SPERM SPECIFIC ANTIGENS 4.1 AND 6.7, DNA ENCODING THEM AND CONTRACEPTIVE VACCINES COMPRISING SAID SPERM ANTIGENS

Abstract: The present invention is directed to mammalian sperm specific proteins 4.1 and 6.7, and nucleotide sequences encoding said proteins, as well as pharmaceutical formulations, in particular vaccines comprising said proteins. The proteins 4.1 and 6.7 are sperm specific and can be used in a vaccine to prevent or reduce fertility in subjects.

Description: Sperm Specific Antigens 4.1 and 6.7, DNA Encoding Them And Contraceptive Vaccines Comprising Said Sperm Antigens The present invention is directed to the isolation and cloning of DNA sequences encoding novel sperm specific antigens, the expression and use of said novel sperm specific antigens, in particular the use of said novel sperm specific antigens in a contraceptive vaccines, and pharmaceutical formulations comprising said novel sperm specific antigens . Contraceptive vaccines provide an alternative approach to the well established use of (steroidal) hormonal contraceptives for the regulation of fertility. Contraceptive vaccines display their mode of action via induction of an immune response to the antigens present in the vaccine. These antigens can be derived from either the zona pellucida, a layer of glycoproteins that surrounds the oocyte, or the antigens carried by the sperm. The resulting antibodies will coat either the oocyte or the spermatozoa and as such interfere with the recognition and/or interaction of the male and female gametes during the fertilization process, thus preventing fertilization. Contraceptive vaccines which are based on one or more glycoproteins from the zona pellucida, however have the serious disadvantage of inducing strong autoimmune reactions, resulting in total depletion of the premordial follicles in the ovaries. Due to this unwanted side effect contraceptive vaccines which target the zona pellucida of the oocyte are unsuitable for the regulation of fertility in females, especially human females. On the other hand, a contraceptive vaccine for females which targets the spermatozoa would not have the disadvantages of a vaccine which targets the zona pellucida. Several antigens for use in a contraceptive anti-sperm vaccine have been suggested. WO 90/09802 describes the use of sperm protein SP10 in a contraceptive vaccine. WO 95/15764 describes the use of the sperm protein Spl7 in contraceptive vaccines. One of the difficulties often observed with contraceptive anti-sperm vaccines is the individual variation in immune response between females. Not every female will elicit an adequate immune response to a specific antigen, and the use of a contraceptive vaccine based on a single sperm antigen has the potential risk that it will not prevent the fusion of oocyte and sperm cell effectively in a small group of women. To reduce the individual variation amongst vaccinated subjects, effective anti-sperm vaccines should be based on multiple antigens, so as to be sure that each individual female will elicit an effective immune response to at least one sperm antigen present in the vaccine. The manufacture of such a multiple vaccine requires the availability of many sperm antigens, thus creating a need to identify a broad range of sperm antigens. In order to be effective, a contraceptive anti-sperm vaccine must elicit a very specific immune response to the spermatozoa, and any cross-reaction with other tissues must be avoided. Hence it is a prerequisite that the antigens which can be used in a contraceptive anti-sperm vaccine are sperm specific. Since the spermatozoa carry numerous antigens, many of which are shared by cells of somatic tissues, the identification of a sperm specific antigen is a careful and important step towards the development of a contraceptive vaccine. Besides being sperm specific, the sperm antigen must be able to raise antibodies that interfere or prevent the fusion of oocyte and sperm cell. If this requirement is not fulfilled, the anti-sperm vaccine will induce an immune response upon administration to the female without subsequently having an effect on the fertility of the female . It is an object of the invention to provide sperm proteins specific for the sperm cell. It is a further object of the invention to provide for sperm specific proteins which induce antibodies that prevent the fusion of oocyte and sperm cell, and which are suitable for use as antigens in a contraceptive vaccine. The present invention provides for such sperm specific antigens . In one aspect the invention provides for a nucleotide sequence encoding mammalian sperm proteins 4.1 and 6.7. Preferably, the nucleotide sequence codes for sperm proteins 4.1 or 6.7 of human or marmoset origin. More preferably, the nucleotide sequence coding for sperm protein 4.1 comprises the nucleotide sequence depicted in SEQ ID NO 1, 11 or 15. The nucleotide sequence coding for sperm protein 6.7 preferably comprises the nucleotide sequence depicted in SEQ ID NO 3, 4, 7, 8 or 13. Two nucleotide sequences were found that code for allelic variants of protein 6.7; these allelic variants result from alternative splicing of the mRNA encoding protein 6.7. In humans the nucleotide sequences depicted in SEQ ID 7 and 8, respectively represent the allelic variants encoding protein 6.7 whereas similar allelic nucleotide sequence variants encoding protein 6.7 in marmoset are depicted in SEQ ID NO 3 and 4, respectively. It is well known that as result of the degeneracy of the genetic code there are various codons which code for a specific amino acid. Therefore, the present invention also encompasses nucleotide sequences which contain alternative codons which code for the same identical amino acid. It must furthermore be understood that the nucleotide sequences of the present invention are not only limited to the specific sequences depicted in the sequence listing, but also encompass nucleotide sequences having a substantial sequence similarity with one of the nucleotide sequences depicted in SEQ ID NO 1, 3, 4, 7, 8, 11, 13 or 15. Such nucleotide sequences are considered "homologous" nucleotide sequences and are characterized by their ability to hybridize to the nucleotide sequences depicted in SEQ ID NO 1, 3, 4, 7, 8, 11, 13 or 15 of the present invention. Such hybridizing techniques are routine practice for the skilled person and standard hybridization techniques are described in "Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989)"; and "Current protocols in Molecular Biology, Volumes 1, 2 and 3.