuvant as a composition with contraceptive effects, such as decreasing or eliminating fertilization of eggs. The peptide desirably decreases sperm-egg binding by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0038] Desirably, the peptide used in a composition with contraceptive effects is derived from the Izumo1 protein of the same species. However, peptides derived from homologous Izumo1 proteins of other species can be used. The species can be a mammal, such as a human, ape, chimpanzee, or rodent, a feline, a canine, or an ungulate (e.g., ruminant or swine). Species of rats include Rattus norvegicus and Rattus rattus. Species of mice include Mus musculus, Mus domesticus, Mus spretus, Mus mecedonicus, Mus hortulanus, Mus molossinus, and Mus castaneous.

[0039] When produced in a heterologous cell, the peptide may exhibit either a “native glycosylation pattern” or a “non-native glycosylation pattern.” The peptide exhibits a native glycosylation pattern if the glycosylation pattern of the peptide is not detectably different from the glycosylation pattern exhibited by the complete Izumo1 protein when that protein is produced in its natural host. The peptide exhibits a non-native glycosylation pattern if the glycosylation pattern of the peptide is detectably different from the glycosylation pattern exhibited by the complete Izumo1 protein when the protein is produced in its natural host. Insect cells and mammalian cells can be used to glycosylate peptides, such that proteins with substantially the same glycosylation patterns as in the host can be obtained.

[0040] A “conservative amino acid substitution” is an amino acid substituted by an alternative amino acid of similar charge density, hydrophilicity / hydrophobicity, size and / or configuration (e.g., Val for Ile). A “nonconservative amino acid substitution” is an amino acid substituted by an alternative amino acid of differing charge density, hydrophilicity / hydrophobicity, size, and or configuration (e.g., Val for Phe). Conservative amino acid substitutions include the exchange of one amino acid for another within one of the following groups:70624-02 (i) small aliphatic, nonpolar, or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (ii) polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; (iii) polar, positively charged residues: His, Arg, and Lys; (iv) large, aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (v) large, aromatic residues: Phe, Tyr and Trp.

[0041] An isolated or purified nucleic acid encoding an above-described peptide is also provided. Desirably, the nucleic acid is substantially free of DNA and RNA that does not encode the Izumo1 peptide, such that the nucleic acid at least 95% or 99% pure. Put another way, the nucleic acid contains no more than 5% or 1% of DNA and / or RNA that does not encode the Izumo1 peptide. In embodiments, the nucleic acid has the nucleotide sequence of nucleotides 1- 765 of any one of SEQ ID NOS: 1, 3, 5, or 14. In other embodiments, the nucleic acid has a nucleotide sequence that is at least 90% identical to the nucleotide sequence of nucleotides 1-765 of any one of SEQ ID NOS: 1, 3, 5, or 14. In embodiments thereof, the nucleotide sequence encoding the Izumo1 domain and the nucleotide sequence encoding the Ig-like domain are identical to the nucleotide sequence encoding the Izumo1 domain and the nucleotide sequence encoding the Ig-like domain, respectively, of any one of SEQ ID NOS: 1, 3, 5, or 14. In embodiments thereof, the nucleotide sequence encodes (i) CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31].

[0042] In other embodiments, the nucleic acid has a nucleotide sequence that is at least 90% identical to the nucleotide sequence of nucleotides 1-765 of any one of SEQ ID NOS: 1, 3, 5, or 14. In embodiments thereof, the nucleotide sequence encoding the Izumo1 domain and the nucleotide sequence encoding the Ig-like domain are identical to the nucleotide sequence encoding the Izumo1 domain and the nucleotide sequence encoding the Ig-like domain, respectively, of any one of SEQ ID NOS: 1, 3, 5, or 14. In embodiments thereof, the nucleotide sequence encodes (i) CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31].

[0043] While specific nucleotide sequences are provided herein, other nucleotide sequences, which encode the same amino acids (or amino acids having at least 90% or at least 95% identity) of the peptide consisting essentially of the Izumo1 domain and the Ig-like domain of the Izumo1 peptide, can be synthesized or obtained due to the degeneracy of the genetic code, with70624-02 consideration given to codon preference (i.e., codon optimization) in host cells for expression of a vector comprising the nucleotide sequence operably linked to a promoter Furthermore, the nucleotide sequence can be modified, e.g., by site-directed mutagenesis, to improve the immunogenicity or the anti-fertility effect of the encoded peptide.

[0044] The nucleic acid can be synthesized using a commercially available DNA synthesis (e.g., such as when the DNA is less than about 200 nucleotides). When the nucleic acid is longer, cDNA can be amplified by PCR.

[0045] The nucleic acids can be used to screen genomic / cDNA libraries to locate Izumo1- encoding nucleotide sequences in other species. Such sequences can be identified by hybridization methods employing high levels of stringency. See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nded. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)).

