CA2296002A1 - Sperm specific antigens 4.1 and 6.7, dna encoding them and contraceptive vaccines comprising said sperm antigens - Google Patents

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 Thesa And Contraceptive Vaccines Comprising Said Speria Antigens s 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 io pharmaceutical formulations comprising said novel sperm specific antigens.
Contraceptive vaccines provide an alternative approach to the well established use of (steroidal) hormonal is 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 zo 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.
2s 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 so 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 s 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 io 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 i5 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 2o 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.
2s 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 3o 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 s 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 to 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.
is 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.
zo 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 2s 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 3o 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 35 genetic code there are various codons which code for a WO 98/58954 _ PCT/EP98/04016 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 _ s 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 io 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 is 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 2o 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 2s depicted by the amino acid sequence of SEQ ID N0. 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 60g, more preferably 70%, highly preferably 80~ with any of the 3o nucleotide sequences shown in SEQ ID NO 1, 3, 4, 7, 8, 11, 13 or I5. 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 s 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 to 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 is 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.
2o 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 2s 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, so 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 35 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 s preferable 80g. 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 io 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 i5 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.
2o 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.85:2149 (1963) and Int. J. Peptide Protein Res. 35:161-214 (1990).
z5 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 so 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, 35 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 s 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 to 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 is 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 zo 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.
2s 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 3o 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 3s result in an auto-immune response to somatic tissues.

_ g _ The sperm specific proteins according to the invention . can be used in a contraceptive vaccine. Pharmaceutical formulations , more specifically vaccines comprising the s 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 so 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 is 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 2o 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 2s 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 so 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, mannitol, starch, sucrosedextrin and WO 98/58954 _ PCT/EP98/04016 _ g _ glucose, proteins such as albumin or casein, and buffers like alkaline phosphates.
Contraception in a female can be obtained by s 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.
io 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 is 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 2o 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 2s the invention.
LEGEND TO THE FIGURES
- Figure 1 Autoradiogram of a Northern blot containing total RNA
- from various marmoset tissues hybridized with a 32P labeled so 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 ~'P-labeled probe from s 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 io 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 is 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 20 (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 zs 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 s The effect of immunization with recombinant 6.7 protein on the fertility immunized female mice.
EXAMP?~E S
Example 1 Production of antiserum against marmoset spermatozoa.
io Antisera to marmoset sperm proteins were raised in male marmosets immunized with octyl-t3-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 i5 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.
zo 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 2s 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 s using Moloney murine leukaemia virus reverse transcriptase.
The cDNA was then loaded onto Size Prep 400 spin columns (Pharmacia) and fragments larger than 400 by were selected.
The cDNA was ligated into the phage-arms of the uni-directional vector via the EcoRI and XhoI sites. The library to was packaged using Giga pack II Gold packaging extract (Stratagene) and plated on E.coli strain XL1-blue MRF'. The resulting library had a titre of 1x10e6 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 is 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 2o 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 2s 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 3o information of the 4.1 cDNA inserts in plasmid pMa4.l 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 s 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 to 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 is pairs are designated 6.7L and 6.75, 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 2o 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 2s 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, 2o SDS and 0.1 mg/ml freshly denatured sheared salmon sperm DNA at 42°C and 3o washed at 50 °C in O.lxSSC, 0.1~ SDS. The results after autoradiography for 2-4 days are shown in Figure lA and 1B
for probes 4.1 and 6.7, respectively.