(ed. board: Ausubel, F.M. et al.) John Wiley and Sons Inc. (1993 and later supplements) . The "homologous" nucleotide sequences according to the invention encode for a protein which has substantially the same physical properties, in particular immunological properties, and structure as the sperm specific proteins 4.1 or 6.7 depicted by the amino acid sequence of SEQ ID NO. 2, 12 or 16, and SEQ ID NO. 5, 6, 9, 10 or 14, respectively. Preferably the nucleotide sequences within the scope of the present invention have a sequence similarity of 60%, more preferably 70%, highly preferably 80% with any of the nucleotide sequences shown in SEQ ID NO 1, 3, 4, 7, 8, 11, 13 or 15. Most preferred the nucleotide sequences have a sequence similarity of 90% or more with any of the nucleotide sequences shown in SEQ ID NO 1, 3, 4, 7, 8, 11, 13 or 15. Fragments of the nucleotide sequences according to the invention are also within the scope of the present invention and the term "fragments" refers to any subset of nucleotides of the nucleotide sequences encoding sperm proteins 4.1 or 6.7. In a second aspect, the invention provides for mammalian sperm proteins 4.1 and/or 6.7. Preferably the sperm protein 4.1 and/or 6.7 are encoded for by any of the nucleotides sequences according to the present invention. More preferably, sperm protein 4.1 comprises the amino acid sequence depicted in SEQ ID NO 2, SEQ ID NO 12 or SEQ ID NO 16 and sperm protein 6.7 comprises the amino acid sequence depicted in SEQ ID NO 5, 6, 9, 10 or 14. Due to alternative splicing two allelic forms of protein 6.7 exist. In humans the two allelic variants of protein 6.7 are depicted in SEQ ID NO 9 and 10, respectively; in marmoset the two allelic forms of protein 6.7 have amino acid sequences shown in SEQ ID NO 5 and 6, respectively. Also included within the scope of the invention are proteins having an amino acid sequence which shows a substantial sequence similarity with one of the amino acid sequences depicted in SEQ ID NO 2, 5, 6, 9, 10, 12, 14 and 16 provided that the physical properties, in particular the immunological properties, of such proteins are substantially the same as the sperm specific proteins 4.1 or 6.7. It is well known, for instance, that conservative substitution of amino acids not necessarily cause a change in the all round properties of a given amino acid sequence. For example, valine, arginine or asparagine can be substituted for leucine, lysine, glutamine respectively in a given amino acid sequence, without resulting in a change of properties of the protein. See Dayhoff, M.O., Atlas of protein sequence and structure, vol.5 (suppl.3); National Biomedical Research Foundation (1978) . Preferably the sequence similarity between the amino acid sequences of the proteins according to the inventions and any of the amino acid sequences shown in SEQ ID NO 2, 5, 6, 9, 10, 12, 14 or 16 is 60%, more preferably 70%, highly preferable 80%. Mostly preferred are proteins which have an amino acid sequence that has a sequence similarity of 90% or more with any of the amino acid sequences depicted in SEQ ID NO 2, 5, 6, 9, 10, 12, 14 or 16. Fragments of the proteins according to the invention are also within the scope of the present invention and the term "fragments" refers to any subset of amino acid residues of the sperm proteins 4.1 or 6.7. The sperm proteins 4.1 and 6.7 according to the invention are sperm specific and can induce the production of antibodies that prevent the fusion of oocyte and sperm cell. Hence the proteins according to the invention can be suitably used as sperm antigens in a contraceptive vaccine. The proteins according to the invention can be prepared by well known organic chemical methods for protein synthesis such as, for example, solid-phase peptide synthesis described fro instance in J. Amer. Chem. Soc.8_5 : 2149 (1963) and Int. J. Peptide Protein Res. ^35:161-214 (1990). The most practical approach is to produce these proteins by expression of the DNA encoding the desired protein. See "Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, latest edition"; and "Current protocols in Molecular Biology, Volumes 1, 2 and 3.(ed. board: Ausubel, F.M. et al.) John Wiley and Sons Inc. (1993 and later supplements). A wide variety of host cell and expression vectors may be usefully employed in cloning the nucleotide sequence coding for the proteins of the invention. For example, useful expression vectors may include chromosomal, non- chromosomal and synthetic DNA sequences such as various known bacterial plasmids and wider host range plasmids and vectors derived from combinations of plasmids and phage or virus DNA. Useful hosts may include prokaryotic host cells such as bacterial cells, eukaryotic host cells such as yeast's and other fungi, insect, plant or mammalian cells, including HeLa cells and Chinese Hamster Ovary (CHO) cells. Expression vectors for use in expression of the sperm specific proteins of the present invention will further comprise control sequences operably linked to the nucleic acid sequence coding for the sperm specific proteins. Such control sequences generally comprise a promoter sequence and sequences which regulate and/or enhance expression levels. Furthermore an origin of replication and/or a dominant selection marker are often present in such vectors. Of course control and other sequences can vary depending on the host cell selected. Recombinant expression vectors which comprise one or more nucleotide sequences according to the invention and host cells which contain one or more nucleotide sequences or an expression vector according to the invention, said host cell being capable of expressing the encoded sperm protein 4.1 and/or 6.7 are also within the scope of the invention. The isolated proteins according to the invention, produced either via organic chemical methods or via recombinant DNA technology have the advantage that these proteins are free from other sperm proteins. If small amounts of other sperm proteins, especially sperm proteins which are not sperm specific, are present in the contraceptive vaccine, antibodies are produced that react with somatic tissues in which those proteins are present as well. Thus the present invention also provides for a safe way of developing a contraceptive vaccine that will not result in an auto-immune response to somatic tissues. The sperm specific proteins according to the invention can