[0046] Reference sequences are as follows: Mouse (Mus musculus) Izumo1coding domain sequence (CDS; nucleotides 266-1459): 266 atggg gccgcatttt acactcttgc tggcagctct 301 tgccaactgc ctgtgtccag ggaggccctg catcaaatgt gaccagtttg tgacagatgc 361 gctaaagact ttcgaaaaca cttacctgaa tgaccacctg ccacacgaca ttcacaaaaa 421 tgtaatgagg atggtgaacc atgaagtatc gagcttcggc gtagtcactt cggctgagga 481 ttcctatttg ggggccgtgg acgagaacac actggaacaa gcaacctgga gttttctgaa 541 ggatctgaag cgtattacag acagtgactt aaaaggagag ctctttataa aggaactatt 601 gtggatgctt cgtcatcaaa aggacatctt taacaatctt gctagacagt tccaaaagga 661 agttctttgt cccaacaaat gcggagtgat gtcgcagact ttgatctggt gtcttaagtg 721 cgaaaagcag ttgcacattt gtcggaaatc cctagattgt ggagagcgcc acatagaggt 781 acatcgctcg gaagacctgg tcctggactg tctgctcagt tggcatcgtg cttctaaggg 841 acttacagat tacagttttt acagggtttg ggagaacagt tctgagacct tgattgccaa 901 ggggaaagaa ccatatctga ccaagtcgat ggtgggtcca gaggatgctg gcaactaccg 961 ctgtgtgcta gataccatca accaaggtca tgccaccgtc atccgctacg atgtcacagt 1021 attgccccca aagcattcag aggaaaacca accaccgaac atcataaccc aagaggagca 1081 cgagactcct gtccacgtga ctccacagac accaccgggg caggagccag agtcggagct 1141 gtacccggag ctgcacccag agttgtaccc ggagctcata cctacagtcg ctcaaaatcc 1201 agagaaaaaa atgaaaactc gcctcttaat actgttgacc cttggctttg tggttcttgt 1261 ggccagcatc attatctcgg tacttcactt taggaaagtc agcgctaaat tgaagaatgc 1321 aagtgacgaa gtcaaaccca ccgcgtcagg atccaagtca gatcagagtt tgtcccaaca 1381 gatgggattg aagaaggcct ctcaggcaga ttttaactct gactactctg gagataagag 1441 cgaggcaaca gaaaactaa [SEQ ID NO: 16] Mouse (Mus musculus) amino acid sequence Izumo1 (Izumo sperm-egg fusion protein 1); UniProt Q9D9J7) with the first 255 amino acids underlined: MGPHFTLLLAALANCLCPGRPCIKCDQFVTDALKTFENTYLNDHLPHDIHKNVMRMVNHEVSSFGVVTSAEDSYLGA VDENTLEQATWSFLKDLKRITDSDLKGELFIKELLWMLRHQKDIFNNLARQFQKEVLCPNKCGVMSQTLIWCLKCEK QLHICRKSLDCGERHIEVHRSEDLVLDCLLSWHRASKGLTDYSFYRVWENSSETLIAKGKEPYLTKSMVGPEDAGNY70624-02 RCVLDTINQGHATVIRYDVTVLPPKHSEENQPPNIITQEEHETPVHVTPQTPPGQEPESELYPELHPELYPELIPTV AQNPEKKMKTRLLILLTLGFVVLVASIIISVLHFRKVSAKLKNASDEVKPTASGSKSDQSLSQQMGLKKASQADFNS DYSGDKSEATEN [SEQ ID NO: 17] Human (Homo sapiens) Izumo1 CDS (nucleotides 407-1459): 407 atgg ggccgcattt 421 taccctcctg tgtgcggcgc tggccggttg cttgcttcct gccgaggggt gtgttatatg 481 tgacccgtct gtcgtgctgg cgctaaagtc cctggagaaa gattacctgc ctggccacct 541 ggatgcgaag catcacaaag ccatgatgga aagggtagag aatgccgtga aggatttcca 601 ggaactgtcg cttaatgagg atgcctatat gggggtcgtt gatgaggcca cactgcaaaa 661 ggggtcctgg agtttgctga aggatctgaa acgcatcaca gacagtgatg taaaaggcga 721 tctctttgtg aaggagctat tttggatgtt gcacttgcaa aaggaaacct ttgccaccta 781 tgttgctcga ttccaaaaag aggcttattg tcccaacaaa tgtggtgtga tgttgcaaac 841 tctgatctgg tgcaagaact gcaaaaagga ggttcacgct tgtcgaaagt cctacgattg 901 cggggagcgg aatgtggaag ttcctcaaat ggaagacatg atcctggact gtgagttaaa 961 ctggcatcag gcttcggaag gcctcactga ttacagcttt tacagggttt gggggaacaa 1021 tacggagacc ttggtgtcca aggggaaaga ggccaccctg accaagccca tggtgggtcc 1081 agaggatgca ggcagctacc gctgcgagct gggctctgtg aattccagcc cagccacgat 1141 catcaatttt cacgtcacag tgttgcccaa aatgatcaag gaggaaaaac cttctccaaa 1201 tatcgtaacc ccgggggagg cgaccacgga gtcgtccata agcctccagc ctctgcagcc 1261 cgagaaaatg ctggcaagcc gccttctggg gctgctgatc tgcggctccc tagcactgat 1321 aaccggcctt acctttgcga tatttcgtcg aaggaaggtg atcgatttca tcaaatcctc 1381 actgtttggc cttggcagtg gagttgccga gcaaacccag gtcccaaaag aaaaggccac 1441 agattcgagg caacaataa [SEQ ID NO: 3] Human (Homo sapiens) amino acid sequence (Izumo1 (Izumo sperm-egg fusion protein 1); UniProt Q8IYV9-1) with the first 255 amino acids underlined: MGPHFTLLCAALAGCLLPAEGCVICDPSVVLALKSLEKDYLPGHLDAKHHKAMMERVENAVKDFQELSLNEDAYMGV VDEATLQKGSWSLLKDLKRITDSDVKGDLFVKELFWMLHLQKETFATYVARFQKEAYCPNKCGVMLQTLIWCKNCKK EVHACRKSYDCGERNVEVPQMEDMILDCELNWHQASEGLTDYSFYRVWGNNTETLVSKGKEATLTKPMVGPEDAGSY RCELGSVNSSPATIINFHVTVLPKMIKEEKPSPNIVTPGEATTESSISLQPLQPEKMLASRLLGLLICGSLALITGL TFAIFRRRKVIDFIKSSLFGLGSGAAEQTQVPKEKATDSRQQ [SEQ ID NO: 4] Rat (Rattus norvegicus) Izumo1 CDS (nucleotides 65-1216): 65 atgggg ctacatttta cactcttgct ggcagctctt gccaactgcc tgtgtccagc 121 aaggctctgc atcatatgtg acccgtttgt ggtggctgca ataaagactt tggagcagaa 181 ttacctgcct acccacctgg cgcccgagca tcacgaagat gtaatgaaga gggtagagca 241 ggaagtgagg aacttcgctg atctgccctt gaatcagaat acctttctgg gggttgtaga 301 tgaggacaca ctggaacaag catcctggag ttttctgaag gatctgaaac gtatcacgga 361 cagtgacgta aaaggagagc tctttgtaaa ggagctattt tggatgcttc gtttgcaaaa 421 ggacatcttt gccacccttg ttgcacgttt ccaaaaggaa gtttattgtc ctaaccaatg 481 tggcacgatg tcgcagactc tgatctggtg taataagtgt gagaagcaga tgcacttctg 541 tcggaaatcc atggattgtg gagagcgcca gatagaggtg catcgcttgg aagacatggt 601 cttggactgc cagctcagtt ggcatcacgc ttctgaggga cttacggatt acagtttcta 661 cagggtttgg gggaacagtt ctgagacctt gatgtccaag ggaaaagaac cttacctgac 721 caagacgatg gtgggtccag aggatgctgg caactaccgc tgtgagctgg acaccgtcaa 781 cgctggtccc gccaccatca tctactacca tgtcacagta ttgcccccaa ggtctgtaga 841 ggaaaaacca ccgccgaaca tcgtaaccca ggaggaggaa gagacccctg tccaggtgat 901 tgtaccgacg ctggagccag agccggagcc agagcccata cctacagtta cccatcgtcc70624-02 961 agaaaaaaaa ttgaaaagcc gcctcttaat actgttgatc cttggcttcg tggttcttgt 1021 ggccagcgtc attgcctcgg tacttcactt tagaaaaacc agagttaaat cgaagaactc 1081 aaatgtggaa aacaaaacct ctgcggcaga attcaagtca gaggcggaat caccccaaaa 1141 gatgggatcg aggaagctct ctcaggcaga gtttcacact gactcttcgg ataaggttga 1201 ggaagcagat aactaa [SEQ ID NO: 5] Rat (Rattus norvegicus) amino acid sequence Izumo1 (Izumo sperm-egg fusion protein 1); UniProt Q6AY06 with the first 255 amino acids underlined: MGLHFTLLLAALANCLCPARLCIICDPFVVAAIKTLEQNYLPTHLAPEHHEDVMKRVEQEVRNFADLPLNQNTFLGV VDEDTLEQASWSFLKDLKRITDSDVKGELFVKELFWMLRLQKDIFATLVARFQKEVYCPNQCGTMSQTLIWCNKCEK QMHFCRKSMDCGERQIEVHRLEDMVLDCQLSWHHASEGLTDYSFYRVWGNSSETLMSKGKEPYLTKTMVGPEDAGNY RCELDTVNAGPATIIYYHVTVLPPRSVEEKPPPNIVTQEEEETPVQVIVPTLEPEPEPEPIPTVTHRPEKKLKSRLL