WO 98/58954 PC'T/EP98/04016 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 s 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 to 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 by in length. Since the Northern blot is 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 zo (Boehringer Mannheim). Briefly, 4 ug of human total testis RNA were reverse transcribed with avian myeloblastosis virus (AMV) reverse transcriptase to produce cDNA (using primer ma6.7r1, which hybridizes to the 3' region of the marmoset 6.7 mRNA and corresponds to nucleotides 779-797 in SEQ ID
2s 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 30 (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 ~f 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
1o 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 1s 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.
ao Example 6 Cloning of the human 4.2 gene The marmoset 9.1 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 25 ZAP-express (Stratagene). Five independent cDNA clones have been isolated that were each individually sequenced. The longest clone contains an insert of 1272 by 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 so 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.
s 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 by was produced upon PCR with a sense primer situated within the to deleted 123 base pairs region indicated in Example 3 and an antisense primer situated approximately 200 by downstream from the deletion (See Figure 3). However two distinct fragments of +380 by and +250 by were produced when primers flanking the deletion were used in the PCR reaction (See 15 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 B
Production of recombinant proteins 2o 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' 2s 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 3o 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 s and 14, respectively.
The insert from plasmid pMa6.7 was isolated after EcoRI
and XhoI (+ klenow to create a blunt end) digestion and subsequently cloned into pRSETA digested with EcoRI and HindIII (+ Klenow) yielding plasmid pRSET-Ma6.7. The io 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.l was isolated after EcoRI and HindIII (+ klenow) digestion and subsequently cloned into pRSETB digested with EcoRI and KpnI (the KpnI site was made is blunt using T4-polymerase treatment), yielding plasmid pRSET-Ma4.I.
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
2o 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).
2s 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 ug of the recombinant antigen s in an emulsion of 500 ul (in the case of antigen 4.1) or 200 ul (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 ug tetanus toxoid (TT) in emulsions of 500 ul or 200 ul made with CFA. The 4.1/control (TT)-immunized io 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 is 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 2o 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~
2s and 50~, respectively.

WO 98/58954 PC'T/EP98/04016 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): 6829 BM
(G) TELEPHONE: 0412 666379 (H) TELEFAX: 0412 650592 (iiy 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 (ivy COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
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Met Glu Ser Ala Gln Gln Leu Pro Pro Gln Leu Leu Ala Ala Ala Ser Asn Leu Ser Asn Phe Gln Ser ThrThr Ser Cys Gly Glu Arg Met Pro Pro Ala Leu Asp Val Gly Thr Pro LysGlu Asp Lys Gly Ser Rla Ala Asn Gln Glu Met Ser Val Tyr Asn AlaVal Lys Ser Ala Ser Leu Asp Arg Lys Ala Val Ala Leu Tyr Gly AlaSer Asn Ile His Pro Val Gly Thr Lys Ala Ser Gly Pro Ser Ser SerPro Ile Ala Thr Ser Gly Gly Ser Pro Thr Thr Thr Pro Lys Pro PheAsn Leu Pro Thr Pro Ser His Pro Ala Pro His Leu Ala Met Leu LysLeu Asn Leu Ser Gln Gln Ser Gln Tyr Gln Gly Met Ala Thr Gly ProGly Glu Ala Ala Pro Gln Ala Gly Pro Leu Gln Asn Asp Gly Gln GlyGly Ala Trp Phe Ala Ala Glu Ser Leu Ser Pro Ser Gly Pro IleIle Asp Ala Ala Ala Ser Gln Ser Asp Pro Val Leu Val Asp Val Glu Glu Leu Glu Gly Val Leu Met Ser Leu Asp Arg Ala Asn Glu Leu Pro Glu Leu Trp Leu Gly Gln Asn Glu Phe Asp Phe Thr Ala Asp Phe Pro Ser Ser Cys (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: NO:6:
SEQ
ID

Val Pro AlaSerGln SerThr GlyLeuGly ValArgGlu ThrTrpLeu Arg Ile ProAlaPro ProLeu SerSerAla ValThrLeu AlaGlnGln Leu Gln LeuAlaAla SerLeu ProAlaAsn LeuSerAsn PheCysGln Gly Ser GluMetPro ThrThr SerArgPro AlaLeuAsp ValLysGly Gly Thr SerProRla LysGlu AspAlaAsn GlnGluMet SerSerVal Ala Tyr SerAsnLeu AlaVal LysAspArg LysAlaVal AlaIleLeu His Tyr ProGlyVal AlaSer AsnGlyThr LysAlaSer GlyAlaPro q0 100 105 110 Thr Ser SerSerGly SerPro IleGlySer ProThrThr ThrProPro 95 Thr Lys ProProSer PheAsn LeuHisPro AlaProHis LeuLeuAla Ser Met Gln Leu Gln Lys Leu Asn Ser Gln Tyr Gln Gly Met Ala Ala Ala Thr Pro Gly Gln Pro Gly Glu Ala Gly Pro Leu Gln Asn Trp Asp Phe Gly Ala Gln Ala Gly Gly Ala Glu Ser Leu Ser Pro Ser Ala Gly Ala Gln Ser Pro Ala Ile Ile Asp Ser Asp Pro Val Asp Glu Glu Val Leu Met Ser Leu Val Val Glu Leu Gly Leu Asp Arg Ala Asn Glu Leu _ 24 _ Pro Glu Leu Trp Leu Gly Gln Asn Glu Phe Asp Phe Thr Ala Asp Phe Pro Ser Ser Cys (2) INFORMATION FOR SEQ ID NO: 7:
' (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 963 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
NO: 7:

TGTTGTAAATAAAAATAAAG TTACTTACAA AGAGAAAAAAAAA~~ ~'~ 960 AAA

(2) INFORMATION
FOR SEQ
ID NO:

8:

(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: 8:

' GAGTCCCTGC TTCGCAGAGC ACCGGACTTG GAGTCAGAGA GACCTGGCTG 60 CGAATCCCGG

GCATCACTTC

AGGCCTGCAC

ATGAGCTCCG

CACTACCCTG

GGATCTCCAA

CACCCCGCCC

GGGATGGCTG

TTTGGGGCCC

GCTATCATCG

GGGTTGGACC

ACTGCGGACT

CACCAATTCT

GTTGTAAATA AAAATAAAGT TACTTACAAA GAGAAAAAAA P.F~7~AAAAP.AP.840 AAAAAAAAAA

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 219 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE NO:9:
DESCRIPTION:
SEQ ID

Met Glu Pro Ala Gln Gln GlnLeu Ala SerLeu Pro Ser Leu Ala Ala Asn Leu Ser Phe Cys Gln SerGlu Met ThrThr Ser Asn Gly Pro Arg Pro Ala Leu Val Lys Gly ThrSer Pro LysGlu Asp Asp Gly Ala Ala Asn GlnGluMet SerSerVal AlaTyr SerAsnLeu AlaValLys Asp Arg LysAlaVal AlaIleLeu HisTyr ProGlyVal AlaSerAsn Gly Thr LysAlaSer GlyAlaPro Thr5er SerSerGly SerProIle Gly Ser ProThrThr ThrProPro ThrLys ProProSer PheAsnLeu His Pro AlaProHis LeuLeuAla SerMet GlnLeuGln LysLeuAsn Ser Gln TyrGlnGly MetAlaAla AlaThr ProGlyGln ProGlyGlu Ala Gly ProLeuGln AsnTrpAsp PheGly AlaGlnAla GlyGlyAla Glu Ser LeuSerPro SerAlaGly AlaGln SerProAla IleIleAsp Ser Asp ProValAsp GluGluVal LeuMet SerLeuVal ValGluLeu Gly Leu AspArgAla AsnGluLeu ProGlu LeuTrpLeu GlyGlnAsn Glu Phe Asp Phe Thr Ala Asp Phe Pro Ser Ser Cys (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 244 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single 90 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: NO:I0:
SEQ
ID

Val ProAla SerGlnSer ThrGly LeuGlyValArg GluThr TrpLeu Arg IlePro AlaProPro LeuSer SerAlaValThr LeuAla GlnGln Leu GlnLeu AlaAlaSer LeuPro AlaAsnLeuSer AsnPhe CysGln Gly SerGlu MetProThr ThrSer ArgProAlaLeu AspVal LysGly Gly ThrSer ProAlaLys GluAsp AlaAsnGlnGlu MetSer Ser~(al Ala Tyr Ser Asn Leu Ala Val Lys Asp Arg Lys Ala Val Ala Ile Leu His Tyr Pro Gly Val Ala Ser Asn Gly Thr Lys Ala Ser Gly Ala Pro Thr Ser Ser Ser Gly Ser Pro Ile Gly Ser Pro Thr Thr Thr Pro Pro Thr Lys Pro Pro Ser Phe Asn Leu His Pro Ala Pro His Leu Leu Ala Ser Met Gln Leu GIn Lys Leu Asn Ser Gln Tyr Gln Gly Met Ala Ala Ala Thr Pro Gly Gln Pro Gly Glu Ala Gly Pro Leu Gln Asn Trp Asp Phe Gly Ala Gln Ala Gly Gly Ala Glu Ser Leu Ser Pro Ser Ala Gly Ala Gln Ser Pro Ala Ile Ile Asp Ser Asp Pro Val Asp Glu Glu Val Leu Met Ser Leu Val Val Glu Leu Gly Leu Asp Arg Ala Asn Glu Leu Pro Glu Leu Trp Leu Gly Gln Asn Glu Phe Asp Phe Thr Ala Asp Phe Pro Ser Ser Cys (2) INFORMATION FOR SEQ ID NO: Il:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1273 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