be used in a contraceptive vaccine. Pharmaceutical formulations , more specifically vaccines comprising the sperm specific proteins according to the invention and a pharmaceutical carrier are also within the scope of the invention. Pharmaceutical formulations and vaccines according to the invention can be administered to subjects in order to induce a systemic as well as a mucosal immune response in said subjects. Particular interesting are vaccines according to the invention which induce a mucosal immune response after administration. Pharmaceutical acceptable carriers are well known to those skilled in the art and include, for example, sterile salin, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil and water. In addition the pharmaceutical composition, more specifically, the vaccine according to the invention may comprise one or more adjuvants. Suitable adjuvants include, amongst others, aluminum hydroxide, aluminum phosphate, amphigen, tocophenols, monophosphenyl lipid A, muramyl dipeptide and saponins such as Quill A. Of particular interest are adjuvants that stimulate a mucosal immune response to the vaccine according to the invention. Suitable mucosal adjuvants are, for example, the B-subunits of cholera toxin and E. coli heat-labile toxin, liposomes, immune-stimulating complexes (ISCOM's)and the likes. The amount of adjuvant depends on the nature of the adjuvant itself. Furthermore the pharmaceutical composition, more specifically vaccine according to the invention may comprise one or more stabilizers such as, for example, carbohydrates including sorbitol, annitol, starch, sucrosedextrin and glucose, proteins such as albumin or casein, and buffers like alkaline phosphates. Contraception in a female can be obtained by administering the sperm antigens according to the invention to a female in suitable amount to induce an immune response. The amount of protein will depend on the route of administration, the time of administration, the age of the subject as well as general health conditions and diet. In general, the vaccines according to the invention are given in a dosage sufficient to either prevent or reduce the fertility in a subject to which the vaccine composition is administered. The dosages in which the vaccines can prevent or reduce fertility can be determined in view of the present teachings by a skilled person by running routine trials with appropriate controls to determine the optimal formulation. Suitable administration routes for the pharmaceutical composition and vaccine compositions according to the invention are parenteral and non-parenteral administration routes. Particular preferable are administration routes such as nasal, oral, and vaginal application. The following examples are illustrative for the invention and should in no way be interpreted as limiting the scope of the invention. LEGEND TO THE FIGURES Figure 1 Autoradiogram of a Northern blot containing total RNA from various marmoset tissues hybridized with a 32 P labeled probe from marmoset cDNA 4.1 (panel A) and 6.7 (panel B) . Figure 2 Autoradiogram from a multiple tissue Northern blot containing 2 micrograms of poly A+ RNA isolated from various human tissues, hybridized with a 32 P-labeled probe from marmoset cDNA 4.1 (panel A) and 6.7 (panel B) . Figure 3 Ethidium bromide stained agarose gel with samples from RT-PCR amplifications with human and marmoset testicular messenger RNA using primer sets either flanking the 123bp deletion (example 3) or within and downstream (3') this deletion. Figure 4 Coomassie Brilliant Blue stained SDS-polyacrylamide gel (left panel) and corresponding Western blot (right panel) of total bacterial lysates following induction of marmoset 4.1 expression. Samples were taken at t=0 and 1, 2 and 3 hours after induction (lanes 1 through 4, respectively) . Figure 5 Coomassie Brilliant Blue stained SDS-polyacrylamide gel (panel A) and corresponding Western blot (panel B) of total bacterial lysates following induction of marmoset 6.7 expression. Samples were taken at t=0 and 1 and 2 hours after induction (lanes 1 through 3, respectively) . Figure 6 Coomassie Brilliant Blue stained SDS polyacrylamide gel of Ni-agarose purified recombinant marmoset 4.1 and 6.7 proteins. Figure 7 The effect of immunization with recombinant 4.1 protein on the fertility immunized female mice. Figure 8 The effect of immunization with recombinant 6.7 protein on the fertility immunized female mice. EXAMPLES Example 1 Production of antiserυm against marmoset spermatozoa . Antisera to marmoset sperm proteins were raised in male marmosets immunized with octyl-β-D-thioglucopyranoside extracted marmoset sperm proteins, in the presence of Freunds (Morris) non-ulcerative adjuvant. The antibodies raised recognized a range of proteins in marmoset spermatozoa determined by Western blotting. Furthermore, by immunocytochemistry it was shown that all regions of the sperm surface were recognized. The generated antisera have been used to screen a marmoset testicular cDNA expression library. Example 2 Construction of marmoset testicular cDNA library Four testes were collected from marmosets and the total RNA was extracted using RNAzol (Biogenesis) . RNA degradation was minimized by using fresh tissue (not frozen) and rapid homogenization. Messenger RNA was purified from the total RNA by binding to oligo (dT) cellulose Type 7 columns (Pharmacia) . The isolated poly A+ mRNA was then used to generate a cDNA library using the ZAP-cDNA synthesis kit (Stratagene) . The kit uses a hybrid oligo (dT) linker primer which contains an Xho I site. First strand synthesis was primed with the linker primer-primer and was transcribed using Moloney murine leukaemia virus reverse transcriptase. The cDNA was then loaded onto Size Prep 400 spin columns (Pharmacia) and fragments larger than 400 bp were selected. The cDNA was ligated into the phage-ar s of the unidirectional vector via the EcoRI and Xhol sites. The library was packaged using Giga pack II Gold packaging extract (Stratagene) and plated on E.coli strain XLl-blue MRF' . The resulting library had a titre of Ixl0e6 plaque forming units (pfu) per ml and contained 99% recombinants . The library was amplified to give a final titer of 3 x 10e9 pfu/ml Example 3 Screening of the marmoset testis cDNA library with antibodies. Duplicate filters lifts from the plates with marmoset testis cDNA expression library were screened with a high titer marmoset antiserum against marmoset sperm lysate. As