ILLILGFVVLVASVIASVLHFRKTRVKSKNSNVENKTSAAEFKSEAESPQKMGSRKLSQAEFHTDSSDKVEEADN [SEQ ID NO: 6] Goat (Capra hircus) Izumo1 CDS: see nucleotide sequence EU627101 (UniProt). Goat (Capra hircus) amino acid sequence Izumo 1 (Izumo sperm-egg fusion protein 1); UniProt C6ZEA3 with the first 255 amino acids underlined: MGPWGLPLLVATLAGCVFPARGCVICDPKVREALNSLEADYLPGHLEANHQKKVMEKIKQAVEDFKDLPIDEDSYMG VVDEATLEKASWSLLKDMKRITDSDAKGELFVKEMLWMLHLAKNTFASYAAQFQKEAFCPNKCGLMLQPLIWCSTCQ KQVHACRKSKNCGKREVKVHQMEDMILDCELNWHKISQGLTDYSFYRVWKNNSETLVSRGKEPILTKTMVRPKDAVL PKRIQEEIPSPNTETQDETALGEVAVDLPHTTVEPQTPKPEQLLRRRLFGVLIWGLVVLTVCVLIA [SEQ ID NO: 7] Sheep (Ovis aries) Izumo1 CDS: see nucleotide sequence EU627102 (UniProt). Sheep (Ovis aries) amino acid sequence Izumo 1 (Izumo sperm-egg fusion protein 1); UniProt C6ZEA6 with the first 255 amino acids underlined: MGPWGLPLLVATLAGCLFPARGCVICDPKVREALNSLEADYLPGHLEANHQKKVMEKIKQAVEDFKDLPIDEDSYMG VVDEATLEKASWSLLKDMKRITDSDAKGELFVKEMLWMLHLAKNTFASYAAQFQKEAFCPNKCGLMLQPLIWCSTCQ KQVHACRKSKNCGEREVKVHQMEDMILDCELNWHKISQGLTDYSFYRVWKNNSETLVSRGKEPILTKTMVRPKDAGT YRCELGSIQSSPATIIYFHVTVLPKRIQEEIPSPNTETQDETALGEVAVDLPHITVEPQTPKPEQLLRRRLFRVLIW GLVVLTVCVLIA [SEQ ID NO: 8] Bovine (Bos taurus) amino acid sequence Izumo 1 (Izumo sperm-egg fusion protein 1); UniProt E1BDA8 with the first 255 amino acids underlined: MGPRGLPLLVATLAGCLFPARGCVICDPKVREALNSLEMDYLPGHLEANHHKKVMEKIKQALEDFKDLPIDEDSYMG VVDEATLEKSSWSLLKDMKRITDSDAKGELFVKEMLWMLHLAKNTFASYAAQFQKEAFCPNKCGLMLQPLIWCSTCQ KQVHACRKSKNCGEREVKVHHMEDMILDCELNWHKISQGLTDYSFYRVWKNNSETLVSKGKEPILTKTMVRPKDAGT70624-02 YRCELGSIQSSPATIIYFHVTVLPKRIQEEIPSPNTETQDETALGEVALDRPHTTTDFEPQTPKPEQLLRRRLLGVL IWGLVVLTVGVLTA [SEQ ID NO: 9] Pig (Sus scrofa) amino acid sequence Izumo 1 (Izumo sperm-egg fusion protein 1); UniProt A0A287BMD8 with the first 255 amino acids underlined: MEPQRLSLLVAALAGCLLPARGCVICDPKVVEELSSLETNYLPGHLEAKHHKDVMKRVKQAVKDFQDLPIDEDSYMG VVDEATLEKASWSLLKDLKRITDSDVKGELFVDELFWTLRLAKDTFASYAAQFQKEVFCPNKCGLMLQPLIWCSTCQ KQVHSCRKSRSCGERKVKVHQMEDMILDCELNWHKVSQGLTDYSFYRVWKNNSETLMAKGQEPTLTKTMVSPKDAGT YRCELGTVQSTPATIIHFHVTVLPKRIVEETPSPNTETQDETAPGEVTLDRPQTSATLQSQSPGPEKVLRHRLVGLL IGAIVVLIAGVVTA [SEQ ID NO: 10] Horse (Equus caballus) amino acid sequence Izumo 1 (Izumo sperm-egg fusion protein 1); UniProt A0A3Q2KUM8 with the first 255 amino acids underlined: MGPRLPLLVAALAGCLLSARGCVTCDSEVMEALNSLEKDYLPGHLAPEHHENVMKRVKEAVQNFKELPFDEDSYMGV VDEPTLKKASWNLLKDLKRITESDVKGELFVKELFWMLSLQKDTFASYAAHFQKEDFCPNKCGMMLQTLIWCSNCEK QVHACRKSTDCGERQVKVHRKEDMILDCELNWHRASEGLTDYSFYRVWGKNDETLVSKGKEPTLTKTMVGPEDQGTY RCELGTVQSDPATIIYFHVTVLPQRVEEEIPSPDIPNQGEVTLGHPQSNTSLQYPLSQSPNPVKMLRGRLLGLLIWG CVVLIAGAATVIFCSRSGKMLDSIKSWFDTGTEAAQHPEVPEGVMKSRSK [SEQ ID NO: 11] Rabbit (Oryctolagus cuniculus) amino acid sequence Izumo 1 (Izumo sperm-egg fusion protein 1); UniProt G1TVX5 with the first 255 amino acids underlined: MGPCFALRVAALAGCLSAVGGCVTCDPRVVTALENLEKDYLPSHLDEKHHQHLMKRVHDALKALQELPFSEDSYMGV LDESTLDQVSWSFLKDLKRITDSDIKGELFVKELFWMLNQQKEEFIRIAAQFHKEAYCPNKCGTMLQVLLWCNTCKK EIHPCRKSQDCGEHRSEVHQMDDLILDCKLSWHQASQGLTDYSFYRVWGNNSETLMAKGKAHLLTKPLVGPEDAGTY RCELGTVSSGPATIMRFQVTVLPPKVMEEEPSSNVITNTDGRPGQATPAGSAPSSTLHAPQPENMLRSRLVGLLICC VAVLIAGIATAMLCRGKAMSKTKSSQLDVGSDDEDEETSKNPKKADSTTQ [SEQ ID NO: 12] Dog (Canis familiaris) amino acid sequence Ig-like domain-containing protein (Izumo1); UniProt A0A8I3MBM0 with the first 255 amino acids underlined: MGPQLPLLVAALAGCLLPARGCVTCDAKVVEALYHFEMEYLPSRSQELQGVLDRIKYLLNDFKKIPDLNDQHLGVVD SPTLQKLSVDFLKNLKRITNSEEQGEAFLSQIFWMLIKQKASILTTITQFQKKGFCPNKCGQMLQTLIWCHNCLKRI HTCRKSIDCGVHEVVVHELEDLVLDCELSWHRISAGLNNYTFYRVLGSNSLLLSVGKEPTLTKTMVRLEDAGTYRCE LANMKTSRAAVTHFRVRVLPQRAGGAGRSSPTTTTATTEYEFSTEWDLSLLFPTALANPSECPKSEDVLRGRLIGLL LWGLLVLLIAFVTAILCFRPGKVINYIKAWRTNQKEAAQQAQTAMEPQAALESQGAQKPQSARKPQAAPKPSVPKGK LSASKLHAGSWGRGKG [SEQ ID NO: 13] Cat (Felis catus) Izumo1 CDS: 1 atgggtcccc aagttgccct cctggtggcg gcgctagccg gctgcctgct tcctgcccgg70624-02 61 agctgtatcg cctgtgatga gagggttggg aagcagttgg acgccttgga aaaagaatac 121 ctgcgaaccc accttgcccc cgggcggcac agggaagtga tggaaaagat aagagagacc 181 gtcgacaatt tcaaggacct gccagatata cagtctacct taaatggggc tgtcgatgag 241 gccacaatga aaatggcagc gtcgagtttt ctgaggagtg tgaaacttat cacaaaccgc 301 ggtttaagag atgacacttt cgtgaaggag ttctccgcga tgttgactcg ggaaaaggaa 361 acgtttcggc gcaatgtcgc tcggtttcac ggaggtgagg attcttgtcc caacaaatgt 421 ggtgtattgt tgcggcttct gaggtggtgc gattcttgcc tgctgcacac gtacccttgt 481 cgaaagtcca cagattgtgg agtgcgccaa atcaacgtcc atgaaacgga agacctggtc 541 ctggactgtg agctcgactg gcacaaaatt gctacaggcg cgacccgtta cagcttttac 601 agggtttggg ggagcaaagc tgacagcttg ttgtacacgg ggaaatggcc caccctgacc 661 aagcaactgg tgaggcccgc ggatgcaggt acctaccgct gcgcgttggg cactccggga 721 gtgagcctat tctcggccct ccgttttgaa gtcacagtgt tgcctcggaa aatcatttcg 781 gtgatggtga catctcccaa gtctgcagcc attgtaaccc agccatcaac cattggaacc 841 cagtcaccaa ccaatggaac gcagtcacca gccattggaa ccgaggctga ggaggaaatc 901 tgggatgacc tttccccaac cctcgtgccc tcagagtgtt caaagcccga aaatgtgcag 961 accggccgtc tgatagggct gcttctctgg tgctttttcc tgctgatcat aggatttttc 1021 actggcgtac tttgctttcg gtctgggatg gtgatcgatt ccataaagtc cagattccgc 1081 accgacaagg cagctgctcc acaggaccag cttccacaga gttggctgtc acagaaccag 1141 ctgtcacatg accagctgtc acagagtccg ctgtcagaaa cccaggttcc gaaagctgag 1201 aaacttccga aggaatag [SEQ ID NO: 14] Cat (Felis catus) amino acid sequence (Izumo sperm-egg fusion protein 1-like protein (Izumo1); UniProt A0AA51YG73 with the first 255 amino acids underlined: MGPQVALLVAALAGCLLPARSCIACDERVGKQLDALEKEYLRTHLAPGRHREVMEKIRETVDNFKDLPDIQSTLNGA VDEATMKMAASSFLRSVKLITNRGLRDDTFVKEFSAMLTREKETFRRNVARFHGGEDSCPNKCGVLLRLLRWCDSCL LHTYPCRKSTDCGVRQINVHETEDLVLDCELDWHKIATGATRYSFYRVWGSKADSLLYTGKWPTLTKQLVRPADAGT YRCALGTPGVSLFSALRFEVTVLPRKIISVMVTSPKSAAIVTQPSTIGTQSPTNGTQSPAIGTEAEEEIWDDLSPTL VPSECSKPENVQTGRLIGLLLWCFFLLIIGFFTGVLCFRSGMVIDSIKSRFRTDKAAAPQDQLPQSWLSQNQLSHDQ LSQSPLSETQVPKAEKLPKE [SEQ ID NO: 15]