_ 28 _ GAACGGAGCC AATAAAGCTT TGTGTGCCTT CAAAAAAAAA AAi4PvAAAAAA AAAAAAAAAA 1260 3 o P,,P~F~AAAAAAA AAA 12 7 3 (2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 288 amino acids (B) TYPE: amino acid tC) STRANDEDNESS: single (D) TOPOLOGY: linear 90 tii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: 5EQ ID NO: 12:
Ala Gly Gly Leu Pro Val Arg His Pro Trp Gly Ala Cys Pro Phe Lys His Glu SerTyr Gln Ser Pro Ser LeuLeu Ala Leu His Arg Val Pro Ala Trp CysArg Leu Thr Phe Leu LeuIle Gln Gln Val Glu Glu Pro Ser Cys LysVal Leu Thr Ala Leu LeuGly Gly Gly Val Gly Leu Gly Leu Met Leu Trp Val Gln Rrg Lys Arg Arg Arg Lys Glu Thr Ser Glu Cys Pro Ser Asp Lys Asp Lys Ser Pro Glu Ser His Lys Ala Lys Asn CGAATCCCGG

WO 98/58954 . PCT/EP98/04016 Glu Ser Trp Ile Lys Ser His Phe Ser Arg Leu Ser Glu Glu Lys Leu Ala Leu Asp Asn Asn Ala Ser Ala Ser Gly Asn Ala Thr Gln Thr Glu ~ Ser Gly Ser Glu Glu Val Ser Ser Thr Val His Ile Glu Thr Phe Thr Thr Arg His Gly Glu Val Gly Ser Ala Leu His Arg Glu Ser Phe Thr Ser Arg Gln LysThrSer Gly Pro ValIle Gln Ile His Ser Glu Gln Glu Ser Gly LysAlaPro Ser Thr AspAla Thr Ala Ala Asp Trp Val Ala Ala Cys ThrLysGlu Ile Asp GlnGly Arg Leu Ala Thr His His Ser Met Leu Gln Arg Ala Ile Ala Tyr Gln His Ser Gly His Leu Glu Ser Lys Asp Ile Asn Gln Glu Glu Leu Arg Ala Leu Glu Glu Val Glu Met Lys Leu Gln Lys Asn Phe Leu Thr Gln Arg Glu Asn Thr Ile Ala Gly Ala Asn His Thr His Thr Phe Tyr Gly His Ser His His Ser His His Gly His Pro Ser His Gln Ser His Ser Leu Pro Asn Arg Arg His 90 (2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 842 base pairs (B) TYPE: nucleic acid 95 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 13:

' ACGCCACTCC GGGCCAACCT GGAGAGGCAG GGCCCCTGCAAAACTGGGAC TTGGGGGCTC540 TCTGTTGTAA ATAAAAATRA AGTTACTTGC AAAGAGACAGGCCAAAAA1'1A F~~AA~AA840 AA

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 240 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCEDES CRIPTION : NO:14:
SEQ
ID

Pro SerArgSer AlaVal TrpIle PheLysPheLeu LeuLeuSer Glu Lys IleAlaIle GluMet GluPro SerAlaGlnGln LeuGlnLeu Ala Ala SerLeuPro AlaAsn LeuSer AsnPheCysGln GlySerGlu Met Pro ThrThrSer ArgPro AlaLeu AspValLysGly GlyThrSer Pro 50 Ala LysGluAsp AlaAsn GlnGlu MetSerSerVal AlaTyrSer Asn Leu AlaValLys AspArg LysAla ValAlaIleLeu HisTyrPro Gly Val RlaSerAsn GlyThr LysAla SerGlyAlaPro ThrSerSer Ser Gly SerProIle GlySer ProThr ThrThrProPro ThrLysPro Pro s Ser PheAsnLeu HisPro AlaPro HisLeuLeuAla SerMetGln Leu Gln Lys Leu Asn Ser Gln Tyr Gln Gly Met Ala Ala Ala Thr Pro Gly Gln Pro Gly Glu Ala Gly Pro Leu Gln Asn Trp Asp Phe Gly Ala Gln Ala Gly Gly Ala Glu Ser Leu Ser Pro Ser Ala Gly Ala Gln Ser Pro Ala Ile Ile Asp Ser Asp Pro Val Asp Glu Glu Val Leu Met Ser Leu Val Val Glu Leu Gly Leu Asp Arg Ala Asn Glu Leu Pro Glu Leu Trp Leu Gly Gln Asn Glu Phe Asp Phe Thr Ala Asp Phe Pro Ser Ser Cys (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:

AAGAAAGGAA ACCTCTGAGT
GTCCATCAGA

. CACGAGGCAC GGAGAAGTGG GCTCCGCTCTGCACCGGGAA TCCTTCACCA GCAGGCAGAA660 2 ATAAAGCTTT GTGTGCCTTC PEA p~~AAAAAAAA p~~A AAAAAAAAAA 14 p~ 1942 (2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 323 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single 30(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 Gln Leu Pro Ser Ser Gln Trp Gly Ser Leu Leu Arg Trp Trp Gly Ala Pro Arg Glu Gly Ala Ala Gly Gly Leu Pro Val Arg His Pro Trp Gly Ala Cys Pro Phe Lys His Glu Ser Tyr His Gln Arg Ser Pro Ser Val Leu Leu Ala Leu Pro Ala Trp Cys Arg Val Leu Glu Thr Phe Leu Glu Leu Ile Gln Gln Pro Ser Cys Lys Val Val Leu Gly Thr Ala Leu Leu Leu Gly 85 90 9'S
Gly Gly Gly Leu Met Leu Trp Val Gln Arg Lys Arg Arg Arg Lys Glu Thr Ser Glu Cys Pro Ser Asp Lys Asp Lys Ser Pro Glu Ser His Lys Ala Lys AsnGluSer TrpIle LysSerHisPhe SerArgLeu SerGlu Glu Lys LeuAlaLeu AspAsn AsnAlaSerAla SerGlyAsn AlaThr Gln Thr GluSerGly SerGlu GluValSerSer ThrValHis IleGlu Thr Phe ThrThrArg HisGly GluValGlySer AlaLeuHis ArgGlu Ser Phe ThrSerArg GlnLys ThrSerGlyPro SerValIle GlnGlu Ile His GlnGluSer GlyLys AlaProSerThr AspAspAla ThrTrp Ala Ala ValAlaAla CysThr LysGluIleAsp ThrGlnGly ArgHis Leu Ala HisSerMet LeuGln ArgAlaIleAla TyrGlnHis SerGly ZS His Leu GluSerLys AspIle AsnGlnGluGlu LeuArgAla LeuGlu Glu Val GluMetLys LeuGln LysAsnPheLeu ThrGlnArg GluAsn Thr Ile AlaGlyAla AsnHis ThrHisThrPhe TyrGlyHis SerHis His Ser HisHisGly HisPro SerHisGlnSer HisSerLeu ProAsn Arg Arg His

Claims (8)

Claims:

1. Nucleotide sequence encoding a mammalian sperm protein 4.1 having the amino acid sequence depicted in SEQ ID NO:2, SEQ ID
NO:12 or SEQ ID NO:16.

2. Nucleotide sequence encoding a mammalian sperm protein 4.1 characterized in that said nucleotide sequence comprises the sequence depicted in SEQ ID NO:1, SEQ ID NO:11, or SEQ ID
NO:15.

3. Nucleotide sequence according to claim 1 or 2, characterized in that the mammalian is marmoset or human.

4. Expression vector comprising a nucleotide sequence according to claim 1 or 2.

5. Host cell comprising a nucleotide sequence according to claim 1 or 2 or an expression vector according to claim 3.

6. Mammalian sperm protein 4.1 characterized in that said protein comprises the amino acid sequence depicted in SEQ ID NO:2, SEQ
ID NO:12 or SEQ ID NO:16.

7. Mammalian sperm protein comprising an 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 for use as a medicament.

8. Contraceptive vaccine comprising one or more sperm proteins which comprise an 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 and a pharmaceutical acceptable carrier.