a second antibody anti-monkey IgG linked to horse radish peroxidase was used in combination with chemiluminescent substrates (ECL, Amersham) . Positive clones were subjected to a second round of screening in order to isolate single phage clones. The pBluescript counterparts of the isolated bacteriophage clones were excised using the exassist/SOLR system (Stratagene). The resulting plasmids, pMA4.1 and pMA6.7 were purified and sequenced. The nucleotide sequence information of the 4.1 cDNA inserts in plasmid pMa .1 is depicted in SEQ ID NO 1 and the corresponding encoded amino acid sequence is shown in SEQ ID NO 2. Using primers derived from the obtained 6.7 nucleotide information addition ' RACE technology (Rapid Amplification of cDNA Ends, Boehringer, Mannheim) was used to obtain additional 5' cDNA fragments. Combining the sequences from the various immuno- screened cDNA clones and the additional 5' RACE-clones revealed the nucleotide sequence as indicated in SEQ ID NO 3. Characterization of the various 6.7 clones revealed cDNAs that are the result of alternative splicing of the 6.7 transcript (see example 7 below) and that are missing nucleotides 91-213 as given in SEQ ID NO 3 were absent. The corresponding shorter 6.7 messenger RNA is represented by the cDNA sequence indicated as SEQ ID NO 4. The amino acid sequences encoded by SEQ ID NO 3 and SEQ ID NO 4 are the proteins given in SEQ ID NO 5 and SEQ ID NO 6, respectively. The 6.7 mRNA with and without the indicated 123 base pairs are designated 6.7L and 6.7S, respectively. Example 4 Multiple Tissue Northern blot analysis To investigate the tissue specific expression of clones 4.1 and 6.7 the complete cDNA inserts of pMA4.1 and pMA6.7 were amplified by PCR and the PCR-product were radiolabeled with 32P-CTP (Rediprime, Amersham) and used to probe multiple tissue Northern blots containing RNA from a variety of marmoset and human tissues. Samples of 30 micrograms of total RNA from various monkey tissues were separated on a formaldehyde-agarose gel and blotted onto nylon membranes (hybond N, Amersham) . The blots were hybridized with the radiolabeled PCR-amplified clone inserts overnight in 5x SSPE, lOx Denharts, 50% formamide, 2% SDS and 0.1 mg/ml freshly denatured sheared salmon sperm DNA at 42°C and washed at 50 °C in O.lxSSC, 0.1% SDS. The results after autoradiography for 2-4 days are shown in Figure 1A and IB for probes 4.1 and 6.7, respectively. The results of the hybridization of human multiple tissues Northern blots (Clontech) with both marmoset cDNAs are given in Figure 2A and 2B, respectively. The 6.7 probe recognizes a testis specific mRNA of ± 1 kb whereas the 4.1 DNA hybridized with a transcript of ± 1.8 kb . Example 5 Cloning of the human 6.7 gene The marmoset 6.7 cDNA insert from plasmid pMA6.7 as described in example 3 was radiolabeled with 32P-CTP and used to screen a human testicular cDNA library cloned into ZAP-express (Stratagene) . Five independent positive clones were isolated of which three were selected for sequence analysis. The longest clone was 878 bp in length. Since the Northern blot experiments revealed a transcript size of approximately 1 Kb the remaining cDNA fragment was isolated by 5'RACE (rapid amplification of cDNA ends) . Amplified DNA fragments were subcloned and three clones were sequenced. Additional 5' sequence information was obtained through 5' RACE technology (Boehringer Mannheim) . Briefly, 4 μg of human total testis RNA were reverse transcribed with avian myeloblastosis virus (AMV) reverse transcriptase to produce cDNA (using primer ma6.7rl, which hybridizes to the 3' region of the marmoset 6.7 mRNA and corresponds to nucleotides 779-797 in SEQ ID 1) . The cDNA was purified (High Pure PCR product purification kit, Boehringer Mannheim) and a homopolymeric tail added to the 3' end of the first strand cDNA using terminal transferase and dATP. The tailed cDNA was amplified by PCR using oligo dT-anchor primer and primer ha6.72r (antisense primer corresponding to nucleotides 549-571 in SEQ ID 7) . A total of 35 cycles were performed using an annealing temperature of 55 °C and an elongation time of 2 minutes for 10 cycles then extending the elongation time for 20 seconds per cycle for the next 25 cycles. Subsequent nested PCR-amplification was carried with the more upstream primer ha6.7r3 (antisense , corresponding to nucleotides 396-418 in SEQ ID 7) . The amplified DNA fragments were cloned into pGEM-T (Promega) and sequenced. By assembling the sequence of the various human 6.7 cDNA clones the nucleotide sequence indicated in SEQ ID 7 was obtained. This human 6.7 sequence could be aligned with the marmoset 6.7S sequence which was lacking a coding segment of 123 bp. Additional RT-PCR experiments with human testicular poly A+ RNA and using primers flanking the 123 deletion allowed amplification of human 6.7 cDNA fragments corresponding to the marmoset 6.7L cDNA and hence containing the extra 123 base pairs. The human sequence for 6.7L and 6.7S are given in SEQ ID 7 and SEQ ID 8, respectively. The amino acid sequences for the corresponding encoded proteins are given in SEQ ID 9 and SEQ ID 10, respectively. Example 6 Cloning of the human 4. 1 gene The marmoset 4.1 cDNA insert from plasmid pMA4.1 as described in example 3 was radiolabeled with 32P-CTP and used to screen a human testicular cDNA library cloned into ZAP-express (Stratagene) . Five independent cDNA clones have been isolated that were each individually sequenced. The longest clone contains an insert of 1272 bp in length. The nucleotide sequence of the human 4.1 cDNA is given in SEQ ID 11 and the amino acid sequence of the encoded 4.1 protein is indicated by SEQ ID 12. A search in gene databases with the sequence depicted in SEQ ID NO 11 resulted in a further nucleotide sequence (SEQ ID NO 15) comprising SEQ ID NO 11. SEQ ID NO 15 encodes a full length protein 4.1 of which the deduced amino acid is depicted in SEQ ID NO 16. Example 7 Alternative splicing of the mammalian 6. 