[0047] An expression vector comprising an above-described nucleic acid operably linked to a promoter is also provided. An expression vector encompasses a DNA molecule, such as a plasmid, bacteriophage, phagemid, virus or other vehicle, which contains a nucleotide sequence encoding the peptide consisting essentially of the Izumo1 domain and the Ig-like domain of the Izumo1 (Izumo sperm-egg fusion protein 1) peptide. The nucleotide sequence is under the control of a functional promoter and, possibly, also an enhancer.

[0048] Examples of phage and viral vectors include, but are not limited to, lambda bacteriophage, EMBL bacteriophage, simian virus 40, bovine papilloma virus, Epstein-Barr virus, adenovirus, herpes virus, vaccinia virus, Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus.

[0049] The expression vector can comprise a selection marker, e.g., an antibiotic resistance gene, and a replication origin for initiation of replication of the vector in a host cell. A promoter is70624-02 present upstream of the peptide-encoding region. A termination codon is present downstream of the peptide-encoding region. Other transcription (e.g., initiation and termination signals), translation (e.g., ribosome entry or binding site and the like), and processing signals (e.g., splice donor or acceptor sites, if necessary, and polyadenylation signals) are properly arranged on the vector.

[0050] The form of the vector can vary with the rationale underlying the introduction of the vector into the host cell. The vector can be closed circular, nicked, or linearized, depending on whether the vector is to be maintained extra-genomically (i.e., as an autonomously replicating vector), integrated as a provirus or prophage, transiently transfected, transiently infected (e.g., replication-deficient or conditionally replicating virus or phage), or stably introduced into the host genome through double or single crossover recombination events.

[0051] A host cell comprising and expressing the expression vector is also provided. Any suitable host cell can be used to produce the peptide. Desirably, the vector is compatible with the cell into which it is introduced, i.e., it can express the peptide in the cell and can be stably maintained or relatively stably maintained in the host cell. Desirably, the host cell is a eukaryotic cell, such as a mammalian cell or mammalian cell line.

[0052] The expression vector can be introduced into a host cell using any suitable method known in the art. Examples of such methods include, but are not limited to, electroporation, transfection, infection, membrane fusion with liposomes, high velocity bombardment with DNA- coated microprojectiles, direct microinjection into single cells, and the like.

[0053] A baculovirus vector can be used to transfer insect cells, such as Sf9 cells, SF21 cells, High FiveTMCells, and MG1 cells. Methods for transfecting and culturing insect cells are described, for example, in USPN 5,278,050 and Summers et al., In: A manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures; Texas Agricultural Experiment Station Bulletin No.1555 (1987).

[0054] A method of producing a peptide consisting essentially of the Izumo1 domain and the Ig- like domain of the Izumo1 (Izumo sperm-egg fusion protein 1) peptide is also provided. The method comprises culturing an above-described host cell in a culture medium. The method can further comprise recovering and purifying the expressed polypeptide.70624-02

[0055] Recovery and purification can be achieved by collecting host cells by centrifugation, disrupting them by conventional means, such as lysozyme and / or ultrasound, extraction, and purification, such as by column chromatography.

[0056] Also provided is a composition comprising (i) an above-described peptide, vector, or host cell, alone or in further combination with (ii) an adjuvant.

[0057] Any suitable adjuvant can be used. Desirably, the adjuvant is NanoST, alone or in further combination with AddaS03 (Invivogen, San Diego, CA). NanoST is an adjuvant comprised of plant-derived nanoparticles (Nano-11) combined with the STING agonist ADU-S100

[0015] . Other examples of adjuvants include, but are not limited to, other oil-in-water emulsion adjuvants such as AS03, MF59 or AddaVax, oil emulsion adjuvants, such as Freund’s complete and incomplete adjuvants, mineral salts, such as silica, kaolin, calcium, aluminum, and carbon, polynucleotides, such as polyIC and poly AU, and other natural substances, such as wax D from M. tuberculosis, substances from C. parvum, B. pertussis, and members of the genus Brucella. See, e.g., Vaccine Design, Powell and Newman, Eds., Plenum Press, NY (1995); pp.821-902, for a general discussion of the preparation of vaccines containing an immunogenic polypeptide.

[0058] Preparations for parenteral administration include sterile and aqueous / non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Carriers for occlusive dressings can be used to increase skin permeability and enhance peptide absorption. Liquid dosage forms for oral administration can comprise a liposomal solution containing a liquid dosage form. Suitable liquid dosage forms include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water. Other ingredients include, but are not limited to, wetting agents, emulsifying agents, suspending agents, stabilizers, and buffers.

[0059] A method of inducing an immune response to sperm expressing Izumo1 is provided. The method comprises administering to a female, which expresses a receptor (Juno, folate receptor 4, folate receptor δ, or IZUMO1R) that binds Izumo1, with an immune response-inducing amount of an above-described composition, whereupon an immune response to sperm introduced into the reproductive tract of the female is induced.70624-02

[0060] The female can be a mammal, such as a human, ape, chimpanzee, a mouse, a rat, a horse, a bovine, a cat, a dog, a deer (e.g., white-tailed deer (Odocoileus virginianus), a rabbit, a goat, a cow, a boar, a pig, a bat, a fox, a raccoon, a squirrel, an opossum, a beaver, a coyote, or a wolf.

[0061] The composition can be administered by any suitable route. A suitable route is injection. Other routes include long-release implants, rapid infusion, intravenous injection / infusion, nasopharyngeal absorption, scarification, dermal absorption, intradermal, subcutaneous, intramuscular, intraperitoneal, intravaginal, and oral, such as in the form of a pharmaceutical composition or as feed (i.e., for non-human animals).

[0062] Desirably, the female only requires a single inoculation. And, while, cross-reactivity is expected between related species, desirably, the female is inoculated with a composition comprising the isolated or purified peptide consisting essentially of the Izumo1 domain and the Ig-like domain of the Izumo peptide from the same species.

[0063] Where necessary and feasible, an initial dose can be supplemented with one, two or more subsequent booster administrations. Many different techniques exist for the timing of administrations when a multiple administration regimen is used. If multiple doses are administered, the doses typically can be spaced 1 month to 6 months apart.

[0064] Another method of inducing an immune response to sperm expressing Izumo1 is also provided. The method comprises administering to a pest population of mammals, the females of which express a receptor (Juno, folate receptor 4, folate receptor δ, or IZUMO1R) that binds Izumo1, an above-described host cell, wherein the host cell is a nonpathogenic or attenuated pathogenic bacterium and the peptide expressed by the bacterium is at least 90% homologous to the Izumo1 expressed by the males of the pest population of mammals and induces an immune response to Izumo1. Examples of bacteria that can be used include, but are not limited to, Salmonella typhimurium (such as strains SL3261 and SL7202), Salmonella typhi, Salmonella flexneri, Listeria monocytogenes, Escherichia coli, Yersinia, Shigella and BCG. The bacteria can be freeze-dried. As an alternative to bacteria, bacteriophage can be used (see, e.g., USPAPN 2011 / 0311565, which published December 22, 2011).

[0065] The pest population of mammals can be mice, rats, horses, donkeys (burros), cats, dogs, deer (e.g., white-tailed deer (Odocoileus virginianus), rabbits, boars, pigs, bats, foxes, raccoons, squirrels, opossums, beavers, coyotes, or wolves.70624-02

[0066] In some embodiments, the peptide expressed by the bacterium can be at least 95% homologous to the Izumo1 expressed by the males of the pest population of mammals and induces an immune response to Izumo1. In other embodiments, the peptide expressed by the bacterium is identical to the Izumo1 expressed by the males of the pest population of mammals and induces an immune response to Izumo1.

[0067] The host cell is administered to the post population of mammals in food pellets. Desirably the food pellets should comprise a stable carrier, be inexpensively and efficiently produced, environmentally safe, and attractive as food. The food pellets can further comprise an adjuvant, such as NanoST, alone or in further combination with AddaS03. See, e.g., USPAPN 2005 / 0191318, published September 1, 2005. Other adjuvants include, but are not limited to, a promiscuous T-cell epitope from tetanus toxin, complete cholera toxin, the beta subunit of cholera toxin, or a genetically or chemically detoxified form of cholera toxin. A promiscuous T- cell epitope is a short sequence that can provide a T-helper response needed for antibody production (see, e.g., O’Hern et al., Vaccine 16: 1761-1766 (1997)). The cholera toxin agents have been shown to be the strongest mucosal adjuvant and can be used in compositions to be administered orally or mucosally. Mucosal adjuvants from bacterial toxins and other sources are described in Piazza, Vaccine 21: 2534-2541 (2001)). EXPERIMENTAL SECTION

[0068] This section serves to illustrate the present disclosure. This section is not intended to limit the scope of the claimed invention in any way. Expression of a recombinant mouse Izumo1 peptide

[0069] A nucleic acid sequence encoding 255 amino acid sequence (amino acids 1-255) of the mouse IZUMO1 peptide with a C-terminal 6-histidine tag was designed and cloned into the pcDNA3.1 plasmid (Invitrogen, Waltham, MA). The plasmid was transformed into Escherichia coli XL 10-gold (Agilent, Santa Clara, CA) following the manufacturer’s instructions. Successful transformants were upscaled to produce large quantities of the plasmid. The plasmid was extracted using PureLink™ HiPure Plasmid Maxiprep Kit (Invitrogen, Waltham, MA). The plasmid was used to transfect Chinese hamster ovary (CHO) cells using the ExpiCHO™ Expression System Kit (Gibco, Waltham, MA). The protein was purified by NiNTA and size70624-02 exclusion column chromatography using the AKTA™ FPLC system (Cytiva, Marlborough, MA). Identity and purity of the protein were determined by SDS-PAGE and immunoblotting. Preparation of immune response-inducing composition

[0070] The mIZUMO1 peptide was dissolved in Tris-saline, pH 7.4, and combined with 50% AddaS03 adjuvant (Invivogen, San Diego, CA) by simple mixing. An alternative adjuvant, NanoST, was prepared by mixing Nano-11 nanoparticles

[0012] with the STING (stimulatory of interferon genes) agonist ADU-S100 (ChemieTek, Indianapolis, IN) for one hour. The IZUMO1 peptide was combined with NanoST by end-over-end rotation for an hour. This resulted in >80% adsorption of ADU-S100 to Nano-11

[0013] . The IZUMO1 / NanoST was then mixed with 50% AddaS03. Administration of immune response-inducing composition to mice and subsequent mating

[0071] Female outbred CD-1 mice (5-week-old) were purchased from Envigo (Indianapolis, IN) and housed in individually ventilated cages with free access to feed and water. Mice were kept for a week to acclimatize with the new environment before any procedures were performed. The mice (n=6 / group) were injected intramuscularly with 50 μl of vaccine in each hindleg (Table 1). This was repeated after 4 weeks. Blood samples were collected 14 days after the second injection. The mice were then mated with male CD-1 mice. At approximately 18 days of pregnancy the female mice were euthanized, and the number of viable pups was counted. Table 1. Experimental groups Group mIZUMO Nano-11 ADU-S100 AddaS03

[0072] Mouse Izumo1 was expressed in Chinese Hamster Ovary cells as previously described. The protein was purified by affinity chromatography. Mice were injected three times at a 3-week interval with 25 μg of mouse Izumo1. The mice developed robust antibody responses against Izumo1 with a significantly stronger titer of IgG2a in mice immunized with NanoST alone or in combination with AddaS03 (Figs.4A-4C).70624-02

[0073] The mice were bred, and the number of pregnancies and the number of live pups were recorded. Table 2. Experimental groups Group # pregnant # of pups % reduction Control 6 / 6 70 -e a a co a acc es o u a e a o a e o e e ective in reducing fertility than vaccines containing AddaS03 as adjuvant. These results further indicate that a 3rdimmunization only gave a marginal improvement over two immunizations. The effect was stronger for AddaS03 adjuvanted vaccines than vaccines formulated with NanoST. It should be noted that a 76% reduction in number of pups is a robust response. It has been stated that “[e]ven immunizations with the whole sperm or their solubilized preparations do not cause a total block in fertility in mice, male or female. The maximum reduction in fertility after immunization with any antigen / sperm preparation is up to 70–75%” (1). Furthermore, computer modeling suggests that at least 50% of female rodents need to be infertile in order to achieve an effect on the population (2). ELISA

[0075] Ninety-six well plates were coated with 2.5 µg / mL of IZUMO1 peptide in carbonate buffer (pH 9.6) at 4 °C overnight. The plates were washed three times with 0.05% Tween 20 in PBS (PBS-T) in a microplate washer (BioTEK, Winooski, VT). The plates were then blocked with 1% BSA in PBS-T (w / v) for 1 hour at 37 °C. After three washes, serum samples diluted 1:10,000 in PBS-T were added to the wells in duplicates, and the plates were incubated for 1 hour. After washing the plates five times, wells were incubated with 1:10,000 peroxidase- conjugated goat anti-mouse IgG (Sigma, St, Louis, MO) in PBS-T for 30 minutes. After five washes, TMB substrate (Sigma) was added. The plates were left at room temperature in the dark for 20 minutes, and 50 µL of 2M sulfuric acid were added to stop the reaction. The absorbance at 450 nm was measured in a microplate reader (BioTEK, Winooski, VT).70624-02

[0076] Genetic deletion of the Izumo1 gene, results in 100% infertility in mice [4]. However, it has been difficult to replicate these results by immunization with the entire protein or selected peptides. We selected a mIZUMO1 peptide that comprises both the Izumo domain and the Ig- like domain of the extracellular portion of the IZUMO1 protein based on the possible role of antibodies against this part of the protein to inhibit fertilization. The mIZUMO1 peptide has a theoretical molecular weight of 30,027. The peptide was successfully expressed and purified as indicated by the correct size and single band on SDS-PAGE and Western blot (Fig.2).

[0077] Mice were immunized with the mIZUMO1 peptide with either AddaS03 adjuvant alone or NanoST and AddaS03. AddaS03 is an oil-in-water emulsion containing squalene oil and α- tocopherol. It is essentially identical to the ASO3 adjuvant that has been used in pandemic H1N1 influenza vaccines in humans. NanoST is comprised of the plant-derived Nano-11 nanoparticles and the synthetic STING agonist ADU-S100. We have shown previously that the adsorption of ADU-S100 or the similar, naturally occurring, STING agonist, cyclic-di-AMP, has a synergistic effect on the immune response [13-15]. We hypothesized that mixing NanoST with AddaS03 would create a safe and effective adjuvant. No adverse effects were observed clinically in the immunized mice.

[0078] Mice immunized with mIZUMO1 with AddaS03 developed a relatively weak antibody response (Fig.3B). In contrast, a stronger response was observed following immunization with mIZUMO1 with NanoST / AddaS03, although with significant variability among individual mice. Five of the six nonimmunized control mice were pregnant, and four of these had a large litter size (14-15 pups) (Fig.3A). In the group immunized with IZUMO1 / AddaS03, two mice did not get pregnant, whereas the number of pups was greatly reduced in the mice that were pregnant (Fig.3A and Table 2). Four of the six mice immunized with mIZUMO1 with NanoST / AddaS03 were not pregnant and the two remaining mice had normal size litters. Overall, there appeared to be a weak correlation between the number of pups and the antibody levels (Fig.3B).