7 messenger RNA. Marmoset and human testicular cDNA was made from purified total RNA using an oligo dT primer and AMV reverse transcriptase (Promega) . Using both marmoset and human testis cDNA, one amplified DNA fragment of 300 bp was produced upon PCR with a sense primer situated within the deleted 123 base pairs region indicated in Example 3 and an antisense primer situated approximately 200 bp downstream from the deletion (See Figure 3) . However two distinct fragments of +380 bp and +250 bp were produced when primers flanking the deletion were used in the PCR reaction (See Figure 3) . From these experiments it can be concluded that two alternatively spliced variants of the 6.7 mRNA exist in both the marmoset and human testis RNA. Example 8 Production of recombinant proteins Recombinant marmoset 4.1 and 6.7 proteins were produced in E.coli using appropriate pRSET-expression plasmids (based on reading frames, pRSETB and pRSETA, Invitrogen) , using routine recombinant DNA techniques. The inserts of both pMa4.1 and pMa6.7 represent 5' truncated partial cDNA clones. The nucleotide sequence of pMa4.1 is given in SEQ ID 1. The insert of plasmid pMa6.7 starts at position 130 in SEQ ID 3 and resides in the alternatively spliced DNA segment of 123 bp. The 5' part of the insert of pMa6.7 encodes an open reading frame which continues in the open reading frame that results in the amino acid sequence given in SEQ ID 5 and in combination encodes a protein of 240 amino acids. The nucleotide sequence of pMa6.7 and the corresponding open reading frame encoded protein are given in SEQ ID 13 and 14, respectively. The insert from plasmid pMa6.7 was isolated after EcoRI and Xhol (+ klenow to create a blunt end) digestion and subsequently cloned into pRSETA digested with EcoRI and HindiII (+ Klenow) yielding plasmid pRSET-Ma6.7. The integrity of the junction between the pRSET-vector and the 4.1 and 6.7 cDNA inserts was confirmed by sequence analysis, the insert from plasmid pMa4.1 was isolated after EcoRI and Hindlll (+ klenow) digestion and subsequently cloned into pRSETB digested with EcoRI and Kpnl (the Kpnl site was made blunt using T4-polymerase treatment) , yielding plasmid pRSET-Ma4.1. Both expression constructs were transformed to E.coli strain BL21 (DE3) pLysS. Bacterial cultures were grown to an optical density of 0.5 and subsequently incubated with 1 mM IPTG to induce expression of the recombinant protein. After several time point samples were taken and the complete bacterial lysate was analysed by SDS polyacrylamide gelelectrophoresis and Western blotting (see Figures 4 and 5 for antigens 4.1 and 6.7, respectively). The expressed recombinant protein were affinity purified using Ni-NTA-resin (Qiagen) and a protocol provided by the distributor. An example of a Coomassie stained polyacrylamide gel with purified recombinant marmoset 4.1 and 6.7 proteins is given in Figure 6. Example 9 Fertility trails Groups of six female Balb/C mice were subcutaneously immunized in the neck with 10 μg of the recombinant antigen in an emulsion of 500 μl (in the case of antigen 4.1) or 200 μl (in the case of antigen 6.7) made with complete Freund' s adjuvants (CFA) . In parallel two groups of control mice were immunized with 10 μg tetanus toxoid (TT) in emulsions of 500 μl or 200 μl made with CFA. The 4.1/control (TT) -immunized mice were boosted with the same amount of antigen/emulsion made in incomplete Freund' s adjuvants (IFA) at weeks 4, 13 and 17, whereas the 6.7/control (TT) -immunized animals were given additional immunizations at weeks 4, 8 and 12. The fertility status of the 4.1/control and the 6.7/control mice was determined at week 20 and 17, respectively. Towards this end groups of 3 immunized females were introduced in the cage of 1 proven fertile male. After 5 and 10 days each group was rotated and introduced in the cage of another male. At day 16 females were housed individually and checked each day for the number pubs. The fertility is expressed as the average number of pubs (litter size) in the groups of 6 immunized animals (see Figures 7 and 8) . Both with the 4.1 and 6.7 antigens a reduction in litter size was observe which could be expressed as a reduction of fertility of 60% and 50%, respectively. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Akzo Nobel N.V. (B) STREET: Velperweg 76 (C) CITY: Arnhem (E) COUNTRY: The Netherlands (F) POSTAL CODE (ZIP) : 6824 BM (G) TELEPHONE: 0412 666379 (H) TELEFAX: 0412 650592 (ii) TITLE OF INVENTION: Sperm specific antigens 4.1 and 6.7, DNA encoding them and contraceptive vaccines comprising said sperm antigens (iii) NUMBER OF SEQUENCES: 16 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 668 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GAATTCGGCA CGAGGGAGAT TGACACCCAG GGGCGCCACC TGGCTCACTC CATGCTGCAG 60 CAGGCCACAG CTTACCAGCA CTCAGGTCAC CTGGAGTCCA AGGACATCAA CCAGGAGGAG 120 CTGAGGGCCC TCGAGGAGGT GGAGATGAAA CTGCAAAGGA ATTTCCTCAT CCAGCAGGAA 180 AACACCATAG CTGGTGCCAA CCACACACAC ACTTTCTATG GCCACGGTCA CCATGGCCAC 240 CTGAGCCACC AGAGCCACAG CCTGCCCAAC CGCAGACACT AGATCTGTGA CTCCTTGGAA 300 GCCACCTAAC CATGGATGGA GATGCCCCTG CCCCCAGCAA GGTGGTCACA CACACAGGCC 360 CATTTTCTCT CCTACGGGGC AATCTACGTA GCCCAGGGTG AGATGCTGCG CCCAAATCCC 420 TGCTTTCTTA GGCTCCCCAC ACGGATCACA GGCCCTTTTG ACTCCATGCG TGGGTTTGCC 480 ACAGACGATG GCCCTTGTCG GAGGGAGAGC TGGGAGAACA CGGCACAGCT GAGACAGACC 540 CTGAGGCTTT GAAAACCCAC CAAGAGAAGA CAGACAGGAG CAGGCTAGAG CCCCACACTG 600 ATGCCAATGC CTGCAATGGA GCCAATAAAG CCTGGTGTGC CCTCCAAAAA AAAAAAAAAA 660 AAAAAAAA 668 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 93 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Glu Phe Gly Thr Arg Glu lie Asp Thr Gin Gly Arg His Leu Ala His 1 5 10 15 Ser Met Leu Gin Gin Ala Thr Ala Tyr Gin His Ser Gly His Leu Glu 20 25 30 Ser Lys Asp lie Asn Gin Glu Glu Leu Arg Ala Leu Tyr Glu Val Glu 35 40 45 Met Lys Leu Gin Arg Asn Phe Leu lie Gin Gin Glu Asn Thr lie Ala 50 55 60 Gly Ala Asn His Thr His Thr Phe Tyr Gly His Gly His His Gly His 65 70 75 80 Leu Ser His Gin Ser His Ser Leu Pro Asn Arg Arg His 85 90 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 964 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GTCCCTGCCT CGCAGAGCTC CGGACTTGGA GTCAGAGAGA CCTGGCTGCG AATCCCGGCT 60 CCGCCACTTT CTAGCGCTGT GACCTTGGTT TTCAGGTGGC GAAACTGACT GGTCTGGTTA 120 GAACGCGTCC AAGCCAAGCC GCAGTGCTGT TTGGATTTTT AAATTTCTCC TTCTGAGTGA 180 GAAAATAGCA ATCGAGATGG AACCATCCGC ACAAGAGCTC CAGCTGGCAG CATCATCTTC 240 TGACAATTTA TCCAACTTCT GCCAAGACTC TGAAATGCCA ACAACTTCAA GGCCTGCACT 300 TGATGTCAAG GGTGGCACCT CACCTGTGAA GGAGGATGCC AACCAAGAGA TGAGCGCCGT 360 GGCCTACTCT AACCTTGCGG TGAAAGATCG CAAAGCAGTG GCCATTCTGC ACTACCCTGG 420 GGTAGCCTCA AATGGAAGCA AGGCCAGTGG GGCTCCCACT AGTCCCTCGG GATCGCCAGT 480 AGGCTCTCCT CCTGCCACCC CTCCCTCTAA ATCCCCATCC TTCAACCTGA ACCCTGCCCC 540 TCACTTGCTG GCCAGTATGC AGCTGCAGAA ACTTAATAGC CAGTATCATG GGATGGCTGA 600 CGCCACTCCG GGCCAACCTG GAGAGGCAGG GCCCCTGCAA AACTGGGACT TGGGGGCTCA 660 GGCGGTGGGA GGGGCGGGAT