[0079] It has been stated that it has not been possible to achieve 100% infertility by immunization with sperm antigens in mice, possibly because of the high fecundity of mice

[0011] . Previous studies used three or four injections with a higher dose of peptide and Freund’s adjuvant to induce up to 50% reduction in fertility [8, 9, 11]. In contrast, we observed a greater reduction of fertility using just two injections with a smaller dose (25 μg / dose) of a mIZUMO1 peptide. For practical application of such a composition for contraception in wildlife it is70624-02 important to limit the number of injections to one or at most two and to minimize the cost of the vaccine. The markedly improved outcome over previously published results may be attributed to the design of a larger peptide that includes multiple epitopes as well as the use of a novel adjuvant. We have demonstrated that immunization of female mice with an IZUMO1-based peptide construct in combination with a nanoparticle and emulsion adjuvant induced a robust antibody response and significantly reduced fertility. References [1] Kirkpatrick JF, Lyda RO, Frank KM. Contraceptive vaccines for wildlife: a review. Am J Reprod Immunol 2011 Jul; 66(1):40-50. [2] Gupta SK. Zona pellucida glycoproteins: Relevance in fertility and development of contraceptive vaccines. Am J Reprod Immunol 2022 Mar 6:e13535. [3] Aydin H, Sultana A, Li S, Thavalingam A, Lee JE. Molecular architecture of the human sperm IZUMO1 and egg JUNO fertilization complex. Nature 2016 Jun 23; 534(7608):562-5. [4] Inoue N, Ikawa M, Isotani A, Okabe M. The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs. Nature 2005 Mar 10; 434(7030):234-8. [5] Ohto U, Ishida H, Krayukhina E, Uchiyama S, Inoue N, Shimizu T. Structure of IZUMO1-JUNO reveals sperm-oocyte recognition during mammalian fertilization. Nature 2016 Jun 23;534(7608):566-9. [6] Clark S, Naz RK. Presence and incidence of izumo antibodies in sera of immunoinfertile women and men. Am J Reprod Immunol 2013 Mar; 69(3):256-63. [7] Wang DG, Huang TH, Xie QD, An G. Investigation of recombinant mouse sperm protein izumo as a potential immunocontraceptive antigen. Am J Reprod Immunol 2008 Mar; 59(3):225-34. [8] Wang M, Lv Z, Shi J, Hu Y, Xu C. Immunocontraceptive potential of the Ig-like domain of Izumo. Mol Reprod Dev 2009 Aug; 76(8):794-801. [9] Xue F, Wang L, Liu Y, Tang H, Xu W, Xu C. Vaccination with an Epitope Peptide of IZUMO1 to Induce Contraception in Female Mice. Am J Reprod Immunol 2016 Apr; 75(4):474-85.

[0010] Inoue N, Hamada D, Kamikubo H, Hirata K, Kataoka M, Yamamoto M, et al. Molecular dissection of IZUMO1, a sperm protein essential for sperm-egg fusion. Development 2013 Aug; 140(15):3221-9.

[0011] Naz RK. Immunocontraceptive effect of Izumo and enhancement by combination vaccination. Mol Reprod Dev 2008 Feb; 75(2):336-44.

[0012] Lu F, Mencia A, Bi L, Taylor A, Yao Y, HogenEsch H. Dendrimer-like alpha-d-glucan nanoparticles activate dendritic cells and are effective vaccine adjuvants. J Control Release 2015 Apr 28; 204:51-9.

[0013] Patil V, Hernandez-Franco JF, Yadagiri G, Bugybayeva D, Dolatyabi S, Feliciano-Ruiz N, et al. A split influenza vaccine formulated with a combination adjuvant composed of alpha-D-glucan nanoparticles and a STING agonist elicits cross-protective immunity in pigs. J Nanobiotechnology 2022 Nov 11; 20(1):477.

[0014] Hernandez-Franco JF, Mosley YC, Franco J, Ragland D, Yao Y, HogenEsch H. Effective and Safe Stimulation of Humoral and Cell-Mediated Immunity by Intradermal Immunization with a Cyclic Dinucleotide / Nanoparticle Combination Adjuvant. J Immunol 2021 Feb 15; 206(4):700-11.

[0015] Hernandez-Franco JF, Yadagiri G, Patil V, Bugybayeva D, Dolatyabi S, Dumkliang E, et al. Intradermal Vaccination against Influenza with a STING-Targeted Nanoparticle Combination Adjuvant Induces Superior Cross-Protective Humoral Immunity in Swine Compared with Intranasal and Intramuscular Immunization. Vaccines (Basel) 2023 Nov 7; 11(11).70624-02

[0016] Naz RK. Antisperm contraceptive vaccine. In: Krause WKH, Naz RK, editors. Immune Infertility. 2 ed. Cham, Switzerland: Springer International Publishing; 2017. p.249-61.

[0017] Jacoblinnert K, Jacob J, Zhang Z, Hinds LA. The status of fertility control for rodents-recent achievements and future directions. Integr Zool.2022; 17(6):964-80. ENUMERATED EMBODIMENTS The following list of enumerated embodiments presents claims with multiply dependent claims depending from multiply dependent claims for presentation in those jurisdictions where such dependencies are allowed as well as additional claims, which may be pursued during the examination of the application or a divisional thereof. EE1. An isolated or purified peptide consisting essentially of the Izumo1 domain and the Ig-like domain of the Izumo1 (Izumo sperm-egg fusion protein 1) peptide. EE2. The isolated or purified peptide of EE1, wherein the Izumo1 domain and the Ig-like domain are at least 90% identical to the Izumo1 domain and the Ig-like domain of the Izumo1 peptide of Mus musculus, Homo sapiens, Rattus norvegicus, Capra hircus, Ovis aries, Bos taurus, Sus scrofa, Equus caballus, Oryctolagus cuniculus, Canis familiaris, or Felis catus. EE3. The isolated or purified peptide of EE1, wherein the Izumo1 domain and the Ig-like domain are at least 95% identical to the Izumo1 domain and the Ig-like domain of the Izumo1 peptide of Mus musculus, Homo sapiens, Rattus norvegicus, Capra hircus, Ovis aries, Bos taurus, Sus scrofa, Equus caballus, Oryctolagus cuniculus, Canis familiaris, or Felis catus. EE4. The isolated or purified peptide of EE1, wherein the Izumo1 domain and the Ig-like domain are identical to the Izumo1 domain and the Ig-like domain of the Izumo1 peptide of Mus musculus, Homo sapiens, Rattus norvegicus, Capra hircus, Ovis aries, Bos taurus, Sus scrofa, Equus caballus, Oryctolagus cuniculus, Canis familiaris, or Felis catus. EE5. An isolated or purified peptide of any one of EE1-EE4, which comprises one or more of the following separately contiguous amino acid sequences (a)-(d): (a) KRIT-X-SD [SEQ ID NO: 18], wherein X is D or E, and which can further comprise N-terminal to the K the contiguous amino acid sequence WSFLKDL [SEQ ID NO: 19] or70624-02 LKDL [SEQ ID NO: 20] and / or which can further comprise C-terminal to the D (i) the contiguous amino acid sequence X-KGELF-X-KEL [SEQ ID NO: 21], wherein the first X is L, V or I and the second X is I or V or (ii) the contiguous amino acid sequence X-KGELF [SEQ ID NO: 22], wherein X is L, A or V; (b) CPNKCG [SEQ ID NO: 23], which can further comprise N-terminal to the C the contiguous amino acid sequence FQKEV-X [SEQ ID NO:24], wherein X is L, Y or F, and / or which can further comprise C-terminal to the G (i) the contiguous amino acid sequence VM-X- QTLISWC [SEQ ID NO: 25], wherein X is S or L, (ii) the contiguous amino acid sequence X- MSQTLIWC-X-KCEKQ [SEQ ID NO: 26], wherein the first X is V or T and the second X is L or N, (iii) the contiguous amino acid sequence LM, TM or VM, or (iv) the amino acid L, T or V; (c) either: GLTDYSFYRVW [SEQ ID NO: 27], which can further comprise N-terminal to the G (i) the contiguous amino acid sequence LSWH-X-AS-X [SEQ ID NO: 28] or (ii) the contiguous amino acid sequence WH-X-AS-X [SEQ ID NO: 29], wherein the first X is R or H and the second X is K or E, and / or which can further comprise C-terminal to the W the contiguous amino acid sequence X-NSSETL [SEQ ID NO: 30], wherein X is E or G; or GATRYSFYRVW [SEQ ID NO: 31], which can further comprise N-terminal to the G (i) the contiguous amino acid sequence LDWHKIAT [SEQ ID NO: 32] and / or which can further comprise C-terminal to the W the contiguous amino acid sequence GSKADSL [SEQ ID NO: 33]; and / or (d) LTK-X-MVGPED-X-G-X-YRC [SEQ ID NO: 34], wherein the first X is S, P or T, the second X is A or Q, and the third X is N, S or T, and which can further comprise N-terminal to the L the contiguous amino acid sequence KGKEPY [SEQ ID NO: 35], KGKEPT [SEQ ID NO: 36] or KGKEAT [SEQ ID NO: 37], wherein: (a) is N-terminal to (b), (c), and / or (d) when present, (b) is C-terminal to (a) and N-terminal to (c) and / or (d) when present, (c) is C-terminal to (a) and / or (b) and N-terminal to (d) when present, and (d) is C-terminal to (a), (b) and / or (c) when present.70624-02 EE6. The isolated or purified peptide of EE5, which comprises (i) CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31]. EE7. The isolated or purified peptide of EE1, which has the amino acid sequence of amino acids 1-255 of any one of SEQ ID NOS: 2, 4, 6, 7, 8, 9, 10, 11, 12, or 13. EE8. An isolated or purified peptide, which has an amino acid sequence that is at least 90% identical to the amino acid sequence of EE7. EE9. The isolated or purified peptide of EE8, wherein the amino acid sequence of the Izumo1 domain and the amino acid sequence of the Ig-like domain are identical to the amino acid sequence of the Izumo1 domain and the amino acid sequence of the Ig-like domain, respectively, of any one of SEQ ID NOS: 2, 4, 6, 7, 8, 9, 10, 11, 12, or 13. EE10. The isolated or purified peptide of EE9, which comprises (i) CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31]. EE11. An isolated or purified peptide, which has an amino acid sequence that is at least 95% identical to the amino acid sequence of EE7. EE12. The isolated or purified peptide of EE11, wherein the amino acid sequence of the Izumo1 domain and the amino acid sequence of the Ig-like domain are identical to the amino acid sequence of the Izumo1 domain and the amino acid sequence of the Ig-like domain, respectively. of any one of SEQ ID NOS: 2, 4, 6, 7, 8, 9, 10, 11, 12, or 13. EE13. The isolated or purified peptide of EE12, which comprises (i) CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31].70624-02 EE14. An isolated or purified nucleic acid encoding the peptide of any one of EE7-EE13. EE15. The isolated or purified nucleic acid of EE14, which has the nucleotide sequence of nucleotides 1-765 of any one of SEQ ID NOS: 1, 3, 5, or 14. EE16. An isolated or purified nucleic acid, which has a nucleotide sequence that is at least 90% identical to the nucleotide sequence of EE15. EE17. The isolated or purified nucleic acid of EE16, wherein the nucleotide sequence encoding the Izumo1 domain and the nucleotide sequence encoding the Ig-like domain are identical to the nucleotide sequence encoding the Izumo1 domain and the nucleotide sequence encoding the Ig-like domain, respectively, of any one of SEQ ID NOS: 1, 3, 5, or 14. EE18. The isolated or purified nucleic acid of EE17, which comprises a nucleotide sequence encoding (i) CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31]. EE19. An isolated or purified nucleic acid, which has a nucleotide sequence that is at least 95% identical to the nucleotide sequence of EE15. EE20. The isolated or purified nucleic acid of EE19, wherein the nucleotide sequence encoding the Izumo1 domain and the nucleotide sequence encoding the Ig-like domain are identical to the nucleotide sequence encoding the Izumo1 domain and the nucleotide sequence encoding the Ig-like domain, respectively, of any one of SEQ ID NOS: 1, 3, 5, or 14. EE21. The isolated or purified nucleic acid of EE20, which comprises a nucleotide sequence encoding (i) CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31]. EE22. An expression vector comprising the isolated or purified nucleic acid of any one of EE14-EE21 operably linked to a promoter.70624-02 EE23. A host cell comprising and expressing the expression vector of EE22. EE24. A method of producing a peptide consisting essentially of the Izumo1 domain and the Ig-like domain of the Izumo1 (Izumo sperm-egg fusion protein 1) peptide, which method comprises culturing the host cell of EE23 in a culture medium. EE25. The method of EE24, which further comprises recovering and purifying the expressed polypeptide. EE26. A composition comprising (i) the isolated or purified peptide of any one of EE1- EE13, the expression vector of EE22, or the host cell of EE23, alone or in further combination with (ii) an adjuvant. EE27. The composition of EE26, wherein the adjuvant is NanoST, alone or in further combination with AddaS03. EE28. A method of inducing an immune response to sperm expressing Izumo1, which method comprises administering to a female, which expresses a receptor (Juno, folate receptor 4, folate receptor δ, or IZUMO1R) that binds Izumo1, with an immune response-inducing amount of a composition of EE26 or EE27, whereupon an immune response to sperm introduced into the reproductive tract of the female is induced. EE29. The method of EE28, wherein the female is a deer, a horse, a dog, a cat, a rabbit, a rat, a mouse, a human, a boar, or a pig. EE30. The method of EE28 or EE29, wherein the composition is administered by injection. EE31. A method of inducing an immune response to sperm expressing Izumo1, which method comprises administering to a pest population of mammals, the females of which express70624-02 a receptor (Juno, folate receptor 4, folate receptor δ, or IZUMO1R) that binds Izumo1, a host cell of EE23, wherein the host cell is a nonpathogenic or attenuated pathogenic bacterium and the peptide expressed by the bacterium is at least 90% homologous to the Izumo1 expressed by the males of the pest population of mammals and induces an immune response to Izumo1. EE32. The method of EE31, wherein the peptide expressed by the bacterium is at least 95% homologous to the Izumo1 expressed by the males of the pest population of mammals and induces an immune response to Izumo1. EE33. The method of EE31, wherein the peptide expressed by the bacterium is identical to the Izumo1 expressed by the males of the pest population of mammals and induces an immune response to Izumo1. EE34. The method of any one of EE31-EE33, wherein the host cell is administered to the post population of mammals in food pellets. EE35. The method of EE34, wherein the food pellets further comprise an adjuvant. EE36. The method of EE35, wherein the adjuvant is NanoST, alone or in further combination with AddaS03.

[0080] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range.

[0081] Values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y”70624-02 has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0082] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation.

[0083] Any use of section headings and subheadings is solely for ease of reference and is not intended to limit any disclosure made in one section to that section only; rather, any disclosure made under one section heading or subheading is intended to constitute a disclosure under each and every other section heading or subheading.

[0084] Various modifications and variations of the described compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection with specific exemplary embodiments, the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims.

[0085] The terms and expressions, which have been employed, are used as terms of description and not of limitation. In this regard, where certain terms are defined and are described or discussed elsewhere, the definitions and all descriptions and discussions are intended to be attributed to such terms. There also is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof.

[0086] Further, all publications and patents mentioned herein are incorporated by reference in their entireties for all purposes. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

Claims

70624-02 WHAT IS CLAIMED IS:

1. A method of inducing an immune response to sperm expressing Izumo1, which method comprises administering to a female, which expresses a receptor (Juno, folate receptor 4, folate receptor δ, or IZUMO1R) that binds Izumo1, with an immune response-inducing amount of a composition comprising (i) (a) an isolated or purified peptide consisting essentially of the Izumo1 domain and the Ig-like domain of the Izumo1 (Izumo sperm-egg fusion protein 1) peptide, (b) an expression vector comprising a nucleic acid encoding a peptide which has the amino acid sequence of amino acids 1-255 of any one of SEQ ID NOS: 2, 4, 6, 7, 8, 9, 10, 11, 12, or 13 operably linked to a promoter, or (c) a host cell comprising and expressing the expression vector, alone or in further combination with (ii) an adjuvant, whereupon an immune response to sperm introduced into the reproductive tract of the female is induced.

2. The method of claim 1, wherein the adjuvant is NanoST, alone or in further combination with AddaS03.

3. The method of claim 1, wherein the female is a deer, a horse, a dog, a cat, a rabbit, a rat, a mouse, a human, a boar, or a pig.

4. The method of claim 1, wherein the composition is administered by injection.

5. A method of inducing an immune response to sperm expressing Izumo1, which method comprises administering to a pest population of mammals, the females of which express a receptor (Juno, folate receptor 4, folate receptor δ, or IZUMO1R) that binds Izumo1, a host cell comprising and expressing an expression vector comprising a nucleic acid encoding a peptide having the amino acid sequence of amino acids 1-255 of any one of SEQ ID NOS: 2, 4, 6, 7, 8, 9, 10, 11, 12, or 13, wherein the nucleic acid is operably linked to a promoter and wherein the host cell is a nonpathogenic or attenuated pathogenic bacterium and the peptide expressed by the bacterium is at least 90% homologous to the Izumo1 expressed by the males of the pest population of mammals and induces an immune response to Izumo1.70624-02 6. The method of claim 5, wherein the peptide expressed by the bacterium is at least 95% homologous to the Izumo1 expressed by the males of the pest population of mammals and induces an immune response to Izumo1.

7. The method of claim 5, wherein the peptide expressed by the bacterium is identical to the Izumo1 expressed by the males of the pest population of mammals and induces an immune response to Izumo1.