CACTCTCTCC TTCTGCTGGT GCCCAGAGCC CTGCTATCAT 720 CGATTCGGAC CCAGTGGATG AGGAGGTGCT GATGTCGCTG GTGGTGGAAC TGGGGTTGGA 780 CCGAGCCAAT GAGCTTCCGG AGCTGTGGCT GGGGCAGAAT GAGTTTGACT TCACTTCGGA 840 CTTTCCATCT AGCTGCTGAT CCCAAATGTC CCTAAAGATG GAGGAATAAA GCCACCAATT 900 CTGTTGTAAA TAAAAATAAA GTTACTTGCA AAGAGACAGG CCAAAAAAAA AAAAAAAAAA 960 AAAA 964 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 843 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GTCCCTGCCT CGCAGAGCTC CGGACTTGGA GTCAGAGAGA CCTGGCTGCG AATCCCGGCT 60 CCGCCACTTT CTAGCGCTGT GACCTTGGCA CAAGAGCTCC AGCTGGCAGC ATCATCTTCT 120 GACAATTTAT CCAACTTCTG CCAAGACTCT GAAATGCCAA CAACTTCAAG GCCTGCACTT 180 GATGTCAAGG GTGGCACCTC ACCTGTGAAG GAGGATGCCA ACCAAGAGAT GAGCGCCGTG 240 GCCTACTCTA ACCTTGCGGT GAAAGATCGC AAAGCAGTGG CCATTCTGCA CTACCCTGGG 300 GTAGCCTCAA ATGGAAGCAA GGCCAGTGGG GCTCCCACTA GTCCCTCGGG ATCGCCAGTA 360 GGCTCTCCTC CTGCCACCCC TCCCTCTAAA TCCCCATCCT TCAACCTGAA CCCTGCCCCT 420 CACTTGCTGG CCAGTATGCA GCTGCAGAAA CTTAATAGCC AGTATCATGG GATGGCTGAC 480 GCCACTCCGG GCCAACCTGG AGAGGCAGGG CCCCTGCAAA ACTGGGACTT GGGGGCTCAG 540 GCGGTGGGAG GGGCGGGATC ACTCTCTCCT TCTGCTGGTG CCCAGAGCCC TGCTATCATC 600 GATTCGGACC CAGTGGATGA GGAGGTGCTG ATGTCGCTGG TGGTGGAACT GGGGTTGGAC 660 CGAGCCAATG AGCTTCCGGA GCTGTGGCTG GGGCAGAATG AGTTTGACTT CACTTCGGAC 720 TTTCCATCTA GCTGCTGATC CCAAATGTCC CTAAAGATGG AGGAATAAAG CCACCAATTC 780 TGTTGTAAAT AAAAATAAAG TTACTTGCAA AGAGACAGGC CAAAAAAAAA AAAAAAAAAA 840 AAA 843 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 219 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Met Glu Pro Ser Ala Gin Gin Leu Gin Leu Ala Ala Ser Leu Pro Ala 1 5 10 15 Asn Leu Ser Asn Phe Cys Gin Gly Ser Glu Met Pro Thr Thr Ser Arg 20 25 30 Pro Ala Leu Asp Val Lys Gly Gly Thr Ser Pro Ala Lys Glu Asp Ala 35 40 45 Asn Gin Glu Met Ser Ser Val Ala Tyr Ser Asn Leu Ala Val Lys Asp 50 55 60 Arg Lys Ala Val Ala lie Leu His Tyr Pro Gly Val Ala Ser Asn Gly 65 70 75 80 Thr Lys Ala Ser Gly Ala Pro Thr Ser Ser Ser Gly Ser Pro lie Gly 85 90 95 Ser Pro Thr Thr Thr Pro Pro Thr Lys Pro Pro Ser Phe Asn Leu His 100 105 110 Pro Ala Pro His Leu Leu Ala Ser Met Gin Leu Gin Lys Leu Asn Ser 115 120 125 Gin Tyr Gin Gly Met Ala Ala Ala Thr Pro Gly Gin Pro Gly Glu Ala 130 135 140 Gly Pro Leu Gin Asn Trp Asp Phe Gly Ala Gin Ala Gly Gly Ala Glu 145 150 155 160 Ser Leu Ser Pro Ser Ala Gly Ala Gin Ser Pro Ala lie lie Asp Ser 165 170 175 Asp Pro Val Asp Glu Glu Val Leu Met Ser Leu Val Val Glu Leu Gly 180 185 190 Leu Asp Arg Ala Asn Glu Leu Pro Glu Leu Trp Leu Gly Gin Asn Glu 195 200 205 Phe Asp Phe Thr Ala Asp Phe Pro Ser Ser Cys 210 215 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 244 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Val Pro Ala Ser Gin Ser Thr Gly Leu Gly Val Arg Glu Thr Trp Leu 1 5 10 15 Arg lie Pro Ala Pro Pro Leu Ser Ser Ala Val Thr Leu Ala Gin Gin 20 25 30 Leu Gin Leu Ala Ala Ser Leu Pro Ala Asn Leu Ser Asn Phe Cys Gin 35 40 45 Gly Ser Glu Met Pro Thr Thr Ser Arg Pro Ala Leu Asp Val Lys Gly 50 55 60 Gly Thr Ser Pro Ala Lys Glu Asp Ala Asn Gin Glu Met Ser Ser Val 65 70 75 80 Ala Tyr Ser Asn Leu Ala Val Lys Asp Arg Lys Ala Val Ala lie Leu 85 90 95 His Tyr Pro Gly Val Ala Ser Asn Gly Thr Lys Ala Ser Gly Ala Pro 100 105 110 Thr Ser Ser Ser Gly Ser Pro lie Gly Ser Pro Thr Thr Thr Pro Pro 115 120 125 Thr Lys Pro Pro Ser Phe Asn Leu His Pro Ala Pro His Leu Leu Ala 130 135 140 Ser Met Gin Leu Gin Lys Leu Asn Ser Gin Tyr Gin Gly Met Ala Ala 145 150 155 160 Ala Thr Pro Gly Gin Pro Gly Glu Ala Gly Pro Leu Gin Asn Trp Asp 165 170 175 Phe Gly Ala Gin Ala Gly Gly Ala Glu Ser Leu Ser Pro Ser Ala Gly 180 185 190 Ala Gin Ser Pro Ala lie lie Asp Ser Asp Pro Val Asp Glu Glu Val 195 200 205 Leu Met Ser Leu Val Val Glu Leu Gly Leu Asp Arg Ala Asn Glu Leu 210 215 220 Pro Glu Leu Trp Leu Gly Gin Asn Glu Phe Asp Phe Thr Ala Asp Phe 225 230 235 240 Pro Ser Ser Cys (2) INFORMATION FOR SEQ ID NO: 7: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 963 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (n) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GAGTCCCTGC TTCGCAGAGC ACCGGACTTG GAGTCAGAGA GACCTGGCTG CGAATCCCGG 60 CTCCGCCACT TTCTAGCGCT GTGACCTTGG TTTTCAGGTG GCGAAACTGA CTGGTCTGGT 120 TAGAGCGCGT CCAAGCCAAG CCGCAGTGCT GTTTGGATTT TTAAATTTCT CCTTCTGAGT 180 GAGAAAATAG CAATCGAGAT GGAACCATCC GCACAACAGC TCCAGCTGGC AGCATCACTT 240 CCCGCCAATT TATCCAACTT CTGCCAAGGC TCTGAAATGC CAACAACGTC GAGGCCTGCA 300 CTTGATGTCA AGGGTGGCAC CTCACCTGCG AAGGAGGATG CCAACCAAGA GATGAGCTCC 360 GTGGCCTACT CCAACCTTGC GGTGAAAGAT CGCAAAGCAG TGGCCATTCT GCACTACCCT 420 GGGGTAGCCT CAAATGGAAC CAAGGCCAGT GGGGCTCCCA CTAGTTCCTC GGGATCTCCA 480 ATAGGCTCTC CTACAACCAC CCCTCCCACT AAACCCCCAT CCTTCAACCT GCACCCCGCC 540 CCTCACTTGC TGGCTAGTAT GCAGCTGCAG AAACTTAATA GCCAGTATCA GGGGATGGCT 600 GCTGCCACTC CAGGCCAACC CGGGGAGGCA GGACCCCTGC AAAACTGGGA CTTTGGGGCC 660 CAGGCGGGAG GGGCAGAATC ACTCTCTCCT TCTGCTGGTG CCCAGAGCCC TGCTATCATC 720 GATTCGGACC CAGTGGATGA GGAAGTGCTG ATGTCGCTGG TGGTGGAACT GGGGTTGGAC 780 CGAGCCAATG AGCTTCCGGA GCTGTGGCTG GGGCAGAATG AGTTTGACTT CACTGCGGAC 840 TTTCCATCTA GCTGCTAATG CCAAGTGTCC CTAAAGATGG AGGAATAAAG CCACCAATTC 900 TGTTGTAAAT AAAAATAAAG TTACTTACAA AGAGAAAAAA AAAAAAAAAA AAAAAAAAAA 960 AAA 963 (2) INFORMATION FOR SEQ ID NO: 8: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 842 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( i) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GAGTCCCTGC TTCGCAGAGC ACCGGACTTG GAGTCAGAGA GACCTGGCTG CGAATCCCGG 60 CTCCGCCACT TTCTAGCGCT GTGACCTTGG CACAACAGCT CCAGCTGGCA GCATCACTTC 120 CCGCCAATTT ATCCAACTTC TGCCAAGGCT CTGAAATGCC AACAACGTCG AGGCCTGCAC 180 TTGATGTCAA GGGTGGCACC TCACCTGCGA AGGAGGATGC CAACCAAGAG ATGAGCTCCG 240 TGGCCTACTC CAACCTTGCG GTGAAAGATC GCAAAGCAGT GGCCATTCTG CACTACCCTG 300 GGGTAGCCTC AAATGGAACC AAGGCCAGTG GGGCTCCCAC TAGTTCCTCG GGATCTCCAA 360 TAGGCTCTCC TACAACCACC CCTCCCACTA AACCCCCATC CTTCAACCTG CACCCCGCCC 420 CTCACTTGCT GGCTAGTATG CAGCTGCAGA AACTTAATAG CCAGTATCAG GGGATGGCTG 480 CTGCCACTCC AGGCCAACCC GGGGAGGCAG GACCCCTGCA AAACTGGGAC TTTGGGGCCC 540 AGGCGGGAGG GGCAGAATCA CTCTCTCCTT CTGCTGGTGC CCAGAGCCCT GCTATCATCG 600 ATTCGGACCC AGTGGATGAG GAAGTGCTGA TGTCGCTGGT GGTGGAACTG GGGTTGGACC 660 GAGCCAATGA GCTTCCGGAG CTGTGGCTGG GGCAGAATGA GTTTGACTTC ACTGCGGACT 720 TTCCATCTAG CTGCTAATGC CAAGTGTCCC TAAAGATGGA GGAATAAAGC CACCAATTCT 780 GTTGTAAATA AAAATAAAGT TACTTACAAA GAGAAAAAAA AAAAAAAAAA AAAAAAAAAA 840 AA 842 (2) INFORMATION FOR SEQ ID NO: 9: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 219 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Met Glu Pro Ser Ala Gin