8. The method of claim 5, wherein the host cell is administered to the post population of mammals in food pellets.

9. The method of claim 8, wherein the food pellets further comprise an adjuvant.

10. The method of claim 9, wherein the adjuvant is NanoST, alone or in further combination with AddaS03.

11. A composition comprising (i) (a) an isolated or purified peptide consisting essentially of the Izumo1 domain and the Ig-like domain of the Izumo1 (Izumo sperm-egg fusion protein 1) peptide, (b) an expression vector comprising a nucleic acid encoding a peptide having the amino acid sequence of amino acids 1-255 of any one of SEQ ID NOS: 2, 4, 6, 7, 8, 9, 10, 11, 12, or 13 operably linked to a promoter, or (c) a host cell comprising and expressing the expression vector, alone or in further combination with (ii) an adjuvant.

12. The composition of claim 11, wherein the adjuvant is NanoST, alone or in further combination with AddaS03.

13. An isolated or purified peptide consisting essentially of the Izumo1 domain and the Ig-like domain of the Izumo1 (Izumo sperm-egg fusion protein 1) peptide.

14. The isolated or purified peptide of claim 13, wherein the Izumo1 domain and the Ig- like domain are at least 90% identical to the Izumo1 domain and the Ig-like domain of the70624-02 Izumo1 peptide of Mus musculus, Homo sapiens, Rattus norvegicus, Capra hircus, Ovis aries, Bos taurus, Sus scrofa, Equus caballus, Oryctolagus cuniculus, Canis familiaris, or Felis catus.

15. The isolated or purified peptide of claim 13, wherein the Izumo1 domain and the Ig- like domain are at least 95% identical to the Izumo1 domain and the Ig-like domain of the Izumo1 peptide of Mus musculus, Homo sapiens, Rattus norvegicus, Capra hircus, Ovis aries, Bos taurus, Sus scrofa, Equus caballus, Oryctolagus cuniculus, Canis familiaris, or Felis catus.

16. The isolated or purified peptide of claim 13, wherein the Izumo1 domain and the Ig- like domain are identical to the Izumo1 domain and the Ig-like domain of the Izumo1 peptide of Mus musculus, Homo sapiens, Rattus norvegicus, Capra hircus, Ovis aries, Bos taurus, Sus scrofa, Equus caballus, Oryctolagus cuniculus, Canis familiaris, or Felis catus.

17. The isolated or purified peptide of claim 13, which comprises one or more of the following separately contiguous amino acid sequences (a)-(d): (a) KRIT-X-SD [SEQ ID NO: 18], wherein X is D or E, and which can further comprise N-terminal to the K the contiguous amino acid sequence WSFLKDL [SEQ ID NO: 19] or LKDL [SEQ ID NO: 20] and / or which can further comprise C-terminal to the D (i) the contiguous amino acid sequence X-KGELF-X-KEL [SEQ ID NO: 21], wherein the first X is L, V or I and the second X is I or V or (ii) the contiguous amino acid sequence X-KGELF [SEQ ID NO: 22], wherein X is L, A or V; (b) CPNKCG [SEQ ID NO: 23], which can further comprise N-terminal to the C the contiguous amino acid sequence FQKEV-X [SEQ ID NO: 24], wherein X is L, Y or F, and / or which can further comprise C-terminal to the G (i) the contiguous amino acid sequence VM-X- QTLISWC [SEQ ID NO: 25], wherein X is S or L, (ii) the contiguous amino acid sequence X- MSQTLIWC-X-KCEKQ [SEQ ID NO: 26], wherein the first X is V or T and the second X is L or N, (iii) the contiguous amino acid sequence LM, TM or VM, or (iv) the amino acid L, T or V; (c) either: GLTDYSFYRVW [SEQ ID NO: 27], which can further comprise N-terminal to the G (i) the contiguous amino acid sequence LSWH-X-AS-X [SEQ ID NO: 28] or (ii) the contiguous amino acid sequence WH-X-AS-X [SEQ ID NO: 29], wherein the first X70624-02 is R or H and the second X is K or E, and / or which can further comprise C-terminal to the W the contiguous amino acid sequence X-NSSETL [SEQ ID NO: 30], wherein X is E or G; or GATRYSFYRVW [SEQ ID NO: 27], which can further comprise N-terminal to the G (i) the contiguous amino acid sequence LDWHKIAT [SEQ ID NO: 32] and / or which can further comprise C-terminal to the W the contiguous amino acid sequence GSKADSL [SEQ ID NO: 33]; and / or (d) LTK-X-MVGPED-X-G-X-YRC [SEQ ID NO: 34], wherein the first X is S, P or T, the second X is A or Q, and the third X is N, S or T, and which can further comprise N-terminal to the L the contiguous amino acid sequence KGKEPY [SEQ ID NO: 35], KGKEPT [SEQ ID NO: 36] or KGKEAT [SEQ ID NO: 37], wherein: (a) is N-terminal to (b), (c), and / or (d) when present, (b) is C-terminal to (a) and N-terminal to (c) and / or (d) when present, (c) is C-terminal to (a) and / or (b) and N-terminal to (d) when present, and (d) is C-terminal to (a), (b) and / or (c) when present.

18. The isolated or purified peptide of claim 17, which comprises (i) CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31].

19. The isolated or purified peptide of claim 13, which has the amino acid sequence of amino acids 1-255 of any one of SEQ ID NOS: 2, 4, 6, 7, 8, 9, 10, 11, 12, or 13.

20. An isolated or purified peptide, which has an amino acid sequence that is at least 90% identical to the amino acid sequence of claim 19.

21. The isolated or purified peptide of claim 20, wherein the amino acid sequence of the Izumo1 domain and the amino acid sequence of the Ig-like domain are identical to the amino acid sequence of the Izumo1 domain and the amino acid sequence of the Ig-like domain, respectively, of any one of SEQ ID NOS: 2, 4, 6, 7, 8, 9, 10, 11, 12, or 13.70624-02 22. The isolated or purified peptide of claim 21, which comprises (i) CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31].

23. An isolated or purified peptide, which has an amino acid sequence that is at least 95% identical to the amino acid sequence of claim 19.

24. The isolated or purified peptide of claim 23, wherein the amino acid sequence of the Izumo1 domain and the amino acid sequence of the Ig-like domain are identical to the amino acid sequence of the Izumo1 domain and the amino acid sequence of the Ig-like domain, respectively. of any one of SEQ ID NOS: 2, 4, 6, 7, 8, 9, 10, 11, 12, or 13.

25. The isolated or purified peptide of claim 24, which comprises (i) CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31].

26. An isolated or purified nucleic acid encoding the peptide of claim 19.

27. The isolated or purified nucleic acid of claim 26, which has the nucleotide sequence of nucleotides 1-765 of any one of SEQ ID NOS: 1, 3, 5, or 14.

28. An isolated or purified nucleic acid, which has a nucleotide sequence that is at least 90% identical to the nucleotide sequence of claim 27.

29. The isolated or purified nucleic acid of claim 28, wherein the nucleotide sequence encoding the Izumo1 domain and the nucleotide sequence encoding the Ig-like domain are identical to the nucleotide sequence encoding the Izumo1 domain and the nucleotide sequence encoding the Ig-like domain, respectively, of any one of SEQ ID NOS: 1, 3, 5, or 14.70624-02 30. The isolated or purified nucleic acid of claim 29, which comprises a nucleotide sequence encoding (i) CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31].

31. An isolated or purified nucleic acid, which has a nucleotide sequence that is at least 95% identical to the nucleotide sequence of claim 27.

32. The isolated or purified nucleic acid of claim 31, wherein the nucleotide sequence encoding the Izumo1 domain and the nucleotide sequence encoding the Ig-like domain are identical to the nucleotide sequence encoding the Izumo1 domain and the nucleotide sequence encoding the Ig-like domain, respectively, of any one of SEQ ID NOS: 1, 3, 5, or 14.

33. The isolated or purified nucleic acid of claim 32, which comprises a nucleotide sequence encoding (i) CPNKCG [SEQ ID NO: 23] and (ii) either GLTDYSFYRVW [SEQ ID NO: 27] or GATRYSFYRVW [SEQ ID NO: 31].

34. An expression vector comprising the isolated or purified nucleic acid of claim 26 operably linked to a promoter.

35. A host cell comprising and expressing the expression vector of claim 34.

36. A method of producing a peptide consisting essentially of the Izumo1 domain and the Ig-like domain of the Izumo1 (Izumo sperm-egg fusion protein 1) peptide, which method comprises culturing the host cell of claim 23 in a culture medium.

37. The method of claim 36, which further comprises recovering and purifying the expressed polypeptide.

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