Gin Leu Gin Leu Ala Ala Ser Leu Pro Ala 1 5 10 15 Asn Leu Ser Asn Phe Cys Gin Gly Ser Glu Met Pro Thr Thr Ser Arg 20 25 30 Pro Ala Leu Asp Val Lys Gly Gly Thr Ser Pro Ala Lys Glu Asp Ala 35 40 45 Asn Gin Glu Met Ser Ser Val Ala Tyr Ser Asn Leu Ala Val Lys Asp 50 55 60 Arg Lys Ala Val Ala lie Leu His Tyr Pro Gly Val Ala Ser Asn Gly 65 70 75 80 Thr Lys Ala Ser Gly Ala Pro Thr Ser Ser Ser Gly Ser Pro lie Gly 85 90 95 Ser Pro Thr Thr Thr Pro Pro Thr Lys Pro Pro Ser Phe Asn Leu His 100 105 110 Pro Ala Pro H s Leu Leu Ala Ser Met Gin Leu Gin Lys Leu Asn Ser 115 120 125 Gin Tyr Gin Gly Met Ala Ala Ala Thr Pro Gly Gin Pro Gly Glu Ala 130 135 140 Gly Pro Leu Gin Asn Trp Asp Phe Gly Ala Gin Ala Gly Gly Ala Glu 145 150 155 160 Ser Leu Ser Pro Ser Ala Gly Ala Gin Ser Pro Ala lie lie Asp Ser 165 170 175 Asp Pro Val Asp Glu Glu Val Leu Met Ser Leu Val Val Glu Leu Gly 180 185 190 Leu Asp Arg Ala Asn Glu Leu Pro Glu Leu Trp Leu Gly Gin Asn Glu 195 200 205 Phe Asp Phe Thr Ala Asp Phe Pro Ser Ser Cys 210 215 (2) INFORMATION FOR SEQ ID NO: 10: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 244 amino acids (B) TYPE: ammo acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Val Pro Ala Ser Gin Ser Thr Gly Leu Gly Val Arg Glu Thr Trp Leu 1 5 10 15 Arg lie Pro Ala Pro Pro Leu Ser Ser Ala Val Thr Leu Ala Gin Gin 20 25 30 Leu Gin Leu Ala Ala Ser Leu Pro Ala Asn Leu Ser Asn Phe Cys Gin 35 40 45 Gly Ser Glu Met Pro Thr Thr Ser Arg Pro Ala Leu Asp Val Lys Gly 50 55 60 Gly Thr Ser Pro Ala Lys Glu Asp Ala Asn Gin Glu Met Ser Ser Val 65 70 75 80 Ala Tyr Ser Asn Leu Ala Val Lys Asp Arg Lys Ala Val Ala lie Leu 85 90 95 H s Tyr Pro Gly Val Ala Ser Asn Gly Thr Lys Ala Ser Gly Ala Pro 100 105 110 Thr Ser Ser Ser Gly Ser Pro lie Gly Ser Pro Thr Thr Thr Pro Pro 115 120 125 Thr Lys Pro Pro Ser Phe Asn Leu His Pro Ala Pro His Leu Leu Ala 130 135 140 Ser Met Gin Leu Gin Lys Leu Asn Ser Gin Tyr Gin Gly Met Ala Ala 145 150 155 160 Ala Thr Pro Gly Gin Pro Gly Glu Ala Gly Pro Leu Gin Asn Trp Asp 165 170 175 Phe Gly Ala Gin Ala Gly Gly Ala Glu Ser Leu Ser Pro Ser Ala Gly 180 185 190 Ala Gin Ser Pro Ala lie lie Asp Ser Asp Pro Val Asp Glu Glu Val 195 200 205 Leu Met Ser Leu Val Val Glu Leu Gly Leu Asp Arg Ala Asn Glu Leu 210 215 220 Pro Glu Leu Trp Leu Gly Gin Asn Glu Phe Asp Phe Thr Ala Asp Phe 225 230 235 240 Pro Ser Ser Cys (2) INFORMATION FOR SEQ ID NO: 11: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1273 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ll) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GCAGGGGGCC TGCCTGTCAG ACATCCTTGG GGAGCCTGCC CTTTCAAGCA TGAATCATAC 60 CACCAGAGAT CACCCAGCGT GCTCTTAGCT CTGCCTGCCT GGTGTAGGGT CCTGGAGACC 120 TTCTTGGAGC TCATCCAGCA GCCATCATGC AAGGTGGTGC TGGGGACTGC CCTCTTGCTA 180 GGAGGTGGAG GTCTCATGCT GTGGGTTCAG AGAAAGAGGA GAAGAAAGGA AACCTCTGAG 240 TGTCCATCAG ACAAGGACAA GTCACCAGAA TCCCATAAAG CAAAGAATGA AAGCTGGATT 300 AAATCCCACT TTAGCCGCCT TTCCGAAGAG AAGCTGGCCC TCGACAACAA TGCCAGCGCT 360 AGTGGCAATG CTACCCAGAC TGAGAGTGGG AGTGAAGAGG TCAGCTCCAC GGTTCACATA 420 GAGACCTTCA CCACGAGGCA CGGAGAAGTG GGCTCCGCTC TGCACCGGGA ATCCTTCACC 480 AGCAGGCAGA AGACATCTGG GCCCTCAGTG ATCCAAGAGA TCCACCAGGA GTCTGGAAAA 540 GCCCCATCCA CTGATGACGC CACGTGGGCC GCTGTGGCTG CCTGCACCAA GGAGATTGAC 600 ACCCAGGGGC GGCACCTGGC TCACTCCATG CTGCAGCGGG CCATAGCTTA CCAGCACTCA 660 GGTCACCTGG AGTCCAAGGA CATCAACCAG GAGGAGCTGA GGGCCCTCGA GGAGGTAGAG 720 ATGAAGCTGC AAAAGAATTT CCTCACCCAG CGGGAAAACA CCATAGCTGG TGCCAATCAC 780 ACACACACCT TCTATGGCCA CAGTCACCAC AGTCACCATG GCCACCCAAG CCACCAGAGC 840 CACAGCCTGC CTAATCGCAG ACACTAGATC TGTGACTTCT TGGAAGCCAC CTAACCATGG 900 ATGGAGACGC CCCTGCCCCC AGCAAGGCTG GCTCACACAC ACGTCTGTTT TCTCTCCTAT 960 GGGGCCATCT ATGCAGGCCA GGGTGAGATG CTGTGCCCAA ATCCCTACTT TCTTAGGCTC 1020 CTCACGGGGA TTATGGGCTC TTTAAACTCC ATGAGTGGGG TCTGCCACAG AAGATGGCCC 1080 CTGTTGGAGG GAGAGCTGGG AGGACACAGC GCAGGTGAGA TAGACCCTGA GGCCCCTGAA 1140 GACCCACCAA GAGAAGACAG ATGGGAGCAG GCAAGAGCCC CACACTGATG TCATTGCCTG 1200 GAACGGAGCC AATAAAGCTT TGTGTGCCTT CAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1260 AAAAAAAAAA AAA 1273 (2) INFORMATION FOR SEQ ID NO: 12: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 288 ammo acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Ala Gly Gly Leu Pro Val Arg His Pro Trp Gly Ala Cys Pro Phe Lys 1 5 10 15 His Glu Ser Tyr His Gin Arg Ser Pro Ser Val Leu Leu Ala Leu Pro 20 25 30 Ala Trp Cys Arg Val Leu Glu Thr Phe Leu Glu Leu lie Gin Gin Pro 35 40 45 Ser Cys Lys Val Val Leu Gly Thr Ala Leu Leu Leu Gly Gly Gly Gly 50 55 60 Leu Met Leu Trp Val Gin Arg Lys Arg Arg Arg Lys Glu Thr Ser Glu 65 70 75 80 Cys Pro Ser Asp Lys Asp Lys Ser Pro Glu Ser His Lys Ala Lys Asn 85 90 95 Glu Ser Trp lie Lys Ser His Phe Ser Arg Leu Ser Glu Glu Lys Leu 100 105 110 Ala Leu Asp Asn Asn Ala Ser Ala Ser Gly Asn Ala Thr Gin Thr Glu 115 120 125 Ser Gly Ser Glu Glu Val Ser Ser Thr Val His lie Glu Thr Phe Thr 130 135 140 Thr Arg His Gly Glu Val Gly Ser Ala Leu His Arg Glu Ser Phe Thr 145 150 155 160 Ser Arg Gin Lys Thr Ser Gly Pro Ser Val lie Gin Glu lie His Gin 165 170 175 Glu Ser Gly Lys Ala Pro Ser Thr Asp Asp Ala Thr Trp Ala Ala Val 180 185 190 Ala Ala Cys Thr Lys Glu lie Asp Thr Gin Gly Arg His Leu Ala His 195 200 205 Ser Met Leu Gin Arg Ala lie Ala Tyr Gin His Ser Gly His Leu Glu 210 215 220 Ser Lys Asp lie Asn Gin Glu Glu Leu Arg Ala Leu Glu Glu Val Glu 225 230 235 240 Met Lys Leu Gin Lys Asn Phe Leu Thr Gin Arg Glu Asn Thr lie Ala 245 250 255 Gly Ala Asn His Thr His Thr Phe Tyr Gly His Ser His His Ser His 260 265 270 His Gly His Pro Ser His Gin Ser His Ser Leu Pro Asn Arg Arg His 275 280 285 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 842 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GAATTCGGCA CGAGCCAAGC CGCAGTGCTG TTTGGATTTT TAAATTTCTC CTTCTGAGTG 60 AGAAAATAGC AATCGAGATG GAACCATCCG CACAAGAGCT CCAGCTGGCA GCATCATCTT 120 CTGACAATTT ATCCAACTTC TGCCAAGACT CTGAAATGCC AACAACTTCA AGGCCTGCAC 180 TTGATGTCAA GGGTGGCACC TCACCTGTGA AGGAGGATGC CAACCAAGAG ATGAGCGCCG 240 TGGCCTACTC TAACCTTGCG GTGAAAGATC GCAAAGCAGT GGCCATTCTG CACTACCCTG 300 GGGTAGCCTC AAATGGAAGC AAGGCCAGTG GGGCTCCCAC TAGTCCCTCG GGATCGCCAG 360 TAGGCTCTCC TCCTGCCACC CCTCCCTCTA AATCCCCATC CTTCAACCTG AACCCTGCCC 420 CTCACTTGCT GGCCAGTATG CAGCTGCAGA AACTTAATAG CCAGTATCAT GGGATGGCTG 480 ACGCCACTCC GGGCCAACCT GGAGAGGCAG GGCCCCTGCA AAACTGGGAC TTGGGGGCTC 540 AGGCGGTGGG AGGGGCGGGA TCACTCTCTC CTTCTGCTGG TGCCCAGAGC CCTGCTATCA 600 TCGATTCGGA CCCAGTGGAT GAGGAGGTGC TGATGTCGCT GGTGGTGGAA CTGGGGTTGG 660 ACCGAGCCAA TGAGCTTCCG GAGCTGTGGC TGGGGCAGAA TGAGTTTGAC TTCACTTCGG 720 ACTTTCCATC TAGCTGCTGA TCCCAAATGT CCCTAAAGAT GGAGGAATAA AGCCACCAAT 780 TCTGTTGTAA ATAAAAATAA AGTTACTTGC AAAGAGACAG GCCAAAAAAA AAAAAAAAAA 840 AA 842 (2) INFORMATION FOR SEQ ID NO: 14: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 240 ammo acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Pro Ser Arg Ser Ala Val Trp lie Phe Lys Phe Leu Leu Leu Ser Glu 1 5 10 15 Lys lie Ala lie Glu Met Glu Pro Ser Ala Gin Gin Leu Gin Leu Ala 20 25 30 Ala Ser Leu Pro Ala Asn Leu Ser Asn Phe Cys Gin Gly Ser Glu Met 35 40 45 Pro Thr Thr Ser Arg Pro Ala Leu Asp Val Lys Gly Gly Thr Ser Pro 50 55 60 Ala Lys Glu Asp Ala Asn Gin Glu Met Ser Ser Val Ala Tyr Ser Asn 65 70 75 80 Leu Ala Val Lys Asp Arg Lys Ala Val Ala He Leu His Tyr Pro Gly 85 90 95 Val Ala Ser Asn Gly Thr Lys Ala Ser Gly Ala Pro Thr Ser Ser Ser 100 105 110 Gly Ser Pro He Gly Ser Pro Thr Thr Thr Pro Pro Thr Lys Pro Pro 115 120 125 Ser Phe Asn Leu His Pro Ala Pro His Leu Leu Ala Ser Met Gin Leu 130 135 140 Gin Lys Leu Asn Ser Gin Tyr Gin Gly Met Ala Ala Ala Thr Pro Gly 145 150 155 160 Gin Pro Gly Glu Ala Gly Pro Leu Gin Asn Trp Asp Phe Gly Ala Gin 165 170 175 Ala Gly Gly Ala Glu Ser Leu Ser Pro Ser Ala Gly Ala Gin Ser Pro 180 185 190 Ala He He Asp Ser Asp Pro Val Asp Glu Glu Val Leu Met Ser Leu 195 200 205 Val Val Glu Leu Gly Leu Asp Arg Ala Asn Glu Leu Pro Glu Leu Trp 210 215 220 Leu Gly Gin Asn Glu Phe Asp Phe Thr Ala Asp Phe Pro Ser Ser Cys 225 230 235 240 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1442 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: CAGCTGTCAG GGTAGGAGAC AGGACCCAGG CCTCAGGCAA TGCCTGCCAG GCTAGACATG 60 AGGGATGCTG CGGCCTCTCC CCACCCCACT CTCCCCACCC CACCCGCAAC TGCCTTCCTC 120 ACAATGGGGT TCATTGTTGA GGTGGTGGGG GGCACCTAGG GAGGGTGCAG CAGGGGGCCT 180 GCCTGTCAGA CATCCTTGGG GAGCCTGCCC TTTCAAGCAT GAATCATACC ACCAGAGATC 240 ACCCAGCGTG CTCTTAGCTC TGCCTGCCTG GTGTAGGGTC CTGGAGACCT TCTTGGAGCT 300 CATCCAGCAG CCATCATGCA AGGTGGTGCT GGGGACTGCC CTCTTGCTAG GAGGTGGAGG 360 TCTCATGCTG TGGGTTCAGA GAAAGAGGAG AAGAAAGGAA ACCTCTGAGT GTCCATCAGA 420 CAAGGACAAG TCACCAGAAT CCCATAAAGC AAAGAATGAA AGCTGGATTA AATCCCACTT 480 TAGCCGCCTT TCCGAAGAGA AGCTGGCCCT CGACAACAAT GCCAGCGCTA GTGGCAATGC 540 TACCCAGACT GAGAGTGGGA GTGAAGAGGT CAGCTCCACG GTTCACATAG AGACCTTCAC 600 CACGAGGCAC GGAGAAGTGG GCTCCGCTCT GCACCGGGAA TCCTTCACCA GCAGGCAGAA 660 GACATCTGGG CCCTCAGTGA TCCAAGAGAT CCACCAGGAG TCTGGAAAAG CCCCATCCAC 720 TGATGACGCC ACGTGGGCCG CTGTGGCTGC CTGCACCAAG GAGATTGACA CCCAGGGGCG 780 GCACCTGGCT CACTCCATGC TGCAGCGGGC CATAGCTTAC CAGCACTCAG GTCACCTGGA. '* 840 GTCCAAGGAC ATCAACCAGG AGGAGCTGAG GGCCCTCGAG GAGGTAGAGA TGAAGCTGCA 900 AAAGAATTTC CTCACCCAGC GGGAAAACAC CATAGCTGGT GCCAATCACA CACACACCTT 960 CTATGGCCAC AGTCACCACA GTCACCATGG CCACCCAAGC CACCAGAGCC ACAGCCTGCC 1020 TAATCGCAGA CACTAGATCT GTGACTTCTT GGAAGCCACC TAACCATGGA TGGAGACGCC 1080 CCTGCCCCCA GCAAGGCTGG CTCACACACA CGTCTGTTTT CTCTCCTATG GGGCCATCTA 1140 TGCAGGCCAG GGTGAGATGC TGTGCCCAAA TCCCTACTTT CTTAGGCTCC TCACGGGGAT 1200 TATGGGCTCT TTAAACTCCA TGAGTGGGGT CTGCCACAGA AGATGGCCCC TGTTGGAGGG 1260 AGAGCTGGGA GGACACAGCG CAGGTGAGAT AGACCCTGAG GCCCCTGAAG ACCCACCAAG 1320 AGAAGACAGA TGGGAGCAGG CAAGAGCCCC ACACTGATGT CATTGCCTGG AACGGAGCCA 1380 ATAAAGCTTT GTGTGCCTTC AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1440 AA 1442 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 323 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Met Leu Arg Pro Leu Pro Thr Pro Leu Ser Pro Pro His Pro Gin Leu 1 5 10 15 Pro Ser Ser Gin Trp Gly Ser Leu Leu Arg Trp Trp Gly Ala Pro Arg 20 25 30 Glu Gly Ala Ala Gly Gly Leu Pro Val Arg His Pro Trp Gly Ala Cys 35 40 45 Pro Phe Lys His Glu Ser Tyr His Gin Arg Ser Pro Ser Val Leu Leu 50 55 60 Ala Leu Pro Ala Trp Cys Arg Val Leu Glu Thr Phe Leu Glu Leu He 65 70 75 80 Gin Gin Pro Ser Cys Lys Val Val Leu Gly Thr Ala Leu Leu Leu Gly 85 90 95 Gly Gly Gly Leu Met Leu Trp Val Gin Arg Lys Arg Arg Arg Lys Glu 100 105 110 Thr Ser Glu Cys Pro Ser Asp Lys Asp Lys Ser Pro Glu Ser His Lys 115 120 125 Ala Lys Asn Glu Ser Trp He Lys Ser His Phe Ser Arg Leu Ser Glu 130 135 140 Glu Lys Leu Ala Leu Asp Asn Asn Ala Ser Ala Ser Gly Asn Ala Thr 145 150 155 160 Gin Thr Glu Ser Gly Ser Glu Glu Val Ser Ser Thr Val His He Glu 165 170 175 Thr Phe Thr Thr Arg His Gly Glu Val Gly Ser Ala Leu His Arg Glu 180 185 190 Ser Phe Thr Ser Arg Gin Lys Thr Ser Gly Pro Ser Val He Gin Glu 195 200 205 He His Gin Glu Ser Gly Lys Ala Pro Ser Thr Asp Asp Ala Thr Trp 210 215 220 Ala Ala Val Ala Ala Cys Thr Lys Glu He Asp Thr Gin Gly Arg His 225 230 235 240 Leu Ala His Ser Met Leu Gin Arg Ala He Ala Tyr Gin His Ser Gly 245 250 255 His Leu Glu Ser Lys Asp He Asn Gin Glu Glu Leu Arg Ala Leu Glu 260 265 270 Glu Val Glu Met Lys Leu Gin Lys Asn Phe Leu Thr Gin Arg Glu Asn 275 280 285 Thr He Ala Gly Ala Asn His Thr His Thr Phe Tyr Gly His Ser His 290 295 300 His Ser His His Gly His Pro Ser His Gin Ser His Ser Leu Pro Asn 305 310 315 320 Arg Arg His

Claims: Claims I. Nucleotide sequence encoding mammalian sperm proteins 4.1 or 6.7. 2. Nucleotide sequence according to claim 1, characterised in that the mammalian is marmoset or human. 3. Nucleotide sequence according to claim 1 or 2 characterised in that said sequence comprises the nucleotide sequence depicted in SEQ ID N0:1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 11, SEQ ID NO 13, or SEQ ID NO 15. 4. Expression vector comprising a nucleotide sequence according to any of the claims 1 to 3. 5. Host cell comprising a nucleotide sequence according to any of the claims 1 to 3 or an expression vector according to claim 4. 6. Mammalian sperm protein 4.1 or 6.7. 7. Recombinant sperm protein encoded by a nucleotide sequence according to any of the claims 1 to 3. 8. Sperm protein according to any of the claims 6 or 7, characterised in that it comprises the amino acid sequence depicted in SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14 or SEQ ID NO 16. 9. Sperm protein 4.1 and/or 6.7 for use as a medicament. 10. Pharmaceutical composition comprising sperm protein 4.1 and/or 6.7 and a pharmaceutical acceptable carrier. II. Contraceptive vaccine comprising sperm protein 4.1 and/or 6.7.

Inventors: AITKEN ROBERT JOHN, GROOTENHUIS ARIJ JAN, VAN DUIN MARCEL, AITKEN, ROBERT, JOHN, GROOTENHUIS, ARIJ, JAN, VAN DUIN, MARCEL
Applicants: AITKEN ROBERT JOHN, AKZO NOBEL NV, DUIN MARCEL VAN, GROOTENHUIS ARIJ JAN, AITKEN, ROBERT, JOHN, AKZO NOBEL N.V., GROOTENHUIS, ARIJ, JAN, VAN DUIN, MARCEL
Application year: 1998
Main classification: C07K14
IPC codes: A61K38, A61K39, A61P15, C07K14, C12N1, C